**Prohepcidin and Hepcidin in Acute Phase Reaction Accompanying Large Cardiac Surgery**

Pavel Maruna, Martin Vokurka and Jaroslav Lindner *Charles University, 1Faculty of Medicine, Institute of Pathological Physiology, 2Department of Surgery - Department of Cardiovascular Surgery, Prague, Czech Republic* 

## **1. Introduction**

238 Inflammatory Diseases – Immunopathology, Clinical and Pharmacological Bases

Zhang, M., Q. Wang, et al. (2004). "Effective induction of immune tolerance by portal venous

of allograft survival." *J Mol Med* 82(4): 240-249.

infusion with IL-10 gene-modified immature dendritic cells leading to prolongation

Hepcidin, a small cystein-rech peptide produced by the liver, was first described as an antimicrobial peptide (hepatic bactericidal protein) (Park et al. 2001) and subsequently discovered as a key regulator of iron homeostasis. Via regulation of ferroportin, hepcidin inhibits intestinal iron absorption and iron release from macrophages and hepatocytes (Ganz, 2006; Papanikolaou et al., 2005).

Hepcidin levels are regulated by at least three independent mechanisms (Nicolas et al., 2002). Whereas both inflammation and iron loading induce hepcidin production, erythropoietic activity suppresses its production. Apart from those factors also endoplasmic reticulum stress can induce hepcidin expression (Vecchi et al., 2009). In general, the decrease of hepcidin due to the iron deficiency or enhanced erythropoiesis stimulates iron intestinal absorption. On the other hand, iron-stimulated hepcidin production prevents iron overload. Increase in hepcidin concentration due to inflammation is considered important for iron sequestration (Ganz & Nemeth, 2009). Studies of humans with severe inflammatory diseases have shown increased levels of hepcidin, suggesting that elevated hepcidin levels play a role in the anemia of inflammation (Nemeth et al., 2004; Krijt et al., 2009). Due to dominant regulation by interleukin-6 (IL-6), hepcidin was classified as a type II acute-phase protein (Nemeth et al., 2003). Increased IL-6 levels cause the binding of signal transducer and activator of transcription 3 (STAT3) to the hepcidin promoter, increasing its activity (Wrighting & Andrews, 2006). Moreover, hepcidin itself has the potential to bind bivalent metal ions (Tselepis et al., 2010) and to mediate transcriptional changes of a wide variety of genes which can play important role in inflammatory response (De Domenico et al., 2010). That is why hepcidin can be considered as important inflammatory mediator, and its measurement can be helpful in many clinical situations. Technical problems prevented reliable and routine measurements of active 25 amino acid (aa) hepcidin in plasma until 2009. Therefore there are only limited data on hepcidin in human subjects, and much of findings come from animal and *in vitro* models.

To evaluate the time course of plasma hepcidin and its precursor prohepcidin in relation to inflammatory parameters, authors used a specific group of patients undergoing large cardiac surgery - pulmonary endarterectomy (PEA) in a deep hypothermic circulatory arrest

Prohepcidin and Hepcidin in Acute Phase Reaction Accompanying Large Cardiac Surgery 241

Radial and femoral artery cannulae, triple lumen central venous cannula, Swan-Ganz catheter, and single lumen jugular bulb catheter were inserted for continuous monitoring of hemodynamic parameters and jugular bulb blood saturation. Left atrial catheter was

Arterial blood samples were drawn from femoral artery catheter before operation, after sternotomy, after DHCA, after separation from CPB, then 12, 18, 24, 36, 48, 72, and 120 h after separation from CPB. For all measurements, 5-ml of arterial blood was drawn into a vacutainer heparin tube and immediately centrifuged at 5000 rpm for 15 min. Plasma was

Plasma hepcidin and prohepcidin concentrations were measured by enzyme-linked immunoassays using a commercially available kits (DRG Diagnostics, Marburg, Germany) in duplicates - the analytical sensitivity of the assay for hepcidin analysis was 0.9 ng/ml, intraassay coefficients of variation (CV) calculated by DRG Diagnostics was below 5%, inter-assay precision was 11% (6,0 ng/ml) and 10% (15,8 ng/ml). Plasma levels of procalcitonin (PCT) were detected by Kryptor test (BRAHMS AG, Hennigsdorf, Germany) in duplicates. The sensitivity of the analytic method was 0.02 ng/ml. Plasma concentrations of tumor necrosis factor (TNF)α, IL-6, IL-8 (ELISA, Immunotech, Paris, France), C-reactive protein (CRP) (Kryptor - TRACE technology, ultrasensitive analysis, BRAHMS AG, Hennigsdorf, Germany), α1-antitrypsin, and ceruloplasmin (nephelometry, BRAHMS AG, Hennigsdorf, Germany)

Plasma iron (colorimetric analysis, Pliva-Lachema a.s., Brno, Czech Republic), ferritin, and transferrin (immunoturbidimetry, Dialab Gmbh, Wr. Neudorf, Austria) were examined

Mean pulmonary artery pressure (MPAP), cardiac index (CI), pulmonary vascular resistance (PVR), and ejection fraction (EF) were followed. The time of norepinephrine support was

Statistical analysis was carried out using SPSS software (version 12.0) for Windows (SPSS, Chicago, USA). The normal distribution of all data was examined using the Kolmogorov-Smirnov normality test to determine subsequent use of tests for statistical comparison. As variables were not normally distributed, the data were reported as medians and interquartile range. Bonferroni correction (multiple-comparison correction) was used to analyze simultaneous measurement at different time points. The Pearson's correlation coefficient and the Spearman's rank correlation evaluated correlation between the

surgically placed for both measurement and norepinephrine administration.

were measured in duplicates, too. The intra- and inter-assay CV were below 5%.

preoperatively and repeatedly within 120 h after the end of surgery.

indicators. For all the tests, p *<* 0.05 was defined as statistically significant.

**2.5 Hepcidin, prohepcidin and inflammatory parameters** 

**2.3 Monitoring** 

**2.4 Blood sample collection** 

stored at -80°C until analysis.

**2.6 Iron, ferritin and transferrin** 

**2.7 Hemodynamic parameters** 

recorded in all patients.

**2.8 Statistical analysis** 

(DHCA). PEA is a potential curative treatment method for patients with chronic thromboembolic pulmonary hypertension (CTEPH). PEA provides a significant survival advantage, compared to the natural prognosis of CTEPH (Roscoe & Klein, 2008). The kinetics of main pro-inflammatory cytokines after PEA were described by Lindner et al. (2009) and Maruna et al. (2011) showing the relations of cytokine network to hemodynamic disturbances post-surgery. PEA leads to a more pronounced activation of cytokines than other surgical procedures (Langer et al., 2004; Martínes-Rosaz, 2006; Maruna et al., 2008 and 2009) with subsequent development of the systemic inflammatory response syndrome. Authors postulated that post-surgery changes of plasma hepcidin and its precursor would be responding to dynamics of other acute-phase proteins and related to IL-6 or other main inflammatory cytokines development.

## **2. Material and methods**

#### **2.1 Patients' group**

A prospective study was approved by local research and ethics committee and a written informed consent was obtained from the subjects. Patients with CTEPH scheduled for isolated PEA on the 2nd Department of Surgery - Department of Cardiovascular Surgery, General Faculty Hospital in Prague were enrolled into study between January 2008 and March 2011. Respecting gender differences in hepcidin basal concentrations with significantly higher levels in men (Grebenchtchikov et al., 2009), only male patients were included into our study. Exclusion criteria were the combination of PEA with other surgical procedure (PEA with maze or PEA with coronary artery bypass grafting), postoperative bleeding, thromboembolic complication, local and systemic infection, defined according to guidelines of the Center for Disease Control and Prevention (Horan & Gaynes, 2004).

#### **2.2 Anesthesia and surgical procedures**

30 minutes before skin incision, the first prophylactic dose of sultamicillin 1.5 g (Unasyn, Pfizer, Italy) was given. The same dose was repeated every 3 hours throughout the procedure and every 6 hours postoperatively until postoperative day 2. A total intravenous anesthesia using combination of benzodiazepines, propofol, opioids and muscle relaxants routinely used in our institution for PEA was given to all study patients.

The *s*tandard approach for PEA was median sternotomy. Cardiopulmonary bypass (CPB) was established with cannulation of the ascending aorta and the inferior and superior vena cava. DHCA (18 – 20 0C) was used to ensure optimum operating conditions and facilitate accurate endarterectomty. Endarterectomy was started with dissection in right level of pulmonary artery and followed to the segmental branches. To achieve accurate visualization during peripheral dissection, repeated periods of DHCA limited to 20 minutes were performed with reestablishment of CPB between them. Arteriotomy on the main pulmonary artery was started on the left side and continued to the left branch. After completion of endarterectomy on the both sides, CPB was recommenced along with controlled rewarming. Weaning from CPB was started with pressure control ventilation with positive endexpiratory pressure, atrio-ventricular epicardial stimulation, stepwise increased filling of the right heart and reduction of pump flow together with low doses of norepinephrine Dobutamine (Dobutrex, Lily, Germany) was administered only if inotropic support was needed during or after weaning of CPB. Before the end of CPB, we used an ultra filtration for hemoconcentration.

## **2.3 Monitoring**

240 Inflammatory Diseases – Immunopathology, Clinical and Pharmacological Bases

(DHCA). PEA is a potential curative treatment method for patients with chronic thromboembolic pulmonary hypertension (CTEPH). PEA provides a significant survival advantage, compared to the natural prognosis of CTEPH (Roscoe & Klein, 2008). The kinetics of main pro-inflammatory cytokines after PEA were described by Lindner et al. (2009) and Maruna et al. (2011) showing the relations of cytokine network to hemodynamic disturbances post-surgery. PEA leads to a more pronounced activation of cytokines than other surgical procedures (Langer et al., 2004; Martínes-Rosaz, 2006; Maruna et al., 2008 and 2009) with subsequent development of the systemic inflammatory response syndrome. Authors postulated that post-surgery changes of plasma hepcidin and its precursor would be responding to dynamics of other acute-phase proteins and related to IL-6 or other main

A prospective study was approved by local research and ethics committee and a written informed consent was obtained from the subjects. Patients with CTEPH scheduled for isolated PEA on the 2nd Department of Surgery - Department of Cardiovascular Surgery, General Faculty Hospital in Prague were enrolled into study between January 2008 and March 2011. Respecting gender differences in hepcidin basal concentrations with significantly higher levels in men (Grebenchtchikov et al., 2009), only male patients were included into our study. Exclusion criteria were the combination of PEA with other surgical procedure (PEA with maze or PEA with coronary artery bypass grafting), postoperative bleeding, thromboembolic complication, local and systemic infection, defined according to

guidelines of the Center for Disease Control and Prevention (Horan & Gaynes, 2004).

routinely used in our institution for PEA was given to all study patients.

30 minutes before skin incision, the first prophylactic dose of sultamicillin 1.5 g (Unasyn, Pfizer, Italy) was given. The same dose was repeated every 3 hours throughout the procedure and every 6 hours postoperatively until postoperative day 2. A total intravenous anesthesia using combination of benzodiazepines, propofol, opioids and muscle relaxants

The *s*tandard approach for PEA was median sternotomy. Cardiopulmonary bypass (CPB) was established with cannulation of the ascending aorta and the inferior and superior vena cava. DHCA (18 – 20 0C) was used to ensure optimum operating conditions and facilitate accurate endarterectomty. Endarterectomy was started with dissection in right level of pulmonary artery and followed to the segmental branches. To achieve accurate visualization during peripheral dissection, repeated periods of DHCA limited to 20 minutes were performed with reestablishment of CPB between them. Arteriotomy on the main pulmonary artery was started on the left side and continued to the left branch. After completion of endarterectomy on the both sides, CPB was recommenced along with controlled rewarming. Weaning from CPB was started with pressure control ventilation with positive endexpiratory pressure, atrio-ventricular epicardial stimulation, stepwise increased filling of the right heart and reduction of pump flow together with low doses of norepinephrine Dobutamine (Dobutrex, Lily, Germany) was administered only if inotropic support was needed during or after weaning of CPB. Before the end of CPB, we used an ultra filtration

inflammatory cytokines development.

**2.2 Anesthesia and surgical procedures** 

**2. Material and methods** 

**2.1 Patients' group** 

for hemoconcentration.

Radial and femoral artery cannulae, triple lumen central venous cannula, Swan-Ganz catheter, and single lumen jugular bulb catheter were inserted for continuous monitoring of hemodynamic parameters and jugular bulb blood saturation. Left atrial catheter was surgically placed for both measurement and norepinephrine administration.

## **2.4 Blood sample collection**

Arterial blood samples were drawn from femoral artery catheter before operation, after sternotomy, after DHCA, after separation from CPB, then 12, 18, 24, 36, 48, 72, and 120 h after separation from CPB. For all measurements, 5-ml of arterial blood was drawn into a vacutainer heparin tube and immediately centrifuged at 5000 rpm for 15 min. Plasma was stored at -80°C until analysis.

## **2.5 Hepcidin, prohepcidin and inflammatory parameters**

Plasma hepcidin and prohepcidin concentrations were measured by enzyme-linked immunoassays using a commercially available kits (DRG Diagnostics, Marburg, Germany) in duplicates - the analytical sensitivity of the assay for hepcidin analysis was 0.9 ng/ml, intraassay coefficients of variation (CV) calculated by DRG Diagnostics was below 5%, inter-assay precision was 11% (6,0 ng/ml) and 10% (15,8 ng/ml). Plasma levels of procalcitonin (PCT) were detected by Kryptor test (BRAHMS AG, Hennigsdorf, Germany) in duplicates. The sensitivity of the analytic method was 0.02 ng/ml. Plasma concentrations of tumor necrosis factor (TNF)α, IL-6, IL-8 (ELISA, Immunotech, Paris, France), C-reactive protein (CRP) (Kryptor - TRACE technology, ultrasensitive analysis, BRAHMS AG, Hennigsdorf, Germany), α1-antitrypsin, and ceruloplasmin (nephelometry, BRAHMS AG, Hennigsdorf, Germany) were measured in duplicates, too. The intra- and inter-assay CV were below 5%.

## **2.6 Iron, ferritin and transferrin**

Plasma iron (colorimetric analysis, Pliva-Lachema a.s., Brno, Czech Republic), ferritin, and transferrin (immunoturbidimetry, Dialab Gmbh, Wr. Neudorf, Austria) were examined preoperatively and repeatedly within 120 h after the end of surgery.

## **2.7 Hemodynamic parameters**

Mean pulmonary artery pressure (MPAP), cardiac index (CI), pulmonary vascular resistance (PVR), and ejection fraction (EF) were followed. The time of norepinephrine support was recorded in all patients.

## **2.8 Statistical analysis**

Statistical analysis was carried out using SPSS software (version 12.0) for Windows (SPSS, Chicago, USA). The normal distribution of all data was examined using the Kolmogorov-Smirnov normality test to determine subsequent use of tests for statistical comparison. As variables were not normally distributed, the data were reported as medians and interquartile range. Bonferroni correction (multiple-comparison correction) was used to analyze simultaneous measurement at different time points. The Pearson's correlation coefficient and the Spearman's rank correlation evaluated correlation between the indicators. For all the tests, p *<* 0.05 was defined as statistically significant.

Prohepcidin and Hepcidin in Acute Phase Reaction Accompanying Large Cardiac Surgery 243

were detected after separation from CPB (p 0.001 in relation to preoperative levels) after which the levels started rise. Concentrations returned to initial levels within 24 – 48 h after the separation from CPB. The following increase was found in samples at 72 h and 120 h,

Fig. 2. Hepcidin plasma concentrations in perioperative period (median values, interquartile

Post-surgical course of plasma hepcidin was associated with a transient initial decline from preoperative level 8.6 ng/ml (6.4 – 10.2) and subsequent significant elevation above preoperative levels (Fig. 2). Minimal hepcidin concentrations were found in blood samples collected after the last DHCA. Initial decline of hepcidin appeared to correlate with the decreased hematocrit due to hemodilution on CPB (r = 0.86, p = 0.002). After it, hepcidin increased to maximum 16.2 ng/ml (13.9 – 18.9) measured 72 h after the end of surgery (e.g., separation from CPB). In following period, the levels started declining but were still higher

As expected, all inflammatory cytokines, PCT and acute-phase proteins increased after surgery. An uncomplicated course after PEA was associated with a transient initial decline of PCT and subsequent elevation. Minimal PCT concentrations were found in blood samples collected after the last DHCA (Fig. 3). Initial decrease of PCT appeared to correlate with the decreased hematocrit due to hemodilution on CPB (r = 0.78, p < 0.01). PCT increased postoperatively from 0.22 ng/ml (0.15 – 0.31) reaching a peak level 24 h after the end of

All measured inflammatory cytokines increased after surgery. TNFα rose from 18 ng/l (10 – 47) to maximum 216 ng/l (136 – 415) in blood sample collected 12 h after separation from CPB. IL-6 rise was maximal 12 hours postoperatively from 26.2 ng/l (21.0 – 36.7) to 544.2 ng/l (411.0 – 641.2) with following decline (Fig. 4). IL-8 with preoperative levels 42.4 ng/l

than pre-operatively until 120 h after the end of surgery.

**3.4 Cytokines and acute-phase proteins** 

surgery (2.05 ng/ml, 1.68 – 2.52).

but without a statistical significance to preoperative levels on p 0.05.

range and full range)

**3.3 Hepcidin** 

## **3. Results**

## **3.1 Patients**

34 patients were enrolled during the 39 months of the trial (Tab. 1). All patients underwent satisfactory clearance of intra-arterial obstruction, and there were no intra-operative deaths. No patients required allogenic blood transfusion. Mean duration of CPB was 334.2 ± 44.6 min.; mean duration of crossclamping time was 121.0 ± 20.5 min. and circulatory arrest time 41.9 ± 7.1 min. Extracorporeal circulation (ECC) time was 330.9 ± 54.4 min.; duration of mechanical ventilation was 51.9 ± 30.6 h. There was considerable improvement in hemodynamic variables. PEA significantly decreased MPAP (from 53.4 ± 8.66 to 25.1 ± 7.04 mm Hg, p < 0.001) as well as pulmonary vascular resistance (from 1095.4 ± 325.3 to 209.7 ± 94.1 dynes.s.cm-5, p < 0.001). CI increased within first 24 h after surgery (from 1.90 ± 0.37 to 3.04 ± 0.49 l.min- ¹m- ², p < 0.001).


Variables are absolute number or mean (standard deviation).

Table 1. Pre-operative data

## **3.2 Prohepcidin**

The time course of prohepcidin in perioperative period is shown in Fig. 1. Cardiac surgery with CPB induced a 49 % fall in plasma prohepcidin. Prohepcidin decreased from preoperative level 94.9 ng/ml (84.5 – 104.9) (median and interquartile range) to minimum 48.5 ng/ml (38.2 – 56.8). The initial decline was revealed after DHCA, and minimal concentrations

Box and whisker plot depicting the median values, interquartile range and full range. … Statistically significant differences to preoperative values, p 0.05. The same arrangement used for Fig 1 – 5.

Fig. 1. Prohepcidin plasma concentrations in perioperative period.

were detected after separation from CPB (p 0.001 in relation to preoperative levels) after which the levels started rise. Concentrations returned to initial levels within 24 – 48 h after the separation from CPB. The following increase was found in samples at 72 h and 120 h, but without a statistical significance to preoperative levels on p 0.05.

Fig. 2. Hepcidin plasma concentrations in perioperative period (median values, interquartile range and full range)

#### **3.3 Hepcidin**

242 Inflammatory Diseases – Immunopathology, Clinical and Pharmacological Bases

34 patients were enrolled during the 39 months of the trial (Tab. 1). All patients underwent satisfactory clearance of intra-arterial obstruction, and there were no intra-operative deaths. No patients required allogenic blood transfusion. Mean duration of CPB was 334.2 ± 44.6 min.; mean duration of crossclamping time was 121.0 ± 20.5 min. and circulatory arrest time 41.9 ± 7.1 min. Extracorporeal circulation (ECC) time was 330.9 ± 54.4 min.; duration of mechanical ventilation was 51.9 ± 30.6 h. There was considerable improvement in hemodynamic variables. PEA significantly decreased MPAP (from 53.4 ± 8.66 to 25.1 ± 7.04 mm Hg, p < 0.001) as well as pulmonary vascular resistance (from 1095.4 ± 325.3 to 209.7 ± 94.1 dynes.s.cm-5, p < 0.001). CI increased within first 24 h after surgery (from 1.90 ± 0.37 to

The time course of prohepcidin in perioperative period is shown in Fig. 1. Cardiac surgery with CPB induced a 49 % fall in plasma prohepcidin. Prohepcidin decreased from preoperative level 94.9 ng/ml (84.5 – 104.9) (median and interquartile range) to minimum 48.5 ng/ml (38.2 – 56.8). The initial decline was revealed after DHCA, and minimal concentrations

**3. Results 3.1 Patients** 

3.04 ± 0.49 l.min-

**3.2 Prohepcidin** 

¹m-

Table 1. Pre-operative data

², p < 0.001).

Variables are absolute number or mean (standard deviation).

Number of patients (males) 34 Age (yr.) 51.4 (9.6) Preoperative New York Heart Association classification 3.4 (0.4) Mean pulmonary artery pressure (mm Hg) 54.2 (6.8)

Box and whisker plot depicting the median values, interquartile range and full range.

… Statistically significant differences to preoperative values, p 0.05.

Fig. 1. Prohepcidin plasma concentrations in perioperative period.

The same arrangement used for Fig 1 – 5.

Post-surgical course of plasma hepcidin was associated with a transient initial decline from preoperative level 8.6 ng/ml (6.4 – 10.2) and subsequent significant elevation above preoperative levels (Fig. 2). Minimal hepcidin concentrations were found in blood samples collected after the last DHCA. Initial decline of hepcidin appeared to correlate with the decreased hematocrit due to hemodilution on CPB (r = 0.86, p = 0.002). After it, hepcidin increased to maximum 16.2 ng/ml (13.9 – 18.9) measured 72 h after the end of surgery (e.g., separation from CPB). In following period, the levels started declining but were still higher than pre-operatively until 120 h after the end of surgery.

#### **3.4 Cytokines and acute-phase proteins**

As expected, all inflammatory cytokines, PCT and acute-phase proteins increased after surgery. An uncomplicated course after PEA was associated with a transient initial decline of PCT and subsequent elevation. Minimal PCT concentrations were found in blood samples collected after the last DHCA (Fig. 3). Initial decrease of PCT appeared to correlate with the decreased hematocrit due to hemodilution on CPB (r = 0.78, p < 0.01). PCT increased postoperatively from 0.22 ng/ml (0.15 – 0.31) reaching a peak level 24 h after the end of surgery (2.05 ng/ml, 1.68 – 2.52).

All measured inflammatory cytokines increased after surgery. TNFα rose from 18 ng/l (10 – 47) to maximum 216 ng/l (136 – 415) in blood sample collected 12 h after separation from CPB. IL-6 rise was maximal 12 hours postoperatively from 26.2 ng/l (21.0 – 36.7) to 544.2 ng/l (411.0 – 641.2) with following decline (Fig. 4). IL-8 with preoperative levels 42.4 ng/l

Prohepcidin and Hepcidin in Acute Phase Reaction Accompanying Large Cardiac Surgery 245

Fig. 4. IL-6 plasma concentrations in perioperative period (median values, interquartile

Fig. 5. CRP plasma concentrations in perioperative period (median values, interquartile

Minimal perioperative prohepcidin concentrations correlated inversely with ECC time (r = -0.84, p < 0.01). When evaluating patients with a quick normalization of prohepcidin into preoperative range within 24 h after separation from CPB (Subgroup 1, n = 16), higher peak plasma levels of IL-6 (620.9 ng/l, 484.2 – 782.0) were revealed in this subgroup compared with patients with delayed prohepcidin normalization (Subgroup 2, n = 18), their IL-6 peak levels were 438.2 ng/l (370.1 – 512.7), p 0.05 between subgroups. Similarly, IL-8 peak postoperative concentrations were higher in Subgroup 1 in relation to Subgroup 2, but

**3.6 Relations among hepcidin, prohepcidin and hemodynamic parameters** 

range and full range)

range and full range)

the differences were not significant on p 0.05.

(22.3 – 82.0) culminated later, 18 h after separation from CPB (446.0 ng/l, 274.2 – 658.1) with following decline.

Three tested acute-phase proteins showed prolonged elevation post-surgery. CRP increased to a peak level 48 h after the end of surgery (53 mg/l, 40 – 72) (Fig. 5). The same dynamics was found in α1-antitrypsin. Ceruloplasmin increase was delayed in relation to other tested acute-phase proteins reaching maximal levels 72 h after separation from CPB.

#### **3.5 Relations among hepcidin, prohepcidin and inflammatory markers**

Postoperative peak values of PCT and IL-6 correlated closely (r = 0.81, p < 0.01), as well as peak values of PCT and CRP (r = 0.72, p < 0.01) and peak values of PCT and TNFα (r = 0.62, p < 0.05). Correlation between PCT and other parameters wasn't significant on p < 0.05. Maximum postoperative concentrations of IL-6 correlated with maximum IL-8 levels (r = 0.81, p < 0.01).

Plasma hepcidin and prohepcidin concentrations didn't correlate preoperatively. Correlation between maximum postoperative hepcidin concentrations 72 h after the end of surgery and prohepcidin concentrations at the same samples (p = 0.056) didn't achieve statistical significance.

No significant correlation was revealed between plasma hepcidin and IL-6 concentrations preoperatively as well as between hepcidin and other tested inflammatory markers, hemodynamic values, plasma iron, transferrin and ferritin before the start of surgery. Similarly plasma prohepcidin did not correlate with any tested inflammatory parameter, plasma iron, transferrin or ferritin preoperatively.

Maximum post-operative concentrations of hepcidin measured 72 h after a separation from CPB correlated with maximum IL-6 levels 12 h after the end of surgery (r = 0.714, p = 0.021) as well as with IL-6 levels measured 18 h (r = 0.644, p = 0.032) and 24 h after the end of surgery (r = 0.614. p = 0.042). Similarly hepcidin and CRP concentrations correlated significantly 72 h after the end of surgery (p = 0.044). No other tested inflammatory parameter correlated with hepcidin post-surgery on p < 0.05.

Fig. 3. PCT plasma concentrations in perioperative period (median values, interquartile range and full range)

(22.3 – 82.0) culminated later, 18 h after separation from CPB (446.0 ng/l, 274.2 – 658.1) with

Three tested acute-phase proteins showed prolonged elevation post-surgery. CRP increased to a peak level 48 h after the end of surgery (53 mg/l, 40 – 72) (Fig. 5). The same dynamics was found in α1-antitrypsin. Ceruloplasmin increase was delayed in relation to other tested

Postoperative peak values of PCT and IL-6 correlated closely (r = 0.81, p < 0.01), as well as peak values of PCT and CRP (r = 0.72, p < 0.01) and peak values of PCT and TNFα (r = 0.62, p < 0.05). Correlation between PCT and other parameters wasn't significant on p < 0.05. Maximum postoperative concentrations of IL-6 correlated with maximum IL-8 levels (r =

Plasma hepcidin and prohepcidin concentrations didn't correlate preoperatively. Correlation between maximum postoperative hepcidin concentrations 72 h after the end of surgery and prohepcidin concentrations at the same samples (p = 0.056) didn't achieve

No significant correlation was revealed between plasma hepcidin and IL-6 concentrations preoperatively as well as between hepcidin and other tested inflammatory markers, hemodynamic values, plasma iron, transferrin and ferritin before the start of surgery. Similarly plasma prohepcidin did not correlate with any tested inflammatory parameter,

Maximum post-operative concentrations of hepcidin measured 72 h after a separation from CPB correlated with maximum IL-6 levels 12 h after the end of surgery (r = 0.714, p = 0.021) as well as with IL-6 levels measured 18 h (r = 0.644, p = 0.032) and 24 h after the end of surgery (r = 0.614. p = 0.042). Similarly hepcidin and CRP concentrations correlated significantly 72 h after the end of surgery (p = 0.044). No other tested inflammatory

Fig. 3. PCT plasma concentrations in perioperative period (median values, interquartile

acute-phase proteins reaching maximal levels 72 h after separation from CPB.

**3.5 Relations among hepcidin, prohepcidin and inflammatory markers** 

following decline.

0.81, p < 0.01).

statistical significance.

range and full range)

plasma iron, transferrin or ferritin preoperatively.

parameter correlated with hepcidin post-surgery on p < 0.05.

Fig. 4. IL-6 plasma concentrations in perioperative period (median values, interquartile range and full range)

Fig. 5. CRP plasma concentrations in perioperative period (median values, interquartile range and full range)

#### **3.6 Relations among hepcidin, prohepcidin and hemodynamic parameters**

Minimal perioperative prohepcidin concentrations correlated inversely with ECC time (r = -0.84, p < 0.01). When evaluating patients with a quick normalization of prohepcidin into preoperative range within 24 h after separation from CPB (Subgroup 1, n = 16), higher peak plasma levels of IL-6 (620.9 ng/l, 484.2 – 782.0) were revealed in this subgroup compared with patients with delayed prohepcidin normalization (Subgroup 2, n = 18), their IL-6 peak levels were 438.2 ng/l (370.1 – 512.7), p 0.05 between subgroups. Similarly, IL-8 peak postoperative concentrations were higher in Subgroup 1 in relation to Subgroup 2, but the differences were not significant on p 0.05.

Prohepcidin and Hepcidin in Acute Phase Reaction Accompanying Large Cardiac Surgery 247

concentrations increased post-operatively reaching a maximum 72 h after the separation from CPB. In a homogenous group of uncomplicated cardiosurgical patients, significant correlation between hepcidin and IL-6 concentrations post-surgery was observed. Our results are in conformity with a recent study by Hoppe et al. (2009). On a limited number of patients undergoing heart surgery, authors found significant alterations in both serum hepcidin and serum prohepcidin. Serum prohepcidin decreased after 48 h compared with preoperative values, whereas serum hepcidin increased within a 144-hour time frame. The human hepcidin gene, located on chromosome 19q13.1, encodes a precursor protein, preprohepcidin of 84 aa. Preprohepcidinundergoes enzymatic cleavage, resulting in the export of a 64 -aa prohepcidin peptide into the endoplasmic reticulum lumen. Next, the 39-aa proregion peptide is probably posttranslationally removed resulting in mature bioactive hepcidin-25 (25-aa form). In human urine, hepcidin-22 and hepcidin-20 were identified, which are Nterminally truncated isoforms of hepcidin-25 (Park et al., 2001). Kemna et al. (2008) results support the hypothesis that the 22-aa peptide is a urinary degradation product of hepcidin-25. An active hepcidin is a 8 cysteine-containing peptide with a distorted β-sheet which is stabilized by four disulfide bridges between the two anti-parallel strands (Hunter et al., 2002). The high cysteine content of the molecule is highly conserved among other species. Structurefunction *in vivo* (mice) and *in vitro* studies on synthetic hepcidin have shown that the iron regulating bioactivity is almost exclusivelydue to the 25 aa peptide, suggesting that the five Nterminal amino acids are essential for this activity. *In vitro* experiments have shown that especially human hepcidin-20 exerts anti-bacterial and anti-fungal activity in a concentration range 10-fold higherthan that measured in healthy individuals (Krause et al., 2000; Park et al., 2001). Nevertheless modeling of a best-fit 3D structure of hepcidin with iron demonstrated significant differences from the previously reported synthetic hepcidin model (Hunter et al., 2002). These new findings suggest a conformational polymorphism for hepcidin as a regulatory mechanismfor iron uptake as part of its role as regulator of iron homeostasis. Hepcidin is produced and secreted predominantly by hepatocytes, circulates in the bloodstream, and is excreted by the kidneys. Expression as studied on mRNA level is also detectable in other tissues (heart, kidney, adipose tissue, pancreas and hematopoietic cells), although the biological relevance of extra-hepatic hepcidin is not well defined yet (Vokurka et al., 2009). Hepcidin expression is regulated in response to iron, erythropoietic demand, hypoxia, and inflammatory signals. Inflammation is a potent inducer of hepcidin expression. Hepcidin was demonstrated to be up-regulated by a set of inflammatory cytokines. The synthesis is up-regulated in intact animals by the injection of lipopolysaccharide (endotoxin) and IL-6 although a direct stimulating effect of other cytokines as IL-1α, and IL-1β was confirmed in *in vitro* studies (Lee et al., 2005). Anemia and hypoxia inhibit hepcidin expression, thus increasing iron availability for erythropoiesis. Probably the activity of erythropoiesis rather than simple anemia was postulated later as an important regulatory factor (Vokurka et al., 2006; Weiss & Goodnough, 2005). Recent studies brought new data about molecular pathways of the regulation of hepcidin gene through

these different stimuli (reviewed by Kemna et al., 2008; Zhang & Enns, 2009).

*In vitro* observations and first animal and clinical studies including our results support a submission of hepcidin into a group of acute-phase proteins (Young et al., 2009). Acute-phase proteins is the generic name of approximately 30 different biochemically and functionally unrelated proteins. These proteins are secreted by hepatocytes and their plasma levels are either increased (positive reactants) or reduced (negative acute-phase reactants) after the onset of a systemic inflammatory reaction (Baumann, 1988). Cell types other than hepatocytes are

Hepcidin maximum concentrations correlated positively with ECC time (p = 0.049). Hepcidin evolution post-surgery was not related to CI, MPAP, PVR and EF on p 0,05. Among inflammatory markers, IL-6 plasma concentrations correlated inversely with CI at the time of separation from CPB (k = -0.644, p = 0.029). Similarly IL-8 concentrations correlated significantly with CI at the same time (k = -0.636, p = 0.017).

## **3.7 Iron, ferritin and transferrin**

48 h postoperatively, there was a significant decrease in serum iron concentration (Table 2). Plasma ferritin levels (44.6 μg/l, 19.4 – 72.9 preoperatively) increased slightly at 36 h after separation from CPB (139.2 μg/l, 44.9 – 172.9) and stayed elevated without significant dynamics until the end of tested period. Plasma ferritin did not significantly correlate with prohepcidin and hepcidin or any other tested inflammatory parameter. Multifactorial analysis did not reveal significant correlation between hepcidin and iron, resp. prohepcidin and iron within a 120-hour time frame after a separation from CPB. Similarly, both parameters did not correlate with plasma transferrin, hemoglobin concentration, alanine aminotransferase or aspartate aminotransferase activities post-surgery. No correlation was found between plasma hepcidin or prohepcidin levels and creatinine levels in postoperative period.


Variables are medians (interquartile range).

\* … Statistically significant differences to preoperative values on p 0,05.

Table 2. Plasma iron, ferritin and transferrin in perioperative period

## **4. Discussion**

This study has demonstrated the large cardiac surgery as an inductor of a deep transient decrease of prohepcidin plasma concentrations. Minimal postoperative prohepcidin levels were related to ECC time and did not correlate with IL-6 or other tested inflammatory parameters. Nevertheless higher IL-6 concentrations advanced prohepcidin normalization after its initial decline. Postoperative changes of plasma iron or transferrin concentrations and hemoglobin concentration did not correlate with plasma prohepcidin levels. Hepcidin

Hepcidin maximum concentrations correlated positively with ECC time (p = 0.049). Hepcidin evolution post-surgery was not related to CI, MPAP, PVR and EF on p 0,05. Among inflammatory markers, IL-6 plasma concentrations correlated inversely with CI at the time of separation from CPB (k = -0.644, p = 0.029). Similarly IL-8 concentrations

48 h postoperatively, there was a significant decrease in serum iron concentration (Table 2). Plasma ferritin levels (44.6 μg/l, 19.4 – 72.9 preoperatively) increased slightly at 36 h after separation from CPB (139.2 μg/l, 44.9 – 172.9) and stayed elevated without significant dynamics until the end of tested period. Plasma ferritin did not significantly correlate with prohepcidin and hepcidin or any other tested inflammatory parameter. Multifactorial analysis did not reveal significant correlation between hepcidin and iron, resp. prohepcidin and iron within a 120-hour time frame after a separation from CPB. Similarly, both parameters did not correlate with plasma transferrin, hemoglobin concentration, alanine aminotransferase or aspartate aminotransferase activities post-surgery. No correlation was found between plasma hepcidin or prohepcidin levels and creatinine levels in postoperative

Time Iron (μmol/l) Ferritin (μg/l) Transferrin (g/l)

This study has demonstrated the large cardiac surgery as an inductor of a deep transient decrease of prohepcidin plasma concentrations. Minimal postoperative prohepcidin levels were related to ECC time and did not correlate with IL-6 or other tested inflammatory parameters. Nevertheless higher IL-6 concentrations advanced prohepcidin normalization after its initial decline. Postoperative changes of plasma iron or transferrin concentrations and hemoglobin concentration did not correlate with plasma prohepcidin levels. Hepcidin

Before sternotomy 21.3 (13.2 – 27.7) 44.6 (19.4 – 72.9) 2.6 (1.8 – 3.2) 12 h after separation from CPB 13.4 \* (7.9 – 22.3) 38.9 \* (20.4 – 69.0) 1.6 \* (1.0 – 2.2) 18 h after separation from CPB 13.7 \* (7.1 – 18.5) 59.9 (28.4 – 88.2) 2.0 (1.4 – 2.6) 24 h after separation from CPB 15.0 (9.6 – 20.7) 112.1 (69.4 – 164.3) 1.8 (1.3 – 2.3) 36 h after separation from CPB 14.1 (9.8 – 19.8) 139.2 \* (44.9 – 172.9) 1.6 \* (1.0 – 2.1) 48 h after separation from CPB 12.7 \* (7.7 – 16.1) 128.0 \* (74.7 – 188.3) 1.6 \* (0.9 – 2.0) 72 h after separation from CPB 13.0 \* (7.0 – 18.8) 122.4 \* (71.2 – 152.1) 2.1 (1.7 – 2.8) 120 h after separation from CPB 12.4 \* (7.4 – 19.1) 116.7 \* (79.0 – 164.2) 2.3 (1.9 – 3.1)

correlated significantly with CI at the same time (k = -0.636, p = 0.017).

**3.7 Iron, ferritin and transferrin** 

Variables are medians (interquartile range).

 … Statistically significant differences to preoperative values on p 0,05. Table 2. Plasma iron, ferritin and transferrin in perioperative period

period.

\*

**4. Discussion** 

concentrations increased post-operatively reaching a maximum 72 h after the separation from CPB. In a homogenous group of uncomplicated cardiosurgical patients, significant correlation between hepcidin and IL-6 concentrations post-surgery was observed. Our results are in conformity with a recent study by Hoppe et al. (2009). On a limited number of patients undergoing heart surgery, authors found significant alterations in both serum hepcidin and serum prohepcidin. Serum prohepcidin decreased after 48 h compared with preoperative values, whereas serum hepcidin increased within a 144-hour time frame.

The human hepcidin gene, located on chromosome 19q13.1, encodes a precursor protein, preprohepcidin of 84 aa. Preprohepcidinundergoes enzymatic cleavage, resulting in the export of a 64 -aa prohepcidin peptide into the endoplasmic reticulum lumen. Next, the 39-aa proregion peptide is probably posttranslationally removed resulting in mature bioactive hepcidin-25 (25-aa form). In human urine, hepcidin-22 and hepcidin-20 were identified, which are Nterminally truncated isoforms of hepcidin-25 (Park et al., 2001). Kemna et al. (2008) results support the hypothesis that the 22-aa peptide is a urinary degradation product of hepcidin-25. An active hepcidin is a 8 cysteine-containing peptide with a distorted β-sheet which is stabilized by four disulfide bridges between the two anti-parallel strands (Hunter et al., 2002). The high cysteine content of the molecule is highly conserved among other species. Structurefunction *in vivo* (mice) and *in vitro* studies on synthetic hepcidin have shown that the iron regulating bioactivity is almost exclusivelydue to the 25 aa peptide, suggesting that the five Nterminal amino acids are essential for this activity. *In vitro* experiments have shown that especially human hepcidin-20 exerts anti-bacterial and anti-fungal activity in a concentration range 10-fold higherthan that measured in healthy individuals (Krause et al., 2000; Park et al., 2001). Nevertheless modeling of a best-fit 3D structure of hepcidin with iron demonstrated significant differences from the previously reported synthetic hepcidin model (Hunter et al., 2002). These new findings suggest a conformational polymorphism for hepcidin as a regulatory mechanismfor iron uptake as part of its role as regulator of iron homeostasis.

Hepcidin is produced and secreted predominantly by hepatocytes, circulates in the bloodstream, and is excreted by the kidneys. Expression as studied on mRNA level is also detectable in other tissues (heart, kidney, adipose tissue, pancreas and hematopoietic cells), although the biological relevance of extra-hepatic hepcidin is not well defined yet (Vokurka et al., 2009). Hepcidin expression is regulated in response to iron, erythropoietic demand, hypoxia, and inflammatory signals. Inflammation is a potent inducer of hepcidin expression. Hepcidin was demonstrated to be up-regulated by a set of inflammatory cytokines. The synthesis is up-regulated in intact animals by the injection of lipopolysaccharide (endotoxin) and IL-6 although a direct stimulating effect of other cytokines as IL-1α, and IL-1β was confirmed in *in vitro* studies (Lee et al., 2005). Anemia and hypoxia inhibit hepcidin expression, thus increasing iron availability for erythropoiesis. Probably the activity of erythropoiesis rather than simple anemia was postulated later as an important regulatory factor (Vokurka et al., 2006; Weiss & Goodnough, 2005). Recent studies brought new data about molecular pathways of the regulation of hepcidin gene through these different stimuli (reviewed by Kemna et al., 2008; Zhang & Enns, 2009).

*In vitro* observations and first animal and clinical studies including our results support a submission of hepcidin into a group of acute-phase proteins (Young et al., 2009). Acute-phase proteins is the generic name of approximately 30 different biochemically and functionally unrelated proteins. These proteins are secreted by hepatocytes and their plasma levels are either increased (positive reactants) or reduced (negative acute-phase reactants) after the onset of a systemic inflammatory reaction (Baumann, 1988). Cell types other than hepatocytes are

Prohepcidin and Hepcidin in Acute Phase Reaction Accompanying Large Cardiac Surgery 249

various modifications of mass spectrometry (Swinkels et al., 2008). Recent methods used for hepcidin measurement comprise mass spectrometry (SELDI-TOF MS, MALDI-TOF MS, LC-MS/MS, IC-TOF-MS) and immunochemical assays (competitive ELISA or RIA). Mass spectrometry-based methods might be superior in detecting bioactive hepcidin-25 and distinguishing other isoforms. This may be valuable mainly in situations with variable presence of hepcidin isoforms like in chronic kidney disease (Kroot et al., 2009; Kroot et al., 2010). The initial decrease of hepcidin and inflammatory cytokines was explained by hemodilution. The effect of hemodilution and hemofiltration on cytokine and acute phase protein concentrations during PEA was well described in our previous study (Maruna et al., 2008) and cleared after correction of cytokine concentrations to hematocrit. Other limitations of this study include possibility of hemolysis impacting the study findings. When using the CPB pump in cardiosurgical procedures taking several hours, there is a degree of

mechanical hemolysis that can affect hematological biomarkers (Hoppe et al., 2009).

factors restraining prohepcidin elevation or inhibiting its production.

CTEPH Chronic thromboembolic pulmonary hypertension

IC-TOF-MS Immunocapture time-of-flight mass-spectrometry

LC-MS/MS Liquid chromatography tandem-MS techniques

MALDI-TOF MS Matrix assisted laser desorption/ionization time-of-flight mass

Our study showed prohepcidin as a negative acute phase reactant with a strong initial decrease after PEA. On the other hand, hepcidin increased after uncomplicated cardiac surgery and this finding is in conformity with recent experimental studies defining hepcidin as a type II acute-phase protein. Relations between IL-6 levels and duration of prohepcidin disturbance as well as between IL-6 and hepcidin post-surgery course were revealed in our study. It remains to be determined whether the initial decrease of prohepcidin documented in our study is due to proteolytic trimming of serum prohepcidin or there are another

The study was supported with a grant MSM0021620819 of the Ministry of Education, Czech

**5. Conclusion** 

**6. Acknowledgement** 

CRP C-reactive protein

EF Ejection fraction

IL Interleukin

PCT Procalcitonin

spectrometry

CV Coefficients of variation

ECC Extracorporeal circulation

BMP Bone morphogenetic protein CPB Cardio-pulmonary bypass

DHCA Deep hypothermic circulatory arrest

MPAP Mean pulmonary artery pressure

PEA Pulmonary endarterectomy

**7. Abbreviations** 

Republic.

known to produce acute-phase proteins in a limited amount. Most of acute-phase proteins are glycoproteins (with exception of CRP and serum amyloid A). Actually about 20 cytokines are known to stimulate acute-phase protein synthesis in liver. The major inducers are IL-6, IL-1β, and TNFα but an essential role of IL-6 in this action is out of doubt (Gabay & Kushner, 1999). Considering a dominant regulation by IL-6, hepcidin was classified as a type II acute-phase protein. Type I acute-phase proteins (CRP, serum amyloid A, α1-acid glycoprotein and other) are those that require the synergistic action of IL-6 and IL-1β for maximum synthesis. Type II acute-phase proteins are those that require IL-6 only for maximal induction. Examples of type II proteins are fibrinogen chains, haptoglobin, and α2-macroglobulin. Expression of genes encoding type II acute-phase proteins is suppressed rather than being enhanced frequently by IL-1β (Ramadori et al., 1999). Additive, synergistic, co-operative, and antagonistic effects between cytokines and other mediator substances influencing the expression of acute phase proteins do occur and have been observed in almost all combinations.

There is a well-characterized mechanism of direct transcriptional activation of hepcidin expression by IL-6 binding to its receptor complex containing gp130 to activate janus kinase (JAK) and activator of transcription STAT3, which binds to a conserved DNA element in the proximal hepcidin promoter (Nemeth et al., 2003 and 2004; Pietrangelo et al., 2007; Truksa et al., 2007, Verga Falzacappa et al., 2007, Wrighting Andrews, 2006). Other proinflammatory cytokines, such as IL-1β, may also play a role in hepcidin induction (Lee et al., 2005). A second mode of hepcidin regulation depends upon signaling through the bone morphogenetic protein/Smad (BMP/Smad) pathway. Babitt et al. (2007) showed that mice with a deletion in the Smad4 gene were unable to synthesize hepcidin in response to inflammatory stimuli or to iron load. In means that induction by IL-6 appears to require an intact BMP/Smad signaling pathway. Hepcidin is also produced in monocytes/macrophages, and is induced in these cells by LPS and certain bacterial pathogens through Toll-like receptors and possibly also the IL-6/STAT3 pathway (Liu et al., 2005).

The dynamic of hepcidin precursor - prohepcidin was different from hepcidin and all tested inflammatory markers including PCT, cytokines and acute-phase proteins. Prohepcidin decreased post-operatively. The decrease of prohepcidin after a heart surgery was firstly documented by Hoppe et al. (2009). However there are some controversies between clinical findings and experimental studies. In a recent experimental lipopolysaccharide model, the investigators did not observe any change in serum prohepcidin during a shorter time frame of 22 h, although there was a tendency toward a decrease at the final observation (Kemta et al., 2005). There is a possibility that serum prohepcidin is a biological nonfunctional precursor of the active hepcidin, as was postulated by Taes et al. (2004) or Brookes et al. (2005). It has been demonstrated by Rivera et al. (2005) that the truncated 25-aa form of hepcidin exercises the hypoferremia effect. Furthermore, the same study assessed the relation between iron absorption and prohepcidin concentrations in healthy women and it did not see any correlation, Iron absorption by healthy women is not associated with either serum or urinary prohepcidin. Thus, a Hoppe's interpretation of the present serum prohepcidin data showing a decrease following surgery is that serum prohepcidin (as insulin and proteases) is synthesized as an inactive precursor that is proteolytically trimmed to be activated (Hoppe et al., 2009).

Technical problems complicated routine measurements of plasma hepcidin until 2009 (Piperno et al., 2009). Substantial clinical results come from the measurement of prohepcidin but its relation to hepcidin remains unclear (Frazer & Anderson, 2009). Hepcidin was measured in serum or urine mainly by the method developed by Ganz (2008) and by various modifications of mass spectrometry (Swinkels et al., 2008). Recent methods used for hepcidin measurement comprise mass spectrometry (SELDI-TOF MS, MALDI-TOF MS, LC-MS/MS, IC-TOF-MS) and immunochemical assays (competitive ELISA or RIA). Mass spectrometry-based methods might be superior in detecting bioactive hepcidin-25 and distinguishing other isoforms. This may be valuable mainly in situations with variable presence of hepcidin isoforms like in chronic kidney disease (Kroot et al., 2009; Kroot et al., 2010). The initial decrease of hepcidin and inflammatory cytokines was explained by

hemodilution. The effect of hemodilution and hemofiltration on cytokine and acute phase protein concentrations during PEA was well described in our previous study (Maruna et al., 2008) and cleared after correction of cytokine concentrations to hematocrit. Other limitations of this study include possibility of hemolysis impacting the study findings. When using the CPB pump in cardiosurgical procedures taking several hours, there is a degree of mechanical hemolysis that can affect hematological biomarkers (Hoppe et al., 2009).

## **5. Conclusion**

248 Inflammatory Diseases – Immunopathology, Clinical and Pharmacological Bases

known to produce acute-phase proteins in a limited amount. Most of acute-phase proteins are glycoproteins (with exception of CRP and serum amyloid A). Actually about 20 cytokines are known to stimulate acute-phase protein synthesis in liver. The major inducers are IL-6, IL-1β, and TNFα but an essential role of IL-6 in this action is out of doubt (Gabay & Kushner, 1999). Considering a dominant regulation by IL-6, hepcidin was classified as a type II acute-phase protein. Type I acute-phase proteins (CRP, serum amyloid A, α1-acid glycoprotein and other) are those that require the synergistic action of IL-6 and IL-1β for maximum synthesis. Type II acute-phase proteins are those that require IL-6 only for maximal induction. Examples of type II proteins are fibrinogen chains, haptoglobin, and α2-macroglobulin. Expression of genes encoding type II acute-phase proteins is suppressed rather than being enhanced frequently by IL-1β (Ramadori et al., 1999). Additive, synergistic, co-operative, and antagonistic effects between cytokines and other mediator substances influencing the expression of acute phase

There is a well-characterized mechanism of direct transcriptional activation of hepcidin expression by IL-6 binding to its receptor complex containing gp130 to activate janus kinase (JAK) and activator of transcription STAT3, which binds to a conserved DNA element in the proximal hepcidin promoter (Nemeth et al., 2003 and 2004; Pietrangelo et al., 2007; Truksa et al., 2007, Verga Falzacappa et al., 2007, Wrighting Andrews, 2006). Other proinflammatory cytokines, such as IL-1β, may also play a role in hepcidin induction (Lee et al., 2005). A second mode of hepcidin regulation depends upon signaling through the bone morphogenetic protein/Smad (BMP/Smad) pathway. Babitt et al. (2007) showed that mice with a deletion in the Smad4 gene were unable to synthesize hepcidin in response to inflammatory stimuli or to iron load. In means that induction by IL-6 appears to require an intact BMP/Smad signaling pathway. Hepcidin is also produced in monocytes/macrophages, and is induced in these cells by LPS and certain bacterial pathogens through Toll-like receptors and possibly also the IL-6/STAT3 pathway (Liu et al., 2005). The dynamic of hepcidin precursor - prohepcidin was different from hepcidin and all tested inflammatory markers including PCT, cytokines and acute-phase proteins. Prohepcidin decreased post-operatively. The decrease of prohepcidin after a heart surgery was firstly documented by Hoppe et al. (2009). However there are some controversies between clinical findings and experimental studies. In a recent experimental lipopolysaccharide model, the investigators did not observe any change in serum prohepcidin during a shorter time frame of 22 h, although there was a tendency toward a decrease at the final observation (Kemta et al., 2005). There is a possibility that serum prohepcidin is a biological nonfunctional precursor of the active hepcidin, as was postulated by Taes et al. (2004) or Brookes et al. (2005). It has been demonstrated by Rivera et al. (2005) that the truncated 25-aa form of hepcidin exercises the hypoferremia effect. Furthermore, the same study assessed the relation between iron absorption and prohepcidin concentrations in healthy women and it did not see any correlation, Iron absorption by healthy women is not associated with either serum or urinary prohepcidin. Thus, a Hoppe's interpretation of the present serum prohepcidin data showing a decrease following surgery is that serum prohepcidin (as insulin and proteases) is synthesized as an inactive precursor that is proteolytically trimmed

Technical problems complicated routine measurements of plasma hepcidin until 2009 (Piperno et al., 2009). Substantial clinical results come from the measurement of prohepcidin but its relation to hepcidin remains unclear (Frazer & Anderson, 2009). Hepcidin was measured in serum or urine mainly by the method developed by Ganz (2008) and by

proteins do occur and have been observed in almost all combinations.

to be activated (Hoppe et al., 2009).

Our study showed prohepcidin as a negative acute phase reactant with a strong initial decrease after PEA. On the other hand, hepcidin increased after uncomplicated cardiac surgery and this finding is in conformity with recent experimental studies defining hepcidin as a type II acute-phase protein. Relations between IL-6 levels and duration of prohepcidin disturbance as well as between IL-6 and hepcidin post-surgery course were revealed in our study. It remains to be determined whether the initial decrease of prohepcidin documented in our study is due to proteolytic trimming of serum prohepcidin or there are another factors restraining prohepcidin elevation or inhibiting its production.

## **6. Acknowledgement**

The study was supported with a grant MSM0021620819 of the Ministry of Education, Czech Republic.

## **7. Abbreviations**


Prohepcidin and Hepcidin in Acute Phase Reaction Accompanying Large Cardiac Surgery 251

Krause, A.; Nietz, S.; Magert, H. J.; Schultz, A.; Forssmann, W. G.; Schultz-Knappe, P. &

Krijt, J.; Fujikura, Y.; Šefc, L.; Vokurka, M.; Hlobeňová, T. & Nečas, E. (2010). Hepcidin

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Kroot, J. J.; Laarakkers, C. M.; Geurts-Moespot, A. J.; Grebenchtchikov, N.; Pickkers, P.; van

Lindner, J.; Maruna, P.; Kunstýř, J.; Jansa, P.; Gürlich, R.; Kubzová, K.; Zakharcenko, M. &

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Martínez Rosas, M. (2006). Cardiac remodeling and inflammation. Arch Cardiol Mex, Vol.

Maruna, P.; Kunstýř, J; Plocová, K. M.; Mlejnský, F.; Hubáček, J. A.; Klein, A. A. & Lindner, J. ,

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Maruna, P.; Lindner, J.; Kunstýř, J.; Plocová, K. & Hubáček, J. (2009). Plasma prohepcidin as

Nemeth, E.; Valore, E. V.; Territo, M.; Schiller, G.; Lichtenstein, A. & Ganz, T. (2003).

Nemeth, E.; Rivera, S.; Gabayan, V.; Keller, C.; Taudorf, S.; Pedersen, B. K. & Ganz, T. (2004).

Nicolas, G.; Chauvet, C.; Viatte, L.; Danan, J. L.; Bigard, X.; Devaux, I.; Beaumont, C.; Kahn

regulatory hormone hepcidin. J Clin Invest, Vol. 113, pp. 1271-1276

pulmonary hypertension. Eur J Cardio-Thorac, Vol. 39, pp. 195-200

exhibits antimicrobial activity. FEBS Lett, Vol. 480, pp. 147-150

standardization. Haematologica, Vol. 94, pp. 1748-1752

hyperstimulation. Eur Surg Res, Vol. 43, pp. 39-46

circulatory arrest. Physiol Res, Vol. 58, pp. 827-833

protein. Blood, Vol. 101, pp. 2461-2463

Blood Cells Mol Dis, Vol. 35, pp. 47–56

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pp. 1906–1910.

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Adermann, K. (2000). LEAP-1, a novel highly disulfide-bonded human peptide,

downregulation by repeated bleeding is not mediated by soluble hemojuvelin.

C.; Koliaraki, V.; Mamalaki, A.; Olbina, G.; Tomosugi, N.; Tselepis, C.; Ward, D. G.; Ganz, T.; Hendriks, J. C. & Swinkels, D. W. (2009). Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for

Ede, A. E.; Peters, H. P.; van Dongen-Lases, E.; Wetzels, J. F.; Sweep, F. C.; Tjalsma H. & Swinkels, D. W. (2010). Immunochemical and mass-spectrometry-based serum hepcidin assays for iron metabolism disorders. Clin Chem, Vol. 56, pp. 1570-1579 Langer, F.; Schramm, R.; Bauer, M.; Tscholl, D.; Kunihara, T. & Schafers, H. J. (2004). Cytokine response to pulmonary thromboendarterectomy. Chest, Vol. 126, pp. 135-141 Lee, P.; Peng, H.; Gelbart, T.; Wang, L. & Beutler, E. (2005). Regulation of hepcidin

transcription by interleukin-1 and interleukin-6. Proc Natl Acad Sci USA, Vol. 102,

Linhart, A. (2009). Hemodynamic instability after pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension correlates with cytokine network

hepcidin and ferroportin expression by lipopolysaccharide in splenic macrophages.

(2011). Predictors of infection after pulmonary endarterectomy for thromboembolic

procalcitonin and cytokines after pulmonary endarterectomy. Prague Med Rep,

a negative acute phase reactant after large cardiac surgery with a deep hypothermic

Hepcidin, a putative mediator of anemia of inflammation, is a type II acute phase

IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron

A. & Vaulont, S. (2002). The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest, Vol. 110, pp. 1037-

PVR Pulmonary vascular resistance

SELDI-TOF MS Surface enhanced laser desorption/ionization time-of-flight mass spectrometry

SPSS Statistical package for social sciences

STAT 3 Signal transducer and activator of transcription 3

TNFα Tumor necrosis factor-α

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**8. References** 


**12** 

*USA* 

**Leukotriene A4 Hydrolase –** 

Y. Michael Shim1 and Mikell Paige2

*University of Virginia, Charlottesville,* 

**An Evolving Therapeutic Target** 

*1Division of Pulmonary & Critical Care Medicine, School of Medicine,* 

*2Georgetown University, Lombardi Comprehensive Cancer Center, Washington,* 

Leukotriene B4 is an important mediator of inflammation derived from successive metabolism of fatty acids by several enzymes including the terminal rate-limiting enzyme called leukotriene A4 hydrolase. Leukotriene A4 hydrolase is a soluble enzyme, and depending on its substrate can function as either an epoxide hydrolase or an aminopeptidase. Over the years, leukotriene B4 has been found to be highly associated with several human diseases, and most of the reported literature has focused on the biology of the epoxide hydrolase activity of the enzyme, which generates the lipid metabolite leukotriene B4. However, emerging data suggests that the aminopeptidase activity of the leukotriene A4 hydrolase enzyme may also play a crucial role in the process of antiinflammatory responses. Previous drug discovery efforts have focused on inhibition of the leukotriene B4 metabolite by indiscriminately blocking both the epoxide hydrolase and aminopeptidase functions of the enzyme. The co-existence of a dichotomous and directly opposing biological function of this enzyme as suggested by recent studies on the aminopeptidase activity of leukotriene A4 hydrolase is a radically paradigm-shifting and relevant concept. This manuscript will review these recent findings in the context of the

The leukotrienes are important downstream effector molecules of inflammatory tissue alterations. Human diseases exhibit dysregulated inflammatory and immune responses in their pathogenesis. Therefore, 5-lipoxygenase (5-LO)-mediated lipid pathways have been investigated as possible pro-inflammatory pathways in the pathogenesis of multiple human diseases. The leukotrienes are lipid mediators of inflammation derived from the metabolism of fatty acids to arachidonic acid by phospholipase A2 (cPLA2), then to leukotriene A4 (LTA4) by 5-lipoxygenase and 5-lipoxygenase activating protein (FLAP). Further downstream metabolism yields two classes of leukotrienes: cysteinyl-leukotrienes (leukotriene C4, D4, and E4) synthesized by leukotriene C4 synthase and leukotriene B4 synthesized by leukotriene A4 hydrolase. Eventually, cysteinyl-leukotrienes and leukotriene B4 bind to a cysteinyl leukotriene receptor or a leukotriene B4 receptor, respectively, to exert final tissue

classical understanding of the leukotriene A4 hydrolase enzyme.

**1. Introduction** 

**2. Background** 

effects **(Figure 1)**.

