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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 28  |  Issue : 2  |  Page : 463-470

Hepcidin and iron regulation in chronic hemolytic anemia


1 Department of pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of pediatrics, El Senbelaween Hospital, El Senbelaween, Egypt

Date of Submission05-Sep-2014
Date of Acceptance14-Jun-2014
Date of Web Publication31-Aug-2015

Correspondence Address:
Maha Z Taha
Department of pediatrics, El Senbelaween Hospital, El Senbelaween
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.163903

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  Abstract 


Objectives
The aim of the study was to investigate the changes in serum hepcidin and ferritin level in chronic hemolytic anemia (β-thalassemia major and sickle cell disease) to clarify the relation between hepcidin level and iron regulation.
Background
The hallmark of hemolytic anemia is reduced life span of RBCs. In β-thalassemia there is excess α-globin chain relative to β-globin chain; α-globin tetramers are formed, and interact with RBC membranes and shorten RBC survival, leading to anemia and increased erythrocyte production. Sickle cell anemia is caused by an abnormal type of hemoglobin called hemoglobin S, which distorts the shape of RBCs, especially with hypoxia. Frequent blood transfusion without proper iron chelation causes hemochromatosis. Recently, the predominant negative regulator of iron absorption and iron release was discovered - namely, hepcidin, the 25-amino acid peptide produced by hepatocytes.
Patients and methods
The study was carried out on 55 children, categorized into 23 children with thalassemia major, 12 with sickle cell disease, and 20 apparently healthy control children, who were matched in age, sex, and socioeconomic standard. Complete blood count, analysis of serum ferritin, hemoglobin electrophoresis, liver and renal function tests, and evaluation of serum hepcidin level using enzyme-linked immunosorbent assay kits were performed.
Results
Ferritin level in thalassemia and sickle cell patients was significantly higher than in controls (P < 0.001). Hepcidin level in thalassemia and sickle cell patients was higher than in controls (P < 0.001). Serum ferritin showed significant negative correlation with serum hepcidin level in thalassemia patients (P < 0.05). Regarding sickle cell, there was no significant correlation between ferritin and hepcidin levels (P = 0.812).
Conclusion
In future, hepcidin level may allow more accurate assessment of the degree of iron overload and iron misdistribution.

Keywords: chronic hemolytic anemia, hepcidin, sickle cell disease, β-thalassemia major


How to cite this article:
Rashidy FH, Abo Elghar HM, Kamal Eldin SM, Taha MZ. Hepcidin and iron regulation in chronic hemolytic anemia. Menoufia Med J 2015;28:463-70

How to cite this URL:
Rashidy FH, Abo Elghar HM, Kamal Eldin SM, Taha MZ. Hepcidin and iron regulation in chronic hemolytic anemia. Menoufia Med J [serial online] 2015 [cited 2020 Feb 27];28:463-70. Available from: http://www.mmj.eg.net/text.asp?2015/28/2/463/163903


  Introduction Top


Anemia in severe thalassemia phenotype necessitates multiple RBC transfusions, which results over time in improper chelation in transfusion-associated iron overload. In addition, ineffective erythropoiesis enhances gastrointestinal iron absorption and can result in iron overload [1] . Sickle cell anemia is caused by an abnormal type of hemoglobin called hemoglobin S, which distorts the shape of red blood cells, especially when there is hypoxia. The distorted red blood cells are shaped like crescents or sickles. These fragile, sickle-shaped cells deliver less oxygen to the tissues. They also clog more easily in small blood vessels, and break into pieces that disrupt the blood flow [2] . Hepcidin, a 25-amino acid peptide produced by hepatocytes, was found to be a key iron-regulatory hormone [3] and the primary regulator of iron homeostasis. Hepcidin modulates iron availability by promoting the internalization and degradation of ferroprotein, a key iron transporter and so far the only identified mammalian iron exporter, which is essential for both iron absorption in the duodenum and recycling of iron/iron efflux by macrophages [4] . The interaction between hepcidin and ferroprotein can explain the systemic regulation of iron metabolism [5] . Hepcidin is a negative regulator of iron absorption and mobilization; high hepcidin levels stop both duodenal iron absorption and release of iron from macrophages, whereas low hepcidin levels promote iron absorption and heme iron recycling/iron mobilization from macrophages. Thus, hepcidin levels are expected to be high in iron-overload states and diminished in iron-deficient states [4] . Serum hepcidin was appropriately correlated with serum ferritin, a finding that was expected, indicating that hepcidin and ferritin levels are both measures of iron stores [6] . Hepcidin and serum ferritin respond similarly to inflammation and changes in iron stores. However, hepcidin responses take place within a few hours, whereas changes in ferritin concentration are much slower [7] .


  Patients and methods Top


Patients

Fifty-five children participated in the study. Their ages ranged from 1 to 6 years with a mean age of 3.5 ± 2.3 years. Patients were selected from the pediatric hematology outpatient clinic and from among inpatients of the pediatric department, Menoufia University Hospital.

Members of this study were categorized into the following three groups:

Group I

Group I comprised 23 β-thalassemia major patients, consisting of 10 male and 13 female patients. Their ages ranged from 1 to 6 years with a mean of 3.5 ± 2.3 years.

Group II

Group II patients comprised 12 sickle cell anemia patients, consisting of eight male and four female patients. Their ages ranged from 2 to 6 years, with a mean of 2.8 ± 1.8 years.

Group III

Group III comprised 20 healthy children of matched age (ranging from 2 to 6 years, with a mean of 3.8 ± 1.9 years), sex (11 male and nine female patients), and socioeconomic standard, who served as the control group.

The study was carried out after obtaining approval from the ethics committee of the faculty of medicine and written consent from patients' parents.

Diagnosis was made on the basis of standard clinical and laboratory investigation (complete blood count, hemoglobin electrophoresis), hepcidin level, and ferritin level.

Liver function and renal function tests were carried out.

Determination of serum hepcidin level was performed by enzyme-linked immunosorbent assay (ELISA) technique.

Inclusion criteria

  1. Presence of β-thalassemia major or sickle cell anemia documented by hemoglobin electrophoresis.


Exclusion criteria

  1. Presence of previous renal pathology.
  2. Presence of acute inflammatory conditions.
  3. Presence of uncompensated hepatitis.
Methods

Sample collection

Blood samples were collected by venipuncture and allowed to clot. After complete clotting of the sample, the serum was separated by centrifugation at room temperature for 5 min at 4000 rpm. The clear supernatant serum was separated from the clot and kept frozen at up to −20°C until analysis, for determining the serum hepcidin level.

Determination of serum hepcidin level

Principle of the test: The DRG Hepcidin ELISA Kit (Biolegend company, USA) is a solid-phase ELISA, based on the principle of competitive binding.

  1. Microtiter wells are coated with a monoclonal antibody coating the microtiter wells and directed toward the antigenic site of the bioactive hepcidin of a patient sample competes with the added hepcidin-biotin conjugate for binding to the coated antibody.
  2. After incubation the unbound conjugate is washed off. This is followed by incubation with a streptavidin-peroxidase enzyme complex and a second wash step.
  3. The addition of substrate solution results in color development, which is stopped after a short incubation. The intensity of color developed is inversely proportional to the concentration of hepcidin in the patient's sample.
Assay procedure

Samples were diluted appropriately.

  1. The desired number of microtiter wells were secured in the frame holder.
  2. A volume of 10 μl sample buffer was added to each of these wells.
  3. A volume of 20 μl of each standard, control, and sample with new disposable tips was dispensed into appropriate wells.
  4. Incubation was performed for 30 min at room temperature on a plate shaker at 500 rpm.
  5. A volume of 150 μl assay buffer and 100 μl enzyme conjugate were added to each of these wells.
  6. Further incubation for 180 min at room temperature was performed on a plate shaker at 500 rpm.
  7. The contents of the wells were shaken out and the wells were rinsed five times with diluted wash solution (400 μl/well).
  8. A volume of 100 μl of substrate solution was added to each well.
  9. The enzymatic reaction was stopped by adding 100 μl of stop solution to each well.
  10. Finally the absorbance of each well was determined at 450 ± 10 nm with a microtiter plate reader.
  11. The average absorbance values for each set of standard, control, and patient samples were calculated using a semilogarithmic graph paper, by constructing a standard curve by plotting the mean absorbance obtained from each standard against its concentration, with absorbance value on the vertical (y) axis and concentration on the horizontal (x) axis. Then by using the mean absorbance value for each sample the corresponding concentration from the standard curve was determined.
Statistical analysis

Results were collected, tabulated, and statistically analyzed using an IBM personal computer and statistical package SPSS version 17 (SPSS Inc., Chicago, Illinois, USA). Two types of statistical analyses were carried out.

Descriptive statistics, which included percentage (%), mean (X), and SD.

Analytic statistics, which included the following:

  1. The χ2 -test: This was used to study the association between two qualitative variables.
  2. The Shapiro-Wilk test: This was used for normality of distribution.
  3. The independent t-test: This was used to compare quantitative data between two groups.
  4. Analysis of variance test: This was used to compare quantitative data among three groups.
  5. Fisher's exact test: This was used to compare qualitative variables.
  6. Spearman correlation curves: This was used to measure the association between two quantitative variables.
  7. The receiver operating characteristic curves: This is used to evaluate the performance of classification schemes where subjects are classified into two categories using one variable. The receiver operating characteristic curves were constructed by calculating the sensitivities and specificities of the variable. The cutoff value with the highest accuracy was selected as the diagnostic cutoff point.
  8. Sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy were calculated according to the following formulas:
Sensitivity = a/(a + c).

Specificity = d/(b + d).

Accuracy = (a + d )/(a + b + c + d).

Negativepredictive value = a 0/(a + b).

Positivepredictive value = a /(a + b).

where a is the number of true positive cases; b, false positive cases; c, false negative cases; and d, true negative cases.

A P value less than 0.05 was considered statistically significant.


  Results Top


Number of participants in the study

The number of participants in the study is shown in [Table 1].
Table 1 Number of participants in the study

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Comparison between the ages of cases and controls in the study

There was no significant difference between the ages of cases and those of controls. This is shown in [Table 2].
Table 2 Comparison between the age of cases and controls in the study

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Comparison of consanguinity among thalassemia and sickle cell cases

There was no significant difference in the percentage of consanguine patients between thalassemia and sickle cell patients. This is shown in [Table 3].
Table 3 Comparison of consanguinity among thalassemia and sickle cell cases

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Comparison between pretransfusion Hb% in cases and Hb% in controls

Pretransfusion Hb% in thalassemia and sickle cell patients was significantly lower than that of controls. This is shown in [Table 4].
Table 4 Comparison between pretransfusion Hb% in cases and Hb% of controls

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Comparison between liver and kidney function tests in cases and controls

This is presented in [Table 5].
Table 5 Comparison between liver and kidney function tests in cases and controls

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Iron chelation among thalassemia and sickle cell cases

The number of thalassemia patients on iron chelation therapy was significantly higher than the number of sickle cell patients (87% of thalassemia patients underwent desferal pump vs. 0% of sickle cell patients). The results of comparison of iron chelation between thalassemia and sickle cell anemia are shown in [Table 6] and [Figure 1].
Figure 1: Comparison of iron chelation among thalassemia and sickle cell case

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Table 6 Comparison of iron chelation among thalassemia and sickle cell cases

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Comparison between surgical splenectomy among thalassemia and sickle cell cases

The number of thalassemia patients who underwent splenectomy was not significantly higher than the number of sickle cell patients who did. This is shown in [Table 7].
Table 7 Comparison between surgical splenectomy among thalassemia and sickle cell cases

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Comparison of complications between thalassemia and sickle cell cases

This is shown in [Table 8].
Table 8 Comparison between complications among thalassemia and sickle cell cases

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Ferritin and hepcidin level in cases and controls

The results of comparison between ferritin and hepcidin levels in thalassemia patients, sickle cell patients, and controls are shown in [Table 9] and [Figure 2] and [Figure 3].
Figure 2: Comparison between ferritin level in cases and controls

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Figure 3: Comparison between hepcidin level in cases and controls

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Table 9 Comparison between ferritin and hepcidin level in cases and controls

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Correlation between ferritin and hepcidin levels in thalassemia

There was significant negative correlation (P < 0.05) between ferritin and hepcidin levels in thalassemia cases; this is demonstrated in [Table 10] and [Figure 4].
Figure 4: Correlation between ferritin and hepcidin levels in tha lassemia

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Table 10 Correlation between ferritin and hepcidin levels in thalassemia patients

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Correlation between ferritin and hepcidin levels in sickle cell anemia

There was no significant correlation between ferritin and hepcidin levels in sickle cell cases; this is demonstrated in [Table 11] and [Figure 5].
Figure 5: Correlation between ferritin and hepcidin levels in sickle cell anaemia

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Table 11 Correlation between ferritin and hepcidin levels in sickle cell patients

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  Discussion Top


The task of the iron-regulatory system is to balance between iron stores and iron consumption. Recently the predominant negative regulator of iron absorption in the small intestine and iron release from macrophages was discovered - namely, hepcidin. Hepcidin, the 25-amino acid peptide produced by hepatocytes, may be a new mediator of innate immunity and the long-sought iron-regulatory hormone [8] .

Hepcidin and serum ferritin respond similarly to inflammation and changes in iron stores. However, hepcidin responses take place within a few hours, whereas changes in ferritin concentration are much slower [7] .

Competitive ELISA assay is a quantitative measurement of bioactive serum hepcidin and is considered the most accurate method to estimate hepcidin level [9] .

The aim of our work was to study changes in serum hepcidin and ferritin level in patients with chronic hemolytic anemia (β-thalassemia major and sickle cell anemia) to clarify the relation between hepcidin level and iron regulation.

The study was carried out on 55 children: 23 patients with thalassemia major, 12 patients with sickle cell disease, and 20 apparently healthy children. The children were diagnosed with b thalassemia and sickle cell disease by complete blood count and HB electrophoresis. The healthy children served as controls and were matched in age, sex, and socioeconomic standard.

There was no significant difference in consanguinity among thalassemia and sickle cell cases, as these diseases are a group of hereditary genetic diseases, although β-thalassemia) is a form of thalassemia due to mutations in the HBB gene on chromosome 11, inherited in an autosomal recessive manner, and sickle cell disease is usually due to gene mutation according to the study by Weizer-Stern et al. [10] .

In our study, there was a significant difference between pretransfusion Hb% in thalassemia cases and sickle cell cases when compared with Hb% of controls. It was found that Hb% in thalassemia and sickle cell patients was significantly lower than Hb% of control (P < 0.001).

This is in agreement with the study by Yaish [11] who documented that symptoms and signs of anemia are the first presentation in the diagnosis of thalassemia and sickle cell disease and that thalassemia should be considered in any child with hypochromic microcytic anemia that does not respond to iron supplementation. These results are also in agreement with the study by Zimmermann et al. [12] , who documented that β-thalassemia and sickle cell patients had significantly lower hemoglobin compared with control subjects.

Unfortunately, repeated blood transfusions lead to a buildup of iron in the body. Iron buildup can damage the heart, liver, and other organs. To prevent organ damage, children and adults who receive regular transfusions are treated with a drug called iron chelator to eliminate excess iron, as in the study by Weizer-Stern et al. [10] .

There was significant difference in iron chelation between thalassemia and sickle cell anemia patients as 87% of thalassemia cases were under chelation therapy in the form of subcutaneous desferoxamine, 8.7% of cases were under oral chelation (deferiprone), and 4.3% of cases were not using chelation therapy as ferritin level did not exceed 1000 ng/ml. In contrast, only one sickle cell patient (8.3% of cases) was under chelation therapy in the form of oral deferiprone because of stroke attack. This is in agreement with the study by Nantasit et al. [13] who documented that desferal subcutaneous infusion is the most widely used iron chelator in thalassemia and is cost-effective when compared with other therapies. In our study only one thalassemia patient was not using iron chelation, as serum ferritin level was less than 1000 ng/ml.

As regards the history of surgical splenectomy, the number of thalassemia patients who had undergone splenectomy was 9 (39.1%), whereas surgical splenectomy had been carried out in two of 12 patients (16.7%) with sickle cell disease (one case was traumatic and the other was due to sequestration crisis).

This comes in agreement with the results of Yaish [11] who stated that splenectomy is the principal surgical procedure used for many patients with β-thalassemia, as the spleen increases RBC destruction and patients are no longer able to maintain adequate Hb level because of hypersplenism.

Most patients with sickle cell disease undergo autosplenectomy, but in rare cases of sequestration crises the indication for splenectomy is mandatory, in agreement with Frenette and Atweh [14] .

Excess iron overload in chronic hemolytic anemia occurs either from the disease itself or from frequent transfusions. Too much iron can result in damage to the heart, liver, and endocrinal system, which includes glands that produce hormones that regulate processes throughout the body causing a lot of complications. This comes in agreement with the results of Schrier [15] .

Splenomegaly in thalassemia patients was found in 21.7% of cases versus 8.3% of sickle cell cases. In thalassemia patients splenomegaly occurs because of the clearance of damaged red cells from the circulation, as stated by Galanello and Origa [16] , whereas in sickle cell anemia the spleen enlarges in the latter part of the first year of life, then undergoes repeated infarctions, and over time the spleen becomes fibrotic and shrinks - called autosplenectomy according to the study by Johnson et al. [17] .

Diabetes mellitus in thalassemia patients was diagnosed in 8.7% of cases, whereas no cases of diabetes mellitus were seen among sickle cell patients. This, according to Brawley et al. [18] , could be because of the absence of regular blood transfusion and iron overload.

Hepatomegaly was seen in 43.4% of thalassemia patients, compared with 50% of sickle cell patients, whereas liver cirrhosis was seen in 8.7% of thalassemia patients compared with 0% of sickle cell patients. This could be attributed to the iron overload in thalassemia patients, which causes damage to the liver and thus hepatomegaly or cirrhosis. The risk of developing blood-borne infection related to blood transfusions, such as hepatitis, increases in chronic hemolytic patients. This is in agreement with the results of Schrier [15] , and also explains the 8.3% of cases among thalassemia patients and hepatitis B virus-positive sickle cell cases in our study.

As for vaso-occlusive crisis, it occurred in 100% of sickle cell cases versus 0% of thalassemia patients. Vaso-occlusive crisis is the most common clinical manifestation of sickle cell disease, as reported by Maciaszek and Lykotrafitis [19] .

Finally, regarding sequestration crises, no cases were seen among thalassemia patients versus 8.3% among sickle cell cases. As reported by Geiger and Yamada [20] , sequestration crises in sickle cell disease may be uncommon but turns fatal rapidly, occurring between 5 and 24 months. It is characterized by splenomegaly and abdominal pain of sudden onset, accompanied by nausea and vomiting.

Serum ferritin levels showed a highly significant increase in thalassemia and sickle cell patients when compared with controls (P < 0.001). This is in agreement with the results of Zimmermann et al. [12] and can be explained by the fact that patients are maintained on regular blood transfusion and each 1 ml of packed red blood cells increases the body's iron load by 1 mg.

There was a significant increase in hepcidin level in thalassemia and sickle cell cases when compared with controls. Hepcidin level in thalassemia was significantly higher than that in sickle cell patients (P < 0.001). This comes in agreement with the studies by Kemna et al. [21] , Kemna et al. [22] , and Nemeth [23] who stated that hepcidin level is much higher in cases of thalassemia major than in sickle cell diseases, as blood transfusions in thalassemia patients decrease the erythropoietic drive and increase the iron overload, resulting in relatively higher hepcidin levels compared with sickle cell patients.

In our study serum ferritin showed significant negative correlation with serum hepcidin level in thalassemia patients, and there was no significant correlation between pretransfusion Hb% and age of years with hepcidin level in thalassemia patients.

Regarding sickle cell cases, there was no significant correlation between ferritin and hepcidin levels in our study. Also, there was no significant difference between pretransfusion Hb% and age in years with hepcidin level.

Ganz [7] documented that hepcidin is a negative regulator of iron absorption and mobilization; high hepcidin levels turn off both duodenal iron absorption and release of iron from macrophages, whereas low hepcidin levels promote iron absorption and heme iron recycling/iron mobilization from macrophages. Thus, hepcidin levels are expected to be high in iron-overload states and low in iron-deficient states. Hepcidin production can be induced by inflammation, which explains the reduced availability of iron in the anemia of chronic disease, whereas anemia and hypoxia have been shown to increase iron absorption and mobilization by decreasing hepcidin production.

However, there is large variability among thalassemia major patients, probably because of several factors that might influence hepcidin production, such as time of transfusion, iron chelation therapy, and amount of iron overload, according to Piperno et al. [4] .


  Conclusion Top


From this study it can be concluded that patients with iron overload (patients with β-thalassemia major and those with sickle cell anemia) have higher hepcidin level, which leads to moderation of dietary iron absorption and iron retention by macrophages. In β-thalassemia patients, a high hepcidin level is considered a result of the chronic blood transfusion given regularly to patients. In the future, hepcidin measurements may allow a more accurate assessment of the degree of iron overload and the maldistribution of iron in iron-overload patients.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Yaish HM. Pediatric thalassemia. 2010; emedicine. Available at: http://www.medscape.com/article/958850-overview [Last accessed on May 2013].  Back to cited text no. 11
    
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Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis 2010; 5:11.  Back to cited text no. 16
    
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Johnson CS, Omata M, Tong MJ. Liver involvement in sickle cell disease. Medicine (Baltimore) 2005; 64 :349-356.  Back to cited text no. 17
    
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Brawley OW, Cornelius LJ, Edwards LR. National Institutes of Health Consensus Development Conference statement hydroxyurea treatment for sickle cell disease. Ann Intern Med 2008; 148 :932-938.  Back to cited text no. 18
    
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Maciaszek JL, Lykotrafitis G. Sickle cell trait human erythrocytes are significantly stiffer than normal. J Biomech 2011; 44 :657-661.  Back to cited text no. 19
    
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Geiger B, Yamada KM. Molecular architecture and function of matrix adhesions. Cold Spring Harb Perspect Biol 2011; 3 :a005033.  Back to cited text no. 20
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11]


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