Body iron content and distribution

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CHAPTER 3 RESULTS

The primary purpose of this study was to investigate the expression of the H-subunit and L-subunit of ferritin in the bone marrow macrophage and cells of the erythron during chronic immune stimulation. In addition to this, various humoral factors were determined to assess the immune and iron status of the patients. In order to perform the H-subunit and L-subunit determinations blind, the diagnosis, immune and iron status of the patients were unknown to the investigator. The confounding factor for the inclusion of patients in this study was a bone marrow biopsy. Biopsies could, in line with the ethical clearance prescriptions for this study, only be obtained during a scheduled bone marrow biopsy for diagnostic purposes or during elective hip replacement surgery. Forty-eight patients attending the Department of Internal Medicine, Kalafong Hospital for treatment of chronic diseases, with a high prevalence of human immunodeficiency virus (HIV) infection were included in the study. Bone marrow tissue was obtained from the 48 patients during the scheduled bone marrow biopsy. All patients gave informed consent for the use of these samples including 25 ml of blood. Ethical clearance for the study was obtained from the Faculty of Health Sciences Research Ethics Committee, University of Pretoria (ethical clearance number 118/2003). Ten patients scheduled for hip replacement at the Department of Orthopaedics at the Pretoria Academic Hospital were included in the study as a group of patients with less severe immune stimulation. These patients were all diagnosed with osteoarthritis and were HIV-negative. Bone marrow tissue from the osteoarthritis patients was taken during hip replacement surgery.
All patients gave informed consent for the use of these samples including 25 ml of blood. Ethical clearance for the study was obtained from the Faculty of Health Sciences Research Ethics Committee, University of Pretoria (ethical clearance number 285/2003).
Table 1 shows the demographics of the patients from the Department of Internal Medicine and from the Department of Orthopaedics. Table 2 shows the diagnosis and HIV status of the patients from the Department of Internal Medicine. The diagnosis of the patients from the Department of Internal Medicine was diverse and included various types of infections (tuberculosis (TB), malaria, HIV), cancers (lung, breast), pancytopenias as a result of bone marrow suppression or peripheral destruction of blood cells, organ failures including renal failure, heart failure and liver failure, anaemias with different etiologies and various other pathologies that resulted in inflammatory reactions. This resulted in an extremely heterogenous group of patients. For the purpose of this study the immune status and iron status, respectively, were used to group these patients. The osteoarthritis patients were treated as a separate group.

Expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron

The expression of the H-subunit and L-subunit of ferritin in the macrophage and cells of the erythron in the bone marrow were determined for both the patients from the Department of Internal Medicine, Kalafong Hospital (Kalafong patient group) and the Department of Orthopaedics, Pretoria Academic Hospital (osteoarthritis group). The results are expressed as count/µm². Table 3 contains the mean gold particle count/µm² for the macrophage, the cells of the erythron and the H-subunit/L-subunit ratios for the macrophages and cells of the erythron.

Serum iron markers for the Kalafong patient group and osteoarthritis patient group

For the purpose of this study it is the iron status and derangements of iron metabolism that were investigated in relation to the expression of the H-subunit and L-subunit of ferritin in the bone marrow macrophage and cells of the erythron. In order to determine the iron status the concentrations of various relevant factors relating to iron metabolism were determined in the blood. These included serum iron, transferrin, transferrin saturation, ferritin and the soluble transferrin receptor (Table 4).

Red blood production for the Kalafong patient group and osteoarthritis patient group

Red blood cell charateristics are useful in evaluating the iron status since the production of red blood cells is directly influenced by the iron status and inflammatory conditions. Red blood cell production is influenced by inflammatory processes as a result of the derangement in iron metabolism but also due to suppression of the bone marrow. The following were measured in relation to red blood cell production, red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red blood cell distribution width and reticulocyte production index (Table 4).

Cytokines, C-reactive protein, neopterin, pro-hepcidin and caeruloplasmin The cytokines investigated included interferon-γ (INF-γ), tumor necrosis factor-α (TNF-

α), interleukin-1β (Il-1β), interleukin-6 (Il-6), interleukin-12 (Il-12), interleukin-2 (Il-2), interleukin-8 (Il-8), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-4 (Il-4), interleukin-5 (Il-5), transforming growth factor-β (TGF-β) and interleukin-10 (Il-10) (Table 5).

Statistical analysis of the study

For the Kalafong patient group three different subdivisions were investigated statistically. The osteoarthritis patients were treated as a separate group and included with each of the subdivisions of the Kalafong patients for statistical evaluation. Kalafong patients were subdivided into two groups based on normal and elevated C-reactive protein levels. Refer to table 6, table 9 and bar diagram figures 1a-33a. Kalafong patients were subdivided into two groups based on normal and elevated neopterin levels. Refer to table 7, table 10 and bar diagram figures 1b-33b. Kalafong patients were subdivided into two groups based on the presence or absence of an iron transfer block. Refer to table 8, table 11 and bar diagram figures 1c-33c.
For the subdivision based on C-reactive protein and the osteoarthritis patients, the subdivision based on neopterin and the osteoarthritis patients and the subdivision based on the presence or absence of an iron transfer block and the osteoarthritis patients the groups were compared using the Welch t-test and since groups were relatively small and variances could be large, use was also made of the ranksum (Mann-whitney) test. The p-values for both tests were reported and when interpreted, preference was given to the p-value of the ranksum test when the Welch t-test was not significant. Testing was done at the 0.05 level of significance. A p-value of 0.05 and less was taken as statistically significant and a p-value of > 0.05 and < 0.1 was considered as marginally different.

Correlations for the study

Pearson’s product-moment correlation coefficient (r) was employed to assess dependence between study parameters.
Electron micrographs and raw data of immunolabelling of H-subunit and L-subunit of ferritin, photographs of the Prussian blue iron stains and the presence or absence of an iron transfer block are contained in volume 2, chapter 6.

CHAPTER 1  INTRODUCTION
1) Body iron content and distribution
2) Ferritin and ferritin isoforms:  Structure-function relationships, synthesis, degradation and secretion
2.1) Structure of ferritin
2.1.1) Structure of the ferritin protein shell
2.1.1.1) Intra-subunit and inter-subunit amino acid side-chain interactions of the ferritin protein shell
2.1.1.2) Channels present in the ferritin protein shell
2.1.1.3) The ferroxidase catalytic center of the H-subunit of the ferritin protein shell
2.1.1.4) The nucleation site of the L-subunit on the inner iron/protein interface of the ferritin protein shell
2.1.2) The iron mineral
2.2) Mechanism of iron sequestration and release: The role of the ferritin protein shell, in iron mineralization and demineralization
2.2.1) Oxidation of ferritin
2.2.1.1) Oxidation of Fe2+ by the ferroxidase center of the H-subunit
2.2.1.2) Oxidation of Fe2+ on the surface of the growing iron core
2.2.2) Hydrolysis and nucleation of the formed Fe3+-compound
2.2.3) Different iron oxidation kinetics and the formation of different reaction products by the ferroxidase center oxidation of iron and oxidation of iron on the mineral surface
2.2.4) Migration of iron between ferritin molecules
2.2.5) Non-specific Fe3+-compound hydrolysis on the outer surface of the ferritin protein shell
2.2.6) The cooperative roles of the H-subunit and L-subunit of the ferritin protein shell in iron mineralization
2.2.7) The release of iron from ferritin
2.3) Isoferritins
2.3.1) Different H-subunit/L-subunit compositions of the ferritin protein shell
2.4) The synthesis of ferritin
2.4.1) Assembly of ferritin from the pool of available H- and L-subunits
2.4.2) Regulation of the expression of the H-subunit and L-subunit genes of ferritin
2.4.3) The gene sequences of the H-subunit and L-subunit of ferritin
2.4.4) Translational regulation of the H-subunit and L-subunit mRNA expression via metabolically available iron
2.4.5) Translational regulation of H-subunit and L-subunit expression irrespective of metabolically available iron
2.5) The degradation of ferritin
2.5.1) The formation of haemosiderin from ferritin
2.5.2) The increased susceptibility of H-subunit rich ferritins to degradation
2.5.3) The reticuloendothelial cell and haemosiderin formation
2.6) Ferritin in cellular organelles
2.6.1) Nuclear ferritin
2.6.2) Mitochondrial ferritin
2.7) Extracellular ferritin
2.7.1) The internalization of ferritin by cells
2.7.2) Other functions of ferritin
2.8) In conclusion
2.9) Figure 1: Heuristic presentation of intracellular ferritin metabolism
3) Ferritin and ferritin isoforms: Protection against uncontrolled cellular proliferation, oxidative damage and inflammatory processes
3.1) Ferritin and the differential expression of the H- and L-subunits of ferritin during uncontrolled cellular proliferation
3.1.1) Cellular proliferation, ferritin subunits and cancer
3.1.2) Cellular differentiation
3.1.3) Programmed cell death (apoptosis)
3.2) The expression of the H- and L-subunits of ferritin in diseases and toxicities associated with an increase in reactive oxygen species (ROS) generation
3.2.1) Oxidative stress and neurodegenerative diseases
3.2.2) Oxidative stress and vascular disorders
3.2.3) UV-induced oxidative damage
3.3) The expression of ferritin and the differential expression of the H- and L-subunits of ferritin in inflammatory conditions
3.3.1) The macrophage, iron metabolism and ferritin in inflammatory conditions
3.3.2) Increased ferritin expression as a result of cytokine activation
3.4) Table 1: The effects of cytokines on the expression of H-subunits and L-subunits of ferritin
3.5) In conclusion
4) Aim of the study
5) References
CHAPTER 2  MATERIALS AND METHODS
1) Funding
2) Investigators
3) Patients
4) Determinations of the study
5) Samples obtained from patients
6) Materials and methods
7) Statistical analysis
8) Study design
9) References
CHAPTER 3 RESULTS
1) Determinations of the study
2) Results of the study
3) Statistical analysis of the study
4) Bar diagrams for variables for the different subdivisions
5) Correlations in the different subgroups of the Kalafong patients and the group of osteoarthritis patients
CHAPTER 4 DISCUSSION
1) INTRODUCTION
2) EXPERIMENTAL GROUPS
3) AIM OF THE STUDY
4) SUBDIVISION OF THE PATIENTS ACCORDING TO THEIR IMMUNE STATUS
5) THE CYTOKINE RESPONSE OF PATIENTS WITH ELEVATED CREACTIVE PROTEIN AND PATIENTS WITH ELEVATED NEOPTERIN
6) EXPRESSION OF THE H-SUBUNIT AND L-SUBUNIT OF FERRITIN IN THE BONE MARROW MACROPHAGE AND CELLS OF THE ERYTHRON IN PATIENTS WITH A PRO-INFLAMMATORY IMMUNE STATUS COMPARED TO PATIENTS WITH NO PRONOUNCED IMMUNE ACTIVATION
7) PREVALENCE OF THE IRON TRANSFER BLOCK IN PATIENTS WITH A PRO-INFLAMMATORY IMMUNE STATUS COMPARED TO PATIENTS WITH NO PRONOUNCED IMMUNE ACTIVATION
8) EXPRESSION OF THE H-SUBUNIT AND L-SUBUNIT OF FERRITIN IN A GROUP OF KALAFONG PATIENTS WITH IRON TRANSFER BLOCK COMPARED TO A GROUP OF KALAFONG PATIENTS WITH NO IRON TRANSFER BLOCK
9) INCREASE IN THE EXPRESSION OF THE H-SUBUNIT OF FERRITIN IN THE MACROPHAGES OF OSTEOARTHRITIS PATIENTS AND IMPLICATIONS
10) References
CHAPTER 5  FINAL SUMMARY AND CONCLUSIONS
References
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