CHAPTER 3 RESULTS. L-subunit of ferritin in the bone marrow macrophage and cells of the erythron during

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1 7 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 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 8/3). 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.

2 8 All patients gave informed consent for the use of these samples including 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 8/3). Table shows the demographics of the patients from the Department of Internal Medicine and from the Department of Orthopaedics. Table 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

3 9 Table. Demographics of the patients from the Department of Internal Medicine, Kalafong Hospital (patients -48) and the Department of Orthopaedics, Pretoria Academic Hospital (patients -) Patient Age Sex Race Patient Age Sex Race 3 Female Black 48 Female Black 8 Female Black 6 Female White 3 7 Female Black 3 63 Male White 4 4 Female Black 4 43 Female White 4 Female Black 6 Male White 6 8 Female White 6 6 Female White 7 46 Male Black 7 77 Female White 8 7 Male Black 8 7 Male White 9 9 Female Black 9 6 Male White Female Black 6 Female White 4 Male Black 4 Female Black 3 33 Female Black 4 44 Female Black 4 Female Black 6 Female Black 7 33 Female Black 8 36 Female White 9 4 Female Black 4 Female Black Female Black 4 Female White 3 3 Female Black 4 33 Female Black 47 Female Black 6 Female Black 7 8 Male Black 8 4 Female Black 9 6 Female Black 3 4 Female White 3 Female Black 3 39 Female Black 33 7 Female Black 34 8 Female Black 3 6 Female Black Female Black 37 4 Female Black 38 3 Female Black Male Black 4 4 Male Black 4 9 Male Black 4 7 Male Black Male Black 44 6 Female Black 4 3 Male Black Female Black Female Black 48 7 Female Black

4 3 Table. Diagnosis and HIV status of the patients from the Department of Internal Medicine, Kalafong Hospital Patient Diagnosis HIV status Pneumonia & sepsis, Escherichia coli & urinary tract infection & acute renal failure positive Heart failure & megaloblastic anaemia & pernicious negative 3 Anaemia & idiopathic thrombocytic purpura & acute haemolytic anaemia vs disseminated intravascular coagulopathy, post mortem positive 4 Carcinoid cancer & pneumonia & metastasis to the brain negative Idiopathic vasculitis & pancytopenia questioning antiphospholipid syndrome & uterine mass negative 6 Malaria & idiopathic thrombocytic purpura negative 7 Cruveilhier-Baumgarten disease & hypersplenism & TB negative 8 Lung cancer & acute renal failure & pneumonia negative 9 Pneumonia, Escherichia coli positive Idiopathic thrombocytic purpura & iron deficient anaemia & questioning systemic lupus erythematosus negative Retro viral disease & renal failure positive Metastatic breast cancer negative 3 Pneumonia & confusion positive 4 Pulmonary TB & effusion positive Megaloblastic anaemia negative 6 Anaemia due to blood loss negative 7 Retro viral disease & anaemia & kaposi's sarcoma & previous pulmonary TB positive 8 Antiphospholipid syndrome & haemolytic anaemia negative 9 Retro viral disease & bicytopenia & mycobacterium avium complex positive Retro viral disease & anaemia & dilated cardiomyopathy & antiphospholipid syndrome & TB & thrombosis positive Retro viral disease & TB & sepsis & anaemia & ascitis positive Chronic obstructive pulmonary disease & liver & kidney failure & pelagra & sepsis & ethanol abuse negative 3 Retro viral disease & pneumonia & pancytopenia positive 4 Idiopathic thrombocytic purpura negative Retro viral disease & diabetes mellitus & heart failure & obesity & splenomegaly & lymphadenopathy & TB, bone marrow positive 6 Ethanol abuse & radial fracture & pulmonary TB & bradycardia & primary hypertension, increase calcium negative 7 Massive splenomegaly & pancytopenia negative 8 Megaloblastic anaemia & syphilis negative 9 Delerium & TB & calcified nodes, post mortem = miliary TB negative 3 OD & thrombocytopenia & macrocytosis & ethanol abuse negative 3 Retro viral disease & pulmonary TB & pancytopenia positive 3 Nephritis: hypertension, edema, proteinuria negative 33 Pancytopenia & ascitis & TB & proteinuria & urinary tract infection & sepsis, ICU negative 34 Retro viral disease & Hodgkin's disease & TB positive 3 Hypertension & diabetes mellitus & leucocytosis, persistent negative 36 Retro viral disease on HAART & pulmonary TB positive 37 Retro viral disease & idiopathic thrombocytic purpura immune, normal spleen positive 38 Megaloblastic anaemia & hypothyroidism negative 39 Anaemia negative 4 Anaemia & pyrexia & miliary TB, bone marrow culture positive 4 Pancytopenia & idiopathic thrombocytic purpura positive 4 Iron deficient anaemia negative 43 Iron deficient anaemia & peptic ulcer disease & pneumonia, Staphylococcus aureus negative 44 Idiopathic 4-limb african gangrene negative 4 Retro viral disease & pneumonia & pancytopenia positive 46 Monoclonal gammopathy of undetermined significance & uterine mass negative 47 Retro viral disease & pancytopenia & pneumonia positive 48 Idiopathic thrombocytic purpura & bicytopenia negative

5 3 ) Determinations of the study.) The expression of the H-subunit and L-subunit in the macrophage of the bone marrow and cells of the erythron (Table 3). The electron micrographs and raw data of these determinations are contained in volume, chapter 6..) Serum iron markers including serum iron, transferrin, transferrin saturation, ferritin and soluble transferrin receptor (Table 4)..3) Red blood cell characteristics including 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)..4) Prussian blue iron stains of bone marrow aspirates and bone marrow core biopsies. Reports by Haematologists from the National Health and Laboratory Services and photographs are contained in volume, chapter 6..) Evaluations of the presence or absence of an iron transfer block are contained in volume, chapter 6..6) Cytokines including Il-, Il-, Il-4, Il-, Il-6, Il-8, Il-, Il-, TNF-α, TGF-β, INF-γ and GM-CSF (Table )..7) Neopterin (Table )..8) C-reactive protein (Table )..9) Pro-hepcidin and caeruloplasmin (Table ).

6 3 ) Results of the study.) 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/µm. Table 3 contains the mean gold particle count/µm for the macrophage, the cells of the erythron and the H-subunit/L-subunit ratios for the macrophages and cells of the erythron.

7 33 Table 3. Expression of the H-subunit and L-subunit of ferritin in the bone marrow macrophage and cells of the erythron for the Kalafong patient group (patients -48) and the osteoarthritis patient group (patients -) Patient H-subunit/Lsubunit macrophages H-subunit/Lsubunit erythron H-subunit macrophages L-subunit macrophages H-subunit erythron L-subunit erythron count/µm count/µm count/µm count/µm

8 34.) 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)..3) 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)..4) Cytokines, C-reactive protein, neopterin, pro-hepcidin and caeruloplasmin The cytokines investigated included interferon-γ (INF-γ), tumor necrosis factor-α (TNFα), interleukin-β (Il-β), interleukin-6 (Il-6), interleukin- (Il-), interleukin- (Il-), interleukin-8 (Il-8), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-4 (Il-4), interleukin- (Il-), transforming growth factor-β (TGF-β) and interleukin- (Il-) (Table ).

9 3 Table 4. Serum iron markers and red blood cell production for the Kalafong patient group (patients -48) and the osteoarthritis patient group (patients -) Trans- Soluble Trans- Soluble Trans- ferrin transferrin ferrin/ transferrin R/ Patient Iron ferrin saturation Ferritin receptor log ferritin log ferritin RCC Hb Hct MCV MCH MCHC RDW RPI µmol/l g/l % µg/l µg/ml x /l g/dl l/l fl pg g/dl %

10 36 Table. Humoral factors for the Kalafong patient group (patients -48) and the osteoarthritis patient group (patients -) C-reactive Pro- Caerulo- Patient Il- Il- Il-4 Il- Il-6 Il-8 Il- Il- TNF-alpha TGF-beta INF-gamma GM-CSF Neopterin protein hepcidin plasmin pg/ml pg/ml ng/ml pg/ml pg/ml ng/ml mg/l ng/ml g/l

11 37 3) 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 a-33a. ) Kalafong patients were subdivided into two groups based on normal and elevated neopterin levels. Refer to table 7, table and bar diagram figures b-33b. 3) Kalafong patients were subdivided into two groups based on the presence or absence of an iron transfer block. Refer to table 8, table and bar diagram figures c- 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. level of significance. A p-value of. and less was taken as statistically significant and a p-value of >. and <. was considered as marginally different. Correlations for the study Pearson s product-moment correlation coefficient (r) was employed to assess dependence between study parameters.

12 38 Table 6. Descriptive statistics and statistical evaluation of expression of the H-subunit and L- subunit of ferritin, serum iron markers, red blood cell production, pro-hepcidin and caeruloplasmin for the C-reactive protein subdivision of the Kalafong patients and the osteoarthritis patients CRP elevated vs CRP elevated vs CRP normal vs CRP normal osteoarthritis osteoarthritis CRP CRP Mann- Osteo- Mann- Mann- Variable Elevated normal t-test Whitney arthritis t-test Whitney t-test Whitney H-subunit Mean macrophages SD count/µm N 9 7 L-subunit Mean macrophages SD count/µm N H-subunit/Lsubunit Mean macrophages SD N 9 6 H-subunit Mean cells of erythron SD count/µm N 9 7 L-subunit Mean cells of erythron SD count/µm N H-subunit/Lsubunit Mean cells of erythron SD N 9 6 Serum iron Mean µmol/l SD N 9 9 Transferrin Mean g/l SD N 8 9 Transferrin Mean saturation SD % N 6 9 Ferritin Mean µg/l SD N 9 9 Soluble transferrin Mean receptor SD µg/ml N 9 9

13 39 Transferrin/ Mean log ferritin SD.3.8 N 8 9 Soluble transferrin Mean receptor/ SD log ferritin N 9 9 Red blood cell count Mean x /l SD N Haemoglobin Mean g/dl SD N 9 9 Haematocrit Mean l/l SD... N 9 9 Mean corpuscular Mean volume SD fl N Mean corpuscular Mean haemoglobin SD pg N Mean corpuscular Mean haemoglobin SD.4. concentration N g/dl Red blood cell Mean distribution width SD % N RPI Mean SD.6.8 N Neopterin Mean ng/ml SD N 9 9 Pro-hepcidin Mean ng/ml SD N 8 9 Caeruloplasmin Mean g/l SD..6.4 N 7 9 CD4 Mean x 6 /l SD N

14 4 Table 7. Descriptive statistics and statistical evaluation of expression of the H-subunit and L- subunit of ferritin, serum iron markers, red blood cell production, pro-hepcidin and caeruloplasmin for the neopterin subdivision of the Kalafong patients and the osteoarthritis patients Neopterin elevated Neopterin elevated Neopterin normal vs neopterin normal vs osteoarthritis vs osteoarthritis Neopterin Neopterin Mann- Osteo- Mann- Mann- Variable elevated normal t-test Whitney arthritis t-test Whitney t-test Whitney H-subunit Mean macrophages SD count/µm N L-subunit Mean macrophages SD count/µm N H-subunit/Lsubunit Mean macrophages SD N H-subunit Mean cells of erythron SD count/µm N L-subunit Mean cells of erythron SD count/µm N H-subunit/Lsubunit Mean cells of erythron SD N Serum iron Mean µmol/l SD N 3 3 Transferrin Mean g/l SD N 34 3 Transferrin Mean saturation SD % N 3 3 Ferritin Mean µg/l SD

15 4 N 3 3 Soluble transferrin Mean receptor SD...4 µg/ml N 3 3 Transferrin/ Mean log ferritin SD N 34 3 Soluble transferrin Mean receptor/ SD log ferritin N 3 3 Red blood cell count Mean x /l SD N Haemoglobin Mean g/dl SD N 3 3 Haematocrit Mean l/l SD.8.. N 3 3 Mean corpuscular Mean volume SD fl N Mean corpuscular Mean haemoglobin SD pg N Mean corpuscular Mean haemoglobin SD. 3. concentration N g/dl Red blood cell Mean distribution width SD % N RPI Mean SD.8.3 N 7 C-reactive Mean protein SD mg/l N 3 3 Pro-hepcidin Mean ng/ml SD N 34 3 Caeruloplasmin Mean g/l SD.6.4.4

16 4 N 33 3 CD4 Mean x 6 /l SD N 7 3 Table 8. Descriptive statistics and statistical evaluation of expression of the H-subunit and L- subunit of ferritin, serum iron markers, red blood cell production, pro-hepcidin and caeruloplasmin for the iron transfer block subdivision of the Kalafong patients and the osteoarthritis patients Fe-block vs Fe-block vs No Fe-block vs no Fe-block osteoarthritis Fe-block No Fe- Mann- Osteo- Mann- Mann- Variable block t-test Whitney arthritis t-test Whitney t-test Whitney H-subunit Mean macrophages SD count/µm N 3 7 L-subunit Mean macrophages SD count/µm N 3 6 H-subunit/Lsubunit Mean macrophages SD N 3 6 H-subunit Mean cells of erythron SD count/µm N 3 7 L-subunit Mean cells of erythron SD count/µm N 3 6 H-subunit/Lsubunit Mean cells of erythron SD N 3 6 Serum iron Mean µmol/l SD N 3 Transferrin Mean g/l SD N 4 3

17 43 Transferrin Mean saturation SD % N 3 Ferritin Mean µg/l SD N 3 Soluble transferrin Mean receptor SD µg/ml N 3 Transferrin/ Mean log ferritin SD N 4 3 Soluble transferrin Mean receptor/ SD log ferritin N 3 Red blood cell count Mean x /l SD N 3 9 Haemoglobin Mean g/dl SD N 3 Haematocrit Mean l/l SD.9.3. N 3 Mean corpuscular Mean volume SD fl N 3 9 Mean corpuscular Mean haemoglobin SD 7.7. pg N 3 9 Mean corpuscular Mean haemoglobin SD.9.7. concentration N 3 9 g/dl Red blood cell Mean distribution width SD % N RPI Mean SD.4.89 N

18 44 Neopterin Mean ng/ml SD N 3 C-reactive Mean protein SD mg/l N 3 Pro-hepcidin Mean ng/ml SD N 3 Caeruloplasmin Mean g/l SD N 3 3 CD4 x 6 /l Mean SD N Table 9. Descriptive statistics and statistical evaluation of all cytokines for the C-reactive protein subdivision of the Kalafong patients and the osteoarthritis patients CRP elevated vs CRP elevated CRP normal CRP normal vs osteoarthritis vs osteoarthritis CRP CRP Mann- Osteo- Mann- Mann- Variable elevated normal t-test Whitney arthritis t-test Whitney t-test Whitney GM-CSF Mean pg/ml SD N 8 9 Interleukin-beta Mean pg/ml SD N 9 9 Interleukin- Mean pg/ml SD N 9 9 Interleukin-4 Mean pg/ml SD.8.. N 9 9 Interleukin- Mean pg/ml SD..6 N 9 9 Interleukin-6 Mean

19 4 pg/ml SD N 9 9 Interleukin-8 Mean pg/ml SD N 9 9 Interleukin- Mean pg/ml SD N 9 9 Interleukin- Mean pg/ml SD N 9 9 TNF-alpha Mean pg/ml SD N 9 9 TGF-beta Mean ng/ml SD N 8 9 INF-gamma Mean pg/ml SD N 8 9 Table. Descriptive statistics and statistical evaluation of all cytokines for the neopterin subdivision of the Kalafong patients and the osteoarthritis patients Neopterin elevated Neopterin elevated Neopterin normal vs neopterin normal vs osteoarthritis vs osteoarthritis Neopterin Neopterin Mann- Osteo- Mann- Mann- Variable elevated normal t-test Whitney arthritis t-test Whitney t-test Whitney GM-CSF Mean pg/ml SD N 34 3 Interleukin-beta Mean pg/ml SD N 3 3 Interleukin- Mean pg/ml SD N 3 3 Interleukin-4 Mean pg/ml SD.8.3. N 3 3

20 46 Interleukin- Mean pg/ml SD N 3 3 Interleukin-6 Mean pg/ml SD N 3 3 Interleukin-8 Mean pg/ml SD N 3 3 Interleukin- Mean pg/ml SD N 3 3 Interleukin- Mean pg/ml SD N 3 3 TNF-alpha Mean pg/ml SD N 3 3 TGF-beta Mean ng/ml SD N 34 3 INF-gamma Mean pg/ml SD N 34 3 Table. Descriptive statistics and statistical evaluation of all cytokines for the iron transfer block subdivision of the Kalafong patients and the osteoarthritis patients Fe-block vs Fe-block No Fe-block no Fe-block vs osteoarthritis vs osteoarthritis Fe-block No Mann- Osteo- Mann- Mann- Variable Fe-block t-test Whitney arthritis t-test Whitney t-test Whitney GM-CSF Mean pg/ml SD N 4 3 Interleukin-beta Mean pg/ml SD N 3 Interleukin- Mean pg/ml SD

21 47 N 3 Interleukin-4 Mean pg/ml SD.9. N 3 Interleukin- Mean pg/ml SD N 3 Interleukin-6 Mean pg/ml SD N 3 Interleukin-8 Mean pg/ml SD N 3 Interleukin- Mean pg/ml SD N 3 Interleukin- Mean pg/ml SD N 3 TNF-alpha Mean pg/ml SD N 3 TGF-beta Mean ng/ml SD N 4 3 INF-gamma Mean pg/ml SD N 4 3 4) Bar diagrams for variables for the different subdivisions Figures a-33a contain bar diagrams for all variables for the C-reactive protein subdivision. Figures b-33b contain bar diagrams for all variables for the neopterin subdivision. Figures c-33c contain bar diagrams for all variables for the iron transfer block subdivision. Error bars refer to the standard deviation.

22 48 Counts/square micrometer H-subunit in macrophages Elevated C-reactive protein Normal C-reactive protein Figure a. Marginally higher for normal CRP and osteoarthritis group normal CRP Counts/square micrometer H-subunit in macrophages Elevated neopterin Normal neopterin Figure b. normal neopterin and osteoarthritis group normal neopterin Counts/square micrometer H-subunit in macrophages Iron transfer block No iron transfer block Figure c. Marginally higher for no iron transfer block iron transfer block group and Marginally higher for osteoarthritis group no iron transfer block Figure a c. Differences in expression of the H-subunit of ferritin in the macrophage for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

23 49 Counts/square micrometer L-subunit in macrophages Elevated C-reactive protein Normal C-reactive protein Figure a. and normal CRP and osteoarthritis group normal CRP Counts/square micrometer L-subunit in macrophages Elevated neopterin Normal neopterin Figure b. and normal neopterin Marginally lower for normal neopterin group and Counts/square micrometer L-subunit in macrophages Iron transfer block No iron transfer block Figure c. iron transfer block group and no iron transfer block iron transfer block group and no and Figure a c. Differences in expression of the L-subunit of ferritin in the macrophage for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

24 Units H-subunit/L-subunit ratio in macrophages Elevated C-reactive protein Normal C-reactive protein Figure 3a. Marginally higher for normal CRP and normal CRP group and Units H-subunit/L-subunit ratio in macrophages Elevated neopterin Normal neopterin Figure 3b. normal neopterin and normal neopterin group and Units H-subunit/L-subunit ratio in macrophages Iron transfer block No iron transfer block Figure 3c. iron transfer block group and no iron transfer block iron transfer block group and no and Figure 3a 3c. Differences in H-subunit/L-subunit ratio in the macrophage for the C- reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

25 Counts/square micrometer H-subunit in cells of the erythron Elevated C-reactive protein Normal C-reactive protein Figure 4a. and normal CRP and normal CRP group and Counts/square micrometer H-subunit in cells of the erythron Elevated neopterin Normal neopterin Figure 4b. and normal neopterin and normal neopterin group and Counts/square micrometer H-subunit in cells of the erythron Iron transfer block No iron transfer block Figure 4c. iron transfer block group and no iron transfer block iron transfer block group and no and Figure 4a 4c. Differences in expression of the H-subunit of ferritin in cells of the erythron for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

26 Counts/square micrometer 3 3 L-subunit in cells of the erythron Elevated C-reactive protein Normal C-reactive protein Figure a. and normal CRP and normal CRP group and Counts/square micrometer 3 3 L-subunit in cells of the erythron Elevated neopterin Normal neopterin Figure b. and normal neopterin and normal neopterin group and Counts/square micrometer 3 3 L-subunit in cells of the erythron Iron transfer block No iron transfer block Figure c. iron transfer block group and no iron transfer block iron transfer block group and no and Figure a c. Differences in expression of the L-subunit of ferritin in cells of the erythron for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

27 3 Units H-subunit/L-subunit ratio in cells of the erythron Elevated C-reactive protein Normal C-reactive protein Figure 6a. and normal CRP and Marginally higher for osteoarthritis group normal CRP Units H-subunit/L-subunit rato in cells of the erythron Elevated neopterin Normal neopterin Figure 6b. Marginally higher for normal neopterin and osteoarthritis group normal neopterin Units H-subunit/L-subunit ratio in cells of the erythron Iron transfer block No iron transfer block Figure 6c. iron transfer block group and no iron transfer block iron transfer block group and no and Figure 6a 6c. Differences in H-subunit/L-subunit ratio in cells of the erythron for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

28 4 micromole/liter Serum iron Elevated C-reactive protein Normal C-reactive protein Figure 7a. and normal CRP Marginally lower for normal CRP group and micromole/liter Serum iron Elevated neopterin Normal neopterin Figure 7b. and normal neopterin Marginally lower for osteaoarthritis Marginally lower for normal neopterin group micromole/liter Serum iron Iron transfer block No iron transfer block Figure 7c. iron transfer block group and no iron transfer block Marginally lower for no and Figure 7a 7c. Differences in serum iron for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

29 gram/liter Transferrin Elevated C-reactive protein Normal C-reactive protein Figure 8a. Significantly lower for normal CRP Significantly lower for normal CRP group and gram/liter Transferrin Elevated neopterin Normal neopterin Figure 8b. Significantly lower for normal neopterin Significantly lower for normal neopterin group and gram/liter Transferrin Iron transfer block No iron transfer block Figure 8c. Significantly lower for no iron transfer block Significantly lower for no and Figure 8a 8c. Differences in transferrin for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

30 6 Percentage Transferrin saturation Elevated C-reactive protein Normal C-reactive protein Figure 9a. and normal CRP and normal CRP group and Percentage Transferrin saturation Elevated neopterin Normal neopterin Figure 9b. Marginally lower for normal neopterin group elevated neopterin and Significantly lower for normal neopterin group Percentage Transferrin saturation Iron transfer block No iron transfer block Figure 9c. iron transfer block group and no iron transfer block iron transfer block group and no and Figure 9a 9c. Differences in transferrin saturation for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

31 7 microgram/liter Serum ferritin Elevated C-reactive protein Normal C-reactive protein Figure a. normal CRP normal CRP group and microgran/liter Serum ferritin Elevated neopterin Normal neopterin Figure b. normal neopterin normal neopterin group and microgram/liter Serum ferritin Iron transfer block No iron transfer block Figure c. Significant higher for no iron transfer block no and Figure a c. Differences in serum ferritin for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

32 8 microgram/milliliter 3 3 Soluble transferrin receptor Elevated C-reactive protein Normal C-reactive protein Figure a. Significantly lower for normal CRP normal CRP group microgram/milliliter 3 Soluble transferrin receptor Elevated neopterin Normal neopterin Figure b. and normal neopterin normal neopterin group microgram/milliliter 3 3 Soluble transferrin receptor Iron transfer block No iron transfer block Figure c. Significantly lower for no iron transfer block no iron transfer block group Figure a c. Differences in soluble transferrin receptor for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

33 9 Units Transferrin/log ferritin Elevated C-reactive protein Normal C-reactive protein Figure a. Significantly lower for normal CRP Significantly lower for normal CRP group Units Transferrin/log ferritin Elevated neopterin Normal neopterin Figure b. Significantly lower for normal neopterin Significantly lower for normal neopterin group and Units Transferrin/log ferritin Iron transfer block No iron transfer block Figure c. Significantly lower for no iron transfer block Significantly lower for Marginally higher for no iron transfer block group Figure a c. Differences in transferrin/log ferritin ratio for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

34 6 Units Soluble transferrin receptor/log ferritin Elevated C-reactive protein Normal C-reactive protein Figure 3a. Significantly lower for normal CRP normal CRP group Units 3 Soluble transferrin receptor/log ferritin Elevated neopterin Normal neopterin Figure 3b. Marginally lower for normal neopterin normal neopterin group Units Soluble transferrin receptor/log ferritin Iron transfer block No iron transfer block Figure 3c. Significantly lower for no iron transfer block iron transfer block group and no iron transfer block group Figure 3a 3c. Differences in soluble transferrin receptor/log ferritin ratio for the C- reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

35 6 x ()/liter Red blood cell count Elevated C-reactive protein Normal C-reactive protein Figure 4a. Significantly lower for normal CRP Significantly lower for Significantly lower for normal CRP group x ()/liter Red blood cell count Elevated neopterin Normal neopterin Figure 4b. Significantly lower for normal neopterin Significantly lower for Marginally lower for normal neopterin group x ()/liter Red blood cell count Iron transfer block No iron transfer block Figure 4c. iron transfer block group and no iron transfer block Significantly lower for Significantly lower for no iron transfer block group Figure 4a 4c. Differences in red blood cell count for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

36 6 femtoliter Mean corpuscular volume Elevated C-reactive protein Normal C-reactive protein Figure a. normal CRP and Significantly lower for normal CRP group femtoliter Mean corpuscular volume Elevated neopterin Normal neopterin Figure b. and normal neopterin Significantly lower for Marginally lower for normal neopterin group femtoliter Mean corpuscular volume Iron transfer block No iron transfer block Figure c. iron transfer block group and no iron transfer block iron transfer block group and Significantly lower for no iron transfer block group Figure a c. Differences in mean corpuscular volume for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

37 63 picogram Mean corpuscular haemoglobin Elevated C-reactive protein Normal C-reactive protein Figure 6a. normal CRP and Significantly lower for normal CRP group picogram Mean corpuscular haemoglobin Elevated neopterin Normal neopterin Figure 6b. and normal neopterin Significantly lower for Marginally lower for normal neopterin group picogram Mean corpuscular haemoglobin Iron transfer block No iron transfer block Figure 6c. iron transfer block group and no iron transfer block iron transfer block group and Significantly lower for no iron transfer block group Figure 6a 6c. Differences in mean corpuscular haemoglobin for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

38 64 gram/deciliter Mean corpuscular haemoglobin concentration Elevated C-reactive protein Normal C-reactive protein Figure 7a. normal CRP and Significantly lower for normal CRP group gram/deciliter Mean corpuscular haemoglobin concentration Elevated neopterin Normal neopterin Figure 7b. and normal neopterin Marginally lower for Significantly lower for normal neopterin group gram/deciliter Mean corpuscular haemoglobin concentration Iron transfer block No iron transfer block Figure 7c. no iron transfer block iron transfer block group and Significantly lower for no iron transfer block group Figure 7a 7c. Differences in mean corpuscular haemoglobin concentration for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

39 6 Percentage 3 3 Red blood cell distribution width Elevated C-reactive protein Normal C-reactive protein Figure 8a. and normal CRP normal CRP group Percentage 3 Red blood cell distribution width Elevated neopterin Normal neopterin Figure 8b. and normal neopterin normal neopterin group Percentage 3 3 Red blood cell distribution width Iron transfer block No iron transfer block Figure 8c. iron transfer block group and no iron transfer block no iron transfer block group Figure 8a 8c. Differences in red blood cell distribution width for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

40 66 Units Elevated C-reactive protein Normal C-reactive protein Figure 9a. normal CRP Reticulocyte production index Units Reticulocyte production index Elevated neopterin Normal neopterin Figure 9b. normal neopterin Units Reticulocyte production index Iron transfer block No iron transfer block Figure 9c. iron transfer block group and no iron transfer block Figure 9a 9c. Differences in reticulocyte production index for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision. The reticulocyte production index was not determined for the osteoarthritis patients.

41 67 nanogram/milliliter Neopterin Elevated C-reactive protein Normal C-reactive protein Figure a. normal CRP normal CRP group nanogram/milliliter Neopterin Elevated neopterin Normal neopterin Figure b. normal neopterin normal neopterin group and nanogram/milliliter Neopterin Iron transfer block No iron transfer block Figure c. no iron transfer block no iron transfer block group Figure a c. Differences in neopterin for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

42 68 milligram/liter C-reactive protein Elevated C-reactive protein Normal C-reactive protein Figure a. normal CRP normal CRP group and milligram/liter C-reactive protein Elevated neopterin Normal neopterin Figure b. normal neopterin normal neopterin group and milligram/liter C-reactive protein Iron transfer block No iron transfer block Figure c. no iron transfer block no iron transfer block group Figure a c. Differences in C-reactive protein for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

43 69 picogram/milliliter Interferon-gamma Elevated C-reactive protein Normal C-reactive protein Figure a. normal CRP normal CRP group and picogram/milliliter Interferon-gamma Elevated neopterin Normal neopterin Figure b. Marginally higher for normal neopterin normal neopterin group and picogram/milliliter Interferon-gamma Iron transfer block No iron transfer block Figure c. no iron transfer block no and Figure a c. Differences in interferon-γ for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

44 7 picogram/milliliter Tumor necrosis factor-alpha Elevated C-reactive protein Normal C-reactive protein Figure 3a. normal CRP normal CRP group and picogram/milliliter Tumor necrosis factor-alpha Elevated neopterin Normal neopterin Figure 3b. normal neopterin normal neopterin group and picogram/milliliter Tumor necrosis factor-alpha Iron transfer block No iron transfer block Figure 3c. iron transfer block group and no iron transfer block Marginally higher for no and Figure 3a 3c. Differences in tumor necrosis factor-α for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

45 7 picogram/milliliter Interleukin-beta Elevated C-reactive protein Normal C-reactive protein Figure 4a. normal CRP normal CRP group and picogram/milliliter Interleukin-beta Elevated neopterin Normal neopterin Figure 4b. Marginally higher for normal neopterin Marginally higher for normal neopterin group and picogram/milliliter Interleukin-beta Iron transfer block No iron transfer block Figure 4c. iron transfer block group and no iron transfer block iron transfer block group and no and Figure 4a 4c. Differences in interleukin-β for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

46 7 picogram/milliliter 3 3 Interleukin-6 Elevated C-reactive protein Normal C-reactive protein Figure a. normal CRP normal CRP group and picogram/milliliter 3 Interleukin-6 Elevated neopterin Normal neopterin Figure b. normal neopterin normal neopterin group and picogram/milliliter 3 Interleukin-6 Iron transfer block No iron transfer block Figure c. no iron transfer block no and Figure a c. Differences in interleukin-6 for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

47 73 picogram/milliliter Interleukin- Elevated C-reactive protein Normal C-reactive protein Figure 6a. and normal CRP normal CRP group picogram/milliliter Interleukin- Elevated neopterin Normal neopterin Figure 6b. and normal neopterin normal neopterin picogram/milliliter Interleukin- Iron transfer block No iron transfer block Figure 6c. iron transfer block group and no iron transfer block no iron transfer block group Figure 6a 6c. Differences in interleukin- for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

48 74 picogram/milliliter Interleukin- Elevated C-reactive protein Normal C-reactive protein Figure 7a. and normal CRP Marginally higher for normal CRP group and picogram/milliliter Interleukin- Elevated neopterin Normal neopterin Figure 7b. and normal neopterin normal neopterin group and picogram/milliliter Interleukin- Iron transfer block No iron transfer block Figure 7c. iron transfer block group and no iron transfer block no and Figure 7a 7c. Differences in interleukin- for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

49 7 picogram/milliliter Interleukin-8 Elevated C-reactive protein Normal C-reactive protein Figure 8a. normal CRP normal CRP group and picogram/milliliter Interleukin-8 Elevated neopterin Normal neopterin Figure 8b. normal neopterin normal neopterin group and picogram/milliliter Interleukin-8 Iron transfer block No iron transfer block Figure 8c. no iron transfer block no and Figure 8a 8c. Differences in interleukin-8 for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

50 76 picogram/milliliter Granulocyte macrophage colony stimulating factor Elevated C-reactive protein Normal C-reactive protein Figure 9a. and normal CRP and normal CRP group and picogram/milliliter Granolocyte macrophage colony stimulating factor Elevated neopterin Normal neopterin Figure 9b. and normal neopterin Marginally higher for normal neopterin group and picogram/milliliter Granulocyte macrophage colony stimulating factor Iron transfer block No iron transfer block Figure 9c. iron transfer block group and no iron transfer block iron transfer block group and Marginally higher for no iron transfer block group Figure 9a 9c. Differences in granulocyte macrophage colony stimulating factor for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.

51 77 picogram/milliliter Interleukin-4 Elevated C-reactive protein Normal C-reactive protein Figure 3a. and normal CRP and normal CRP group and picogram/milliliter 4 3 Interleukin-4 Elevated neopterin Normal neopterin Figure 3b. and normal neopterin and normal neopterin group and picogram/milliliter Interleukin-4 Iron transfer block No iron transfer block Figure 3c. iron transfer block group and no iron transfer block iron transfer block group and no and Figure 3a 3c. Differences in interleukin-4 for the C-reactive protein subdivision, the neopterin subdivision and the iron transfer block subdivision.