Ferritin in erythrocytes and plasma of patients with iron overload

Erythrocyte and plasma ferritin was followed in 13 patients with iron overload undergoing phlebotomies for at least 6 months in comparison with untreated patients and normal males. Plasma ferritin was widely scattered with an average of only twice the normal, whereas erythrocyte ferritin was highly elevated to about twelve times the normal (p less than 0.0001). - The time course of plasma and erythrocyte ferritin during phlebotomy therapy was analyzed in 3 patients with idiopathic hemochromatosis. Three stages were established: 1. plasma ferritin dropped gradually into the normal range while erythrocyte ferritin remained high, 2. appropriate phlebotomies maintained normal plasma ferritin and high erythrocyte ferritin, and indicated a monthly uptake of dietary iron of 150-200 mg at a steady state, 3. at low plasma ferritin levels, erythrocyte ferritin was rapidly decreased by further intensive phlebotomy therapy. Based on the presumed net removal of iron, 1 microgram/l plasma ferritin was equivalent to 3-6 mg of body iron and 1 microgram/l erythrocyte ferritin to somewhat less than 1 mg of body iron. - An elevated erythrocyte ferritin during phlebotomy therapy in iron overload not only depends on body iron stores like plasma ferritin but may also be regulated by the activity of erythropoiesis.     

Solid phase immunoradiometric assay for porcine serum ferritin

1. A solid phase immunoradiometric assay using anti-serum coated polystyrene tubes, is described for the assay of porcine serum ferritin. 2. The mean concentration of ferritin in the serum of both male and female pigs (Sus scrofa) was 12.1 micrograms/l +/- 8.7 micrograms (range less than 1-35 micrograms/l) and no sex differences were observed in 40 pigs from 1 day to 4 years old. 3. Serum ferritin increased with increasing body iron stores in iron loaded pigs as assessed by hepatic iron concentration. 4. The assay is sensitive (detecting less than 1 microgram/l), reproducible, specific and it does not cross-react with human or rat ferritin.     

Red cell ferritin content: a re-evaluation of indices for iron deficiency in the anaemia of rheumatoid arthritis.

In iron deficiency anaemia basic red cell content of ferritin is appreciably reduced. This variable was determined in 62 patients with rheumatoid arthritis to evaluate conventional laboratory indices for iron deficiency in the anaemia of rheumatoid arthritis. For 23 patients with rheumatoid arthritis and normocytic anaemia irrespective of plasma ferritin concentration, red cell ferritin content did not differ significantly from that for non-anaemic patients with rheumatoid arthritis. For 27 patients with rheumatoid arthritis and microcytic anaemia, the mean red cell ferritin content for patients with a plasma ferritin concentration in the 13-110 micrograms/l range was appreciably reduced. It was indistinguishable from that for patients with rheumatoid arthritis and classical iron deficiency anaemia, indicated by plasma ferritin concentrations of less than 12 micrograms/l. In contrast, the mean red cell ferritin content for patients with rheumatoid arthritis, microcytic anaemia, and plasma ferritin concentrations above 110 micrograms/l did not differ from that for patients with rheumatoid arthritis and normocytic anaemia. Oral treatment with iron in patients with rheumatoid arthritis, microcytic anaemia, and appreciably reduced red cell ferritin concentrations was accompanied by significant increases in haemoglobin concentration (p less than 0.01), mean corpuscular volume (p less than 0.01), and red cell ferritin contents (p less than 0.05). This treatment, however, did not produce any appreciable change in haemoglobin concentration in patients with rheumatoid arthritis, normocytic anaemia, and normal red cell ferritin contents. These findings suggest that the indices for iron deficiency in patients with rheumatoid arthritis and anaemia should include peripheral blood microcytosis together with a plasma ferritin concentration of less than 110 micrograms/l.     

Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and of incorporation of acetate into cholesterol in homozygous hypercholesterolemic fibroblasts by ferritin-low density lipoprotein conjugates.

Conjugates of ferritin with low density lipoproteins (LDL) were prepared and separated by sucrose gradient centrifugation. These conjugates, at cholesterol concentration of 100--132 microgram/ml, caused a greater than 90% suppression of hydroxymethylglutaryl coenzyme A reductase activity and of acetate incorporation into cholesterol in cultured skin fibroblasts from a normal subject as well as from a subject with homozygous familial hypercholesterolemia. The half maximal inhibition concentration was approx. 10 microgram/ml cholesterol for LDL and ferritin . (LDL)2 and 5 microgram/ml for (ferritin)2 . LDL in both cell lines. In contrast, native low density lipoproteins have only a minimal inhibitory effect in homozygous cells. The ability of the conjugates to stimulate the incorporation of oleate into cholesteryl esters was also equal in the two cell lines, although the conjugates were only 10% as active as low density lipoproteins in the normal cells. LDL reduced the ferritin . (LDL)2-mediated suppression of hydroxymethylglutaryl-CoA reductase activity in homozygous cells while ferritin . (LDL)2 reduced the LDL-mediated stimulation of cholesteryl ester formation in normal cells.     

Serum hyaluronan concentration determined by radiometric assay in patients with pretibial myxedema and Graves' ophthalmopathy

The serum concentration of hyaluronan (HA) was measured by radiometric assay in patients with pretibial myxedema (PTM) and Graves' ophthalmopathy (GO). The mean HA concentration in the patients (n = 8) was 21.2 +/- 15.3 (mean +/- SD) microgram/l, while that of Graves' disease without skin or eye involvement (n = 7) was 23.5 +/- 11.0 (mean +/- SD) microgram/l and that of the control (n = 8) was 25.5 +/- 16.4 (mean +/- SD) microgram/l. We conclude that local accumulation of glycosaminoglycan in PTM or GO is not associated with an increase in the serum HA concentration.     

Elevation in Inflammatory Serum Biomarkers Predicts Response to Trastuzumab-Containing Therapy

Approximately half of all HER2/neu-overexpressing breast cancer patients do not respond to trastuzumab-containing therapy. Therefore, there remains an urgent and unmet clinical need for the development of predictive biomarkers for trastuzumab response. Recently, several lines of evidence have demonstrated that the inflammatory tumor microenvironment is a major contributor to therapy resistance in breast cancer. In order to explore the predictive value of inflammation in breast cancer patients, we measured the inflammatory biomarkers serum ferritin and C-reactive protein (CRP) in 66 patients immediately before undergoing trastuzumab-containing therapy and evaluated their progression-free and overall survival. The elevation in pre-treatment serum ferritin (>250 ng/ml) or CRP (>7.25 mg/l) was a significant predictor of reduced progression-free survival and shorter overall survival. When patients were stratified based on their serum ferritin and CRP levels, patients with elevation in both inflammatory biomarkers had a markedly poorer response to trastuzumab-containing therapy. Therefore, the elevation in inflammatory serum biomarkers may reflect a pathological state that decreases the clinical efficacy of this therapy. Anti-inflammatory drugs and life-style changes to decrease inflammation in cancer patients should be explored as possible strategies to sensitize patients to anti-cancer therapeutics.     

Inflammation associated anemia and ferritin as disease markers in SLE

Introduction

In a recent screening to detect biomarkers in systemic lupus erythematosus (SLE), expression of the iron storage protein, ferritin, was increased. Given that proteins that regulate the storage, transfer and release of iron play an important role in inflammation, this study aims to determine the serum and urine levels of ferritin and of the iron transfer protein, transferrin, in lupus patients and to correlate these levels with disease activity, inflammatory cytokine levels and markers of anemia.

Methods

A protein array was utilized to measure ferritin expression in the urine and serum of SLE patients and healthy controls. To confirm these results as well as the role of the iron transfer pathway in SLE, ELISAs were performed to measure ferritin and transferrin levels in inactive or active SLE patients and healthy controls. The relationship between ferritin/transferrin levels and inflammatory markers and anemia was next analyzed.

Results

Protein array results showed elevated ferritin levels in the serum and urine of lupus patients as compared to controls, which were further validated by ELISA. Increased ferritin levels correlated with measures of disease activity and anemia as well as inflammatory cytokine titers. Though active SLE patients had elevated urine transferrin, serum transferrin was reduced.

Conclusion

Urine ferritin and transferrin levels are elevated significantly in SLE patients and correlate with disease activity, bolstering previous reports. Most importantly, these changes correlated with the inflammatory state of the patients and anemia of chronic disease. Taken together, altered iron handling, inflammation and anemia of chronic disease constitute an ominous triad in SLE.     

The etiological relation between serum iron level and infection incidence in hemodialysis uremic patients

Through the treatment of anaemia in dialysis patients part of the iron ions remain free in the serum which is at the bacterias disposal for growth and the strengthening of their virulence. The linear relation of the increased serum iron level and tissue iron stores in the body and the infection incidence in dialysed patients has become more emphasised. The need of a clearly defined upper threshold of the serum iron concentration limit has been mentioned in scientific journals intensely, and consequently the demand for more precise professional instructions for anaemia treatment. For the purpose of participating in these professional and scientific discussions, we have observed the relation between the iron overload of the organism and complication incidence in 120 of our haemodialysis uremic patients, with special emphasis on infections. It has been established that the sepses incidence is much higher in patients with a serum ferritin concentration above 500 microg/L, than in those patients with a ferritin level lower than the mentioned value ( 2 = 7.857, p = 0.005). The incidence of vascular access infection is significantly higher in those patients with a serum ferritin level above 500 microg/L than in those patients with a ferritin level lower than the mentioned value (Chi2 = 23.186, p = 0.001). Furthermore, it has been determined that the incidence of total infection in patients is 3.8 episodes per 100 patients months, which is in accordance to the referral values of other authors. CONCLUSION--In the analysis of the achieved results, it has been determined that the infection incidence is significantly higher in dialysed patients with a serum iron level higher than 500 g/L, than in those patients with lower values.     

Integrated optical surface plasmon resonance immunoprobe for simazine detection.

This paper presents the detailed design and characterisation of a regenerable integrated optical surface plasmon resonance immunoprobe as a detector for the triazine herbicide simazine. A sensor design theoretically optimised for use in the aqueous environment is presented and its fabrication described. Experimental results on the sensitivity to changes in bulk refractive index of the analyte and on non-specific binding of ovalbumin are presented. Binding inhibition immunoassays were conducted for simazine and the lower limit of detection determined to be 0.16 microgram/l using anti-simazine IgG antibodies and 0.11 microgram/l using anti-simazine Fab fragments. A sample test cycle of 20 min was established.     

Diagnostic value of ferritin in the differential diagnosis of malignant effusions

The diagnostic value of ferritin in pleural effusions or ascites was studied in 151 samples from 147 patients (four patients had both kind of effusions). Samples (99 pleural effusions, 52 ascites) were evaluated in 4 groups: benign transudate (27 cases), benign nontuberculous exudate (26 cases), tuberculous exudate (47 cases) and malignant exudate (51 cases). Median ferritin levels in effusions were 67 ng/ml, 805 ng/ml, 889 ng/ml, 998 ng/ml and median effusion/serum (E/S) ratios were 0.7. 2.0, 4.9, 3.2 respectively. There was a significant difference between the concentrations of ferritin in malignant (51 cases) and nonmalignant effusions (100 cases) (p < 0.001), but the specificity and positive predictive value were low (43% and 45% respectively). Ferritin levels in transudate group were significantly lower than those in the others (p < 0.001). However, ferritin concentrations in three exudate groups were similar (p > 0.05). When compared the all inflammatory effusions (malignant, tuberculous, nontuberculous inflammatory exudates) with noninflammatory effusions (transudate and exudate), we determined a significant difference (p < 0.001). CONCLUSIONS: 1) Elevated ferritin concentration in effusions is significant indicators of exudates; 2) It is not good a parameter to discriminate the malignant effusions from the benign ones; 3) They can be useful in the differential diagnosis of the inflammatory exudations from the noninflammatory ones.     

Determination of kanamycin in serum by solid-phase extraction,pre-capillary derivatization and capillary electrophoresis

An effective method based on solid-phase extraction (SPE) and capillary electrophoresis (CE) for the determination of kanamycin in human serum was developed and validated. Off-line SPE was employed for the isolation of kanamycin from serum on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge. A mixture of 0.2 M borate (pH 10.5)-methanol (50:50, v/v) was used as analyte eluting solvent. After pre-capillary derivatization with o-phthalaldehyde/mercaptoacetic acid reagent, the sample was analyzed by CE with a separation buffer of 30 mM borax, pH 10.0, containing 16% (v/v) methanol. A linear response over the concentration range 5-40 microgram/ml was obtained with a detection limit of 2 microgram/ml. Intra-day and inter-day precision were 6.2 and 10.3% RSD, respectively. Recoveries of approximately 90% were found. For the determination of lower levels of kanamycin (<5 microgram/ml), NH(4)OH (25%, w/v)-methanol (30:70, v/v) was used for analyte elution. After evaporation, reconstitution and derivatization, the sample was analyzed by on-line field-amplified sample stacking (FASS) CE. Good linearity in the concentration range 0.4-5 microgram/ml was obtained with a detection limit of 0.1 microgram/ml. Intra-day and inter-day RSD were 3.4 and 11.2%, respectively. Recoveries of approximately 60% were found. The method was successfully applied to the analysis of kanamycin in sera of tuberculosis patients at peak level and trough level concentrations.     

Serum Ferritin in Acute Leukaemia at Presentation and during Remission

In patients with acute myeloblastic leukaemia the mean serum ferritin concentration showed a twenty-five-fold increase compared with normal people, and in children with acute lymphoblastic leukaemia (A.L.L.) there was a thirteen-fold increase. The high concentration of circulating ferritin seemed to be related to increased synthesis by leukaemic cells. The return of serum concentrations to normal in A.L.L. patients after successful chemotherapy suggested that ferritin concentration may be a useful index of active disease and may help in prognosis.     

Serum ferritin, iron and transferrin in women with thyrotoxic Graves' disease before and after methimazole treatment

We investigated iron metabolism in 47 women with thyrotoxic Graves' disease. Serum iron, ferritin, transferrin, triiodothyronine and thyroxine concentrations were RIA measured before and after methimazole treatment when patients became euthyroid. The control group consisted of 52 healthy women. We noted that serum ferritin levels and the ferritin to transferrin ration were significantly lower while the iron to ferritin ratio was higher in patients before and after methimazole therapy. Iron concentration as well as the iron to transferrin and the iron to thyroid hormone ratios were decreased only before treatment.     

Pulsatile secretion pattern of growth hormone during the luteal phase and mid-anoestrus in beagle bitches

The pulsatile secretion pattern of growth hormone was investigated during four stages of the luteal phase and during mid-anoestrus in six cyclic beagle bitches. Plasma samples were obtained via jugular venepuncture at 10 min intervals for 12 h at 19 +/- 2 (mean +/- SEM; luteal phase 1), 38 +/- 2 (luteal phase 2), 57 +/- 2 (luteal phase 3), 78 +/- 2 (luteal phase 4) and 142 +/- 4 days (mid-anoestrus) after ovulation. During all stages, growth hormone was secreted in a pulsatile fashion. The mean basal plasma growth hormone concentration during luteal phase 1 (2.2 +/- 0.3 microgram l(-1)) was significantly higher than that during luteal phase 4 (1.5 +/- 0.1 microgram l(-1)) and mid-anoestrus (1.4 +/- 0.2 microgram l(-1)). The mean area under the curve (AUC) above zero during luteal phase 1 (27.3 +/- 2.7 microgram l(-1) in 12 h) tended to be higher than that during luteal phase 4 (20.8 +/- 1.8 microgram l(-1) in 12 h) and mid-anoestrus (19.2 +/- 2.5 microgram l(-1) in 12 h). In contrast, the mean AUCs above the baseline during luteal phase 1 (1.1 +/- 0.5 microgram l(-1) in 12 h) and luteal phase 2 (1.2 +/- 0.5 microgram l(-1) in 12 h) were significantly lower than that during luteal phase 4 (2.8 +/- 0.5 microgram l(-1) in 12 h). In conclusion, the pulsatile secretion pattern of growth hormone changes during the luteal phase in healthy cyclic bitches: basal growth hormone secretion is higher and less growth hormone is secreted in pulses during stages in which the plasma progesterone concentration is high. It is hypothesized that this change is caused by a partial suppression of pituitary growth hormone release by progesterone-induced growth hormone production in the mammary gland. The progesterone-induced production of growth hormone in the mammary gland may promote the physiological proliferation and differentiation of mammary gland tissue during the luteal phase of the bitch by local autocrine-paracrine effects. In addition, progesterone-induced mammary growth hormone production may exert endocrine effects, such as hyperplastic changes in the uterine epithelium and insulin resistance.     

The role of folic acid and Vitamin B12 in genomic stability of human cells

Folic acid plays a critical role in the prevention of chromosome breakage and hypomethylation of DNA. This activity is compromised when Vitamin B12 (B12) concentration is low because methionine synthase activity is reduced, lowering the concentration of S-adenosyl methionine (SAM) which in turn may diminish DNA methylation and cause folate to become unavailable for the conversion of dUMP to dTMP. The most plausible explanation for the chromosome-breaking effect of low folate is excessive uracil misincorporation into DNA, a mutagenic lesion that leads to strand breaks in DNA during repair. Both in vitro and in vivo studies with human cells clearly show that folate deficiency causes expression of chromosomal fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation and DNA hypomethylation. In vivo studies show that Vitamin B12 deficiency and elevated plasma homocysteine are significantly correlated with increased micronucleus formation. In vitro experiments indicate that genomic instability in human cells is minimised when folic acid concentration in culture medium is >227nmol/l. Intervention studies in humans show: (a) that DNA hypomethylation, chromosome breaks, uracil misincorporation and micronucleus formation are minimised when red cell folate concentration is >700nmol/l folate; and (b) micronucleus formation is minimised when plasma concentration of Vitamin B12 is >300pmol/l and plasma homocysteine is <7.5micromol/l. These concentrations are achievable at intake levels in excess of current RDIs i.e. more than 200-400microgram folic acid per day and more than 2microgram Vitamin B12 per day. A placebo-controlled study with a dose-response suggests that based on the micronucleus index in lymphocytes, an RDI level of 700microgram/day for folic acid and 7microgram/day for Vitamin B12 would be appropriate for genomic stability in young adults. Dietary intakes above the current RDI may be particularly important in those with extreme defects in the absorption and metabolism of these Vitamins, for which ageing is a contributing factor.     

Insulin-induced lipoatrophy: evidence for an immune pathogenesis.

Skin biopsy samples from 14 diabetic patients with lipoatrophy at injection sites and from five insulin-treated diabetic patients without such lipoatrophy (controls) were examined by immunofluorescence for the deposition of immunological components. Also sera from 13 of the patients with lipoatrophy and from all of the controls were assayed for insulin-binding capacity. Biopsy samples from the edge of lipoatrophic areas (eight cases) invariably showed abnormal deposition of immunological components in dermal vessel walls, whereas no such deposition was seen in the control samples. Mean serum insulin-binding capacity was 33.1 microgram/l in the patients with lipoatrophy compared with only 4.6 microgram/l in the controls. These findings suggest that insulin-induced lipoatrophy results from the local formation of immune complexes, complement fixation, and release of inflammatory mediators from the cellular infiltrate.     

Erythrocyte ferritin content in idiopathic haemochromatosis and alcoholic liver disease with iron overload.

The erythrocyte ferritin content was measured in patients with either idiopathic haemochromatosis or alcoholic liver disease and iron overload to define its value as a marker for an excess of tissue iron. The mean erythrocyte ferritin content in patients with untreated idiopathic haemochromatosis was increased 60-fold and fell with phlebotomy. After phlebotomy many patients had an increased red cell ferritin content despite normal serum ferritin concentrations. That this reflected persistent iron overload with inadequate phlebotomy was suggested by the higher serum iron concentrations, percentage transferrin saturation, and urinary excretion of iron after administration of desferrioxamine, together with a lower annual iron loss by phlebotomy in this group compared with patients with treated disease and normal red cell ferritin content. The mean erythrocyte ferritin content in patients with alcoholic liver disease and iron overload was increased only sevenfold, and the ratio of erythrocyte to serum ferritin clearly discriminated these patients from those with idiopathic haemochromatosis. The determination of erythrocyte ferritin content is a useful non-invasive test for diagnosing idiopathic haemochromatosis, monitoring the effect of phlebotomy in this disorder, and distinguishing patients with this disorder from those with alcoholic liver disease with iron overload.     

Plasma ferritin concentrations: their clinical significance and relevance to patient care.

In healthy persons the plasma ferritin concentration is a sensitive index of the size of body iron stores. It has been successfully applied to large-scale surveys of the iron status of populations. It has also proved useful in the assessment of clinical disorders of iron metabolism. A low plasma ferritin level has a high predictive value for the diagnosis of uncomplicated iron deficiency anemia. It is of less value, however, in anemia associated with infection, chronic inflammatory disorders, liver disease and malignant hematologic diseases, for which a low level indicates iron deficiency and a high level excludes it, but intermediate levels are not diagnostic. Measuring the plasma ferritin concentration is also useful for the detection of excess body iron, particularly in idiopathic hemochromatosis, but again it lacks specificity in the presence of active hepatocellular disease. If iron overload is suspected in these circumstances determination of the iron content of a percutaneous liver biopsy specimen is required. In families with idiopathic hemochromatosis the combined determination of the plasma ferritin concentration and the transferrin saturation is a sufficient screen to identify affected relatives; however, estimation of the hepatic iron concentration is required to establish the diagnosis.     

Diagnostic efficacy of tests for the detection of iron overload in chronic liver disease.

The value of tests for the detection of body iron overload was investigated in 8 aptients with clinically manifest primary hemochromatosis, 12 patients with cirrhosis and iron overload and 20 patients with liver disease and low or normal iron stores. Iron overload was defined as the presence of stainable iron in more than 50% of hepatocytes in a liver biopsy specimen. The percentages of patients with a true-positive (abnormal) or true-negative (normal) result were: serum iron concentration 65%, transferin saturation 85%, serum ferritin concentration 78%, serum ferritin:serum glutamic oxaloacetic transaminase (SGOT) index 78%, percent iron absorption 58%, percent iron absorption in relation to serum ferritin concetration 80% and percent iron absorption in relation to serum ferritin:SGOT index 93%. The calculated predictive value of a normal test result for the exclusion of iron overload in patients with liver disease, a group with an assumed prevalence of iron overload of 10%, was 98% to 99% for transferrin saturation and serum ferritin concentration used alone and 100% for these measures used together; the predictive value of an abnormal result for the diagnosis of iron overload was less than 50% for all of the above measures used alone or in combination. Hence, in patients with an increased serum ferritin concentration or transferrin saturation, or both, determination of the hepatocellular iron content of a specimen from a percutaneous liver biopsy is required for the diagnosis of iron overload.