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.     

Association between recombinant human erythropoietin and quality of life and exercise capacity of patients receiving haemodialysis. Canadian Erythropoietin Study Group.

OBJECTIVE--To determine whether recombinant human erythropoietin improves the quality of life and exercise capacity of anaemic patients receiving haemodialysis. DESIGN--A double blind, randomised, placebo controlled study. SETTING--Eight Canadian university haemodialysis centres. PATIENTS--118 Patients receiving haemodialysis aged 18-75 with haemoglobin concentrations less than 90 g/l, no causes of anaemia other than erythropoietin deficiency, and no other serious diseases. INTERVENTIONS--Patients were randomised to three groups to receive placebo (n = 40), erythropoietin to achieve a haemoglobin concentration of 95-110 g/l (n = 40), or erythropoietin to achieve a haemoglobin concentration of 115-130 g/l (n = 38). Erythropoietin was given intravenously thrice weekly, initially at 100 units/kg/dose. The dose was subsequently adjusted to achieve the target haemoglobin concentration. All patients with a serum ferritin concentration less than 250 micrograms/l received oral or intravenous iron for one month before the study and as necessary throughout the trial. MAIN OUTCOME MEASURES--Scores obtained with kidney disease questionnaire, sickness impact profile, and time trade off technique; and results of six minute walk test and modified Naughton stress test. RESULTS--The mean (SD) haemoglobin concentration at six months was 74 (12) g/l in patients given placebo, 102 (10) g/l in those in the low erythropoietin group, and 117 (17) g/l in those in the high erythropoietin group. Compared with the placebo group, patients treated with erythropoietin had a significant improvement in their scores for fatigue, physical symptoms, relationships, and depression on the kidney disease questionnaire and in the global and physical scores on the sickness impact profile. The distance walked in the stress test increased in the group treated with erythropoietin, but there was no improvement in the six minute walk test, psychosocial scores on the sickness impact profile, or time trade off scores. There was no significant difference in the improvement in quality of life or exercise capacity between the two groups taking erythropoietin. Patients taking erythropoietin had a significantly increased diastolic blood pressure despite an increase in either the dose or number of antihypertensive drugs used. Eleven of 78 patients treated with erythropoietin had their sites of access clotted compared with only one of 40 patients given placebo. CONCLUSIONS--Patients receiving erythropoietin were appreciably less fatigued, complained of less severe physical symptoms, and had moderate improvements in exercise tolerance and depression compared with patients not receiving erythropoietin. At the doses used in this trial there was a higher incidence of hypertension and clotting of the vascular access in patients treated with erythropoietin.     

Serum ferritin in patients with various haemolytic disorders.

174 serum ferritin assays in 121 patients with various haemolytic disorders have been performed. The mean serum ferritin levels were significantly increased in all these disorders in contrast to healthy controls. The highest serum ferritin levels were found in pyruvate kinase (PK) deficiency, moderate increase was observed in hereditary sphaerocytosis (HS) and in autoimmune haemolytic anaemia (AIHA) with massive haemolysis and in glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. Mild elevation of serum ferritin levels was depicted in paroxysmal nocturnal haemoglobinuria (PNH), in beta thalassaemia minor and in other types of haemoglobinopathies. The range of values was associated with a degree of haemolysis and its relation to duration of the disease was not apparent in most cases. Highly significant differences between serum ferritin levels in splenectomized and non-splenectomized patients with HS and between serum ferritin levels in patients with AIHA with massive haemolysis or in remission were found. As compared to normal controls, significant increase of serum ferritin levels was observed even in patients with AIHA in remission or in splenectomized patients with HS. In two patients with PK deficiency the levels exceeding 2,000 micrograms/l indicated manifest iron overload. A reliability of serum ferritin assay as an index of iron stores in haemolytic disorders has been discussed.     

Changes in transferrin during the red cell replacement in amphibia

Transferrin, a plasma glycoprotein, carries iron from storage sites to immature erythroid cells for hemoglobin synthesis. The replacement of larval red cells by adult red cells, which occurs during metamorphosis in bullfrogs, requires extensive formation of hemoglobin and new red cells. Large changes in red cell iron storage also occur during the red cell replacement. Both the concentration and the level of iron saturation of plasma transferrin were measured during metamorphosis to determine if there were changes in plasma transferrin which coincided with the changes in red cell iron storage and ferritin content. Plasma transferrin concentrations increased from 0.96 to 2.6 mg/ml during the period when red cell storage iron and ferritin decreased. Plasma iron concentrations also increased when the transferrin concentration increased, suggesting that the additional transferrin may be involved in moving iron from the larval red cell stores. At the end of metamorphosis, the plasma iron concentration decreased to premetamorphic levels but the transferrin concentration remained high, resulting in a decrease in saturation to 18% compared to 45% in the larvae. In addition to differences in iron saturation, adult transferrin had different electrophoretic properties from larval transferrin. The results support the hypotheses that during early ontogeny plasma transferrin and red cell iron storage are coordinated to provide iron for the formation of the first generation of adult red cells and that transferrin may participate in the control of red cell ferritin synthesis.     

Ferritin in liver, plasma and bile of the iron-loaded rat

Rats were loaded with iron. With overload, up to a 10-fold increase of the iron and ferritin protein content of the livers was measured. The plasma ferritin concentration increased gradually with the ferritin concentration in the liver. The ferritin concentration in the bile increased also and was in the same range as in the plasma. The ratio plasma ferritin concentration to bile ferritin concentration in individual rats decreased in the case of considerable iron overload. After intravenous injection of liver ferritin, less than 2% of the ferritin concentration that disappeared from the blood was found to be in the bile. Isoelectric focussing revealed that the microheterogeneity of liver and bile ferritin were identical, but slightly different from plasma ferritin. These results indicate that ferritin was not solely leaking from the plasma to the bile. Together with ferritin, iron accumulated in the bile. The iron content of the bile ferritin was in the same range as in fully iron-loaded liver ferritin. It is likely that ferritin in the bile is excreted by the liver and consists of normal iron-loaded liver ferritin molecules. In all circumstances, the amount of iron in the bile was much higher than could be accounted for by transport by the bile ferritin. The ferritin protein to iron ratio in the bile was 0.1-1.2, which was in the same range as was measured in isolated lysosomal fractions of the liver. Those results agree with the supposition that ferritin and iron in the bile are excreted by the liver though lysosomal exocytosis.     

Iron in bone marrow

In the bone-marrow, non-haemoglobin iron can predominantly be found in the reticulum. Slight granules containing iron can also be observed in parts of erythroblasts by means of the Berlin blue reaction. These cells are called sideroblasts. In chemical respect, non-haemoglobin iron consists of ferritin soluble in water and haemosiderin insoluble in water. Erythroblasts will only take their iron from plasma transferrin. For the most part, this iron uptake is being regulated by erythropoietin adapting erythropoiesis to the oxygen requirements of the tissue. The iron contained in erythroblasts is predominantly utilized for haemoglobin synthesis in these cells. A slight part is being taken up by ferritin. The bone-marrow reticulum will phagocytise aged erythrocytes and store liberated iron as ferritin and haemosiderin. Part of the iron is being delivered again to plasma transferrin. With constant serum iron level the liberation of iron from the reticulo-endothelial tissue must correspond to the iron uptake by erythropoiesis. The absence of iron capable of being coloured in the bone-marrow reticulum is considered to be a reliable parameter of iron deficiency. It enables the diagnosis of iron deficiency anaemia to be made even in those patients with serum iron level and a total iron binding capacity lying within the normal range and no hypochromia of erythrocytes being present. It enables iron deficiency anaemia to be separated from sideropenic anaemia with reticulo-endothelial siderosis in differential-diagnostic manner. Even in patients with sideroblastic anaemia, iron colouring of bone-marrow smears is required for ensuring the diagnosis. Recently, a separation has also been made for idiopathic anaemia with abnormal sideroblasts. In these patients there is an increased risk for acute leukemia to develop.     

Disparity between dietary iron intake and iron status of children aged 10-12 years

Iron status was assessed in a representative sample of 188 adolescents living in a medium-sized city in Poland. Dietary intakes were evaluated using records of diet over a period of seven consecutive days. Subjects were considered to be iron deficient when two or more of the following parameters were abnormal: serum ferritin, transferrin saturation or mean corpuscular haemoglobin concentration. Based on this definition, the prevalence of iron deficiency in the investigated sample of children aged from ten to twelve years was 12.7%. Iron deficiency anaemia was defined using the following criteria: haemoglobin values less than 12.0 g. dl (-1) in girls or less than 12.2 g. dl(-1) in boys, combined with an iron deficiency. With such a definition, the prevalence of iron deficiency anaemia in all subjects was 6.3%. Four boys (3.9%) and six girls (6.8%) were diagnosed as anaemic. The values for Hb in the anaemic boys ranged from 10.9 to 12.2 g. dl (-1) and in anaemic girls from 8.7 to 12.0 g. (-1). It was found that the majority of the individuals studied had a dietary haem-iron intake lower than that recommended. No relationship was found between the level of serum ferritin and total iron and vitamin C dietary intake, but there was positive correlation between serum ferritin and intake of haem iron. A seven-day dietary history questionnaire correctly identified children at risk of iron deficiency anaemia.     

Ferritin in bone marrow and serum in iron deficiency and iron overload

Nonheme iron and ferritin in the bone marrow and serum ferritin was investigated in patients with iron deficiency anaemia or iron overload. As controls served patients without any disturbance of the iron metabolism. There is a precise correlation between the nonheme iron and ferritin in the bone marrow of patients with and without disturbance of iron metabolism. A correlation was also found between the ferritin in the bone marrow and the serum. Nonheme iron and ferritin in the bone marrow and serum ferritin was decreased in patients with iron deficiency anaemia. Conversely, the same parameters were increased in patients with iron overload.     

The clinical utility of discriminant functions for the differential diagnosis of microcytic anemias

Several groups of authors have derived discriminant functions (DFs) based on red cell indices (primarily MCH, MCV, and RDW) that can be used to differentiate iron deficiency from thalassemia minor. The Technicon H*1 analyzer provides a direct MCHC measurement (termed the CHCM), in addition to the conventional computed value (Hgb/PCV). To evaluate the clinical utility of red cell discriminant analysis, chart review was performed in 176 cases for which hemoglobin characterization and quantitation studies had been requested. Six published discriminants were evaluated for cases of clearly defined iron deficiency anemia and thalassemia minor. Overall diagnostic efficiency ranged from 50%-82%, and the diagnostic performance of three of the discriminants failed to achieve statistical significance. Mean values for both MCHC and CHCM were significantly lower in patients with iron deficiency than in patients with other causes of microcytic anemia. It was also observed that MCHC was significantly greater than CHCM in patients with iron deficiency anemia, but not in patients with other causes of microcytic anemia. Both MCHC and the difference between MCHC and CHCM showed potential value as parameters for the differential diagnosis of iron deficiency from other causes of microcytic anemia. It was noted, however, that in 67% of the cases studied, the use of a DF could not have resolved the diagnosis to the extent that hemoglobin characterization and quantitation studies were no longer indicated.     

Iron stores in female blood donors evaluated by serum ferritin

Iron stores were evaluated by serum ferritin determinations in 948 menstruating and 141 non-menstruating female blood donors. Blood donation was associated with a decrease in ferritin. First-time donors (n = 163) had a geometric mean ferritin of 24 micrograms/l and multiple-time donors a value of 19 micrograms/l (p less than 0.01). In the donating population 31.5% had ferritin values less than 15 micrograms/l (i.e. depleted iron stores). Menstruating donors had lower mean serum ferritin than non-menstruating donors (p less than 0.001), and a higher frequency of ferritin values less than 15 micrograms/l (p less than 0.05). There was no relationship between ferritin levels and the number of pregnancies. The frequency of donations was more predictive of ferritin levels than the number of donations. Mean ferritin displayed a moderate fall up to the 2nd donation, and was hereafter relatively constant, whereas an increase in donation frequency was accompanied by a significant decrease in ferritin. Female donors, especially when phlebotomised greater than or equal to 3 times per year, should have their iron status checked at appropriate intervals by measurement of serum ferritin and should be advised regular iron supplementation.     

Erythrocyte ferritin in patients with normal and abnormal iron metabolism

Erythrocyte and serum ferritin was determined in 36 patients with different pictures of diseases and under bleeding therapy in idiopathic haemochromatosis (IH). With latent iron deficiency there was no significant difference in the ferritin content of erythrocytes in comparison with the control group. The intraerythrocyte ferritin content in idiopathic haemochromatosis is always increased and will normalize under bleeding therapy as well serum ferritin. After disrupting the therapy the ferritin content in erythrocytes will more rapidly increase again than in the serum. Increased erythrocyte ferritin could be identified in patients with anaemia due to ineffective erythropoiesis.     

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.     

Consequences of 6 weeks of strength training on red cell O2 transport and iron status

Effects of endurance training on O2 transport and on iron status are well documented in the literature. Only a few data are available concerning the consequences of strenuous anaerobic muscular exercise on red cell function. This study was performed to test the influence of strength training alone on parameters of red cell O2 transport and iron status. Twelve healthy untrained males participated in a strength-training programme of 2-h sessions four times a week lasting 6 weeks. After 6 weeks a small but significant reduction of haemoglobin (Hb; -5.4 g.l-1) was found (p less than 0.05). Mean red cell volume did not change, but a pronounced decrease of mean cell Hb concentration (from 329.2 g.l-1, SE 2.5 to 309.8 g.l-1, SE 1.2; p less than 0.001) and mean corpuscular Hb (from 29.6 pg, SE 0.4 to 27.7 pg, SE 0.3; p less than 0.01) was observed. Serum ferritin decreased significantly by 35% (p less than 0.01); transferrin, serum iron and iron saturation of transferrin were unaltered. Serum haptoglobin concentration was diminished significantly by 30.5% (p less than 0.01). The reticulocyte count had already increased after 3 weeks of training (p less than 0.05) and remained elevated during the following weeks. Strength training had no significant influence on the O2 partial pressure at which Hb under standard conditions was 50% saturated, red cell 2,3-diphosphoglycerate and ATP concentration as well as on erythrocytic glutamate-oxalacetate transaminase activity. The data demonstrate that mechanical stress of red cells due to the activation of large muscle masses led to increased intravascular haemolysis, accompanied by a slightly elevated erythropoiesis, which had no detectable influence on Hb-O2 affinity. Training caused an initial depletion of body iron stores (prelatent iron deficiency). Although Hb had decreased by the end of the training phase a true "sports anaemia" could not be detected.     

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.     

Utilization of Iron Dextran

The utilization of iron dextran was investigated in normal subjects, in patients with iron-deficiency anaemia, and in anaemias associated with rheumatoid arthritis, reticulosis, and uraemia. Utilization of iron for haemoglobin formation at 14 days was found to be depressed in patients with rheumatoid arthritis, reticulosis, and uraemia, but when a concomitant iron-deficiency anaemia was present utilization was significantly increased. When iron dextran is used to treat anaemias in such conditions an optimum therapeutic response will be obtained only when bone marrow iron stores are absent.     

A long term survey of plasma and tissue ferritin in mice fed on iron deficient diets

1. Changes in plasma and tissue ferritin concentrations were surveyed in mice fed on iron deficient diets (Fe, 2.4 micrograms/g) from the age of 8 to 30 weeks by an enzyme-immunoassay for mouse ferritin. Values were compared with those in mice on control diets (Fe, 110 micrograms/g) and mice with blood loss. 2. In mice on iron deficient diets, while plasma ferritin remained at normal level, ferritin in the spleen and kidney decreased, but that in the liver increased as much as twice of that in the control. 3. Mice with blood loss revealed a drastic reduction of ferritin in every tissue and plasma examined.     

Association between NAD+ levels and anaemia among women in community‐based study

Nicotinamide adenine dinucleotide (NAD⁺) level is the protective factor of cardiovascular diseases (CVDs). In addition, anaemia is a risk factor of adverse cardiovascular outcomes in women. However, there are limited data about the association between NAD⁺ and anaemia. The aim of this study was to evaluate association of NAD⁺ with anaemia among women. A total of 727 females from Jidong community were included in the current analysis. NAD⁺ levels were tested by the cycling assay and HPLC assay using whole blood samples. Anaemia was determined by haemoglobin (Hb) concentration, and the subtypes of anaemia were further defined according to mean corpuscular volume (MCV) in blood. Multivariable logistic analysis was used to analyse the association between NAD⁺ levels and anaemia or its subtypes. The mean age of recruited subjects was 42.7 years. The proportion of anaemia by NAD⁺ levels quartiles were 19.7% (35/178), 4.8% (9/189), 3.4% (6/178) and 2.7% (5/182). Haematological parameters including haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and red blood count (RBC) increased over NAD⁺ quartiles. Red cell volume distribution width (RDW) decreased over NAD⁺ quartiles. Compared with the lowest quartile of NAD⁺ levels (<27.6μM), the adjusted odds ratios with 95% confidence intervals of the top quartile were 0.15 (0.06–0.41) for anaemia, 0.05 (0.01–0.36) for microcytic anaemia and 0.37 (0.10–1.36) for normocytic anaemia respectively. Higher NAD⁺ levels were significantly associated with lower prevalence of anaemia among women, especially microcytic anaemia and normocytic anaemia. Haematological parameters might serve as a predictor of the blood NAD⁺ levels.     

The course of serum ferritin in blood donors

In the course of 18 months there occurred a decrease in the serum ferritin concentration from 58 +/- 2 micrograms/l to 32 +/- 1 micrograms/l in male donors after 5-6 donations of 400 ml of blood each. Female permanent donors showed a constantly lowered content of an average of 15 +/- 1 micrograms/l. Female first donors attained the serum ferritin level of permanent female donors with the fourth donation, whereas it was not until the fifth donation that male first donors declined to the level of permanent donors. This decrease of serum ferritin content in blood donors points to a depletion of iron stores. This process should be counteracted by exerting an increased influence on nutritional habits supported by oral iron substitution and diminishing the frequency of donation, particularly for female blood donors.     

Indices of iron and copper status during experimentally induced,marginal zinc deficiency in humans

This study examined the effect of diet-induced, marginal zinc deficiency for 7 wks in 15 men (aged 25.3 +/- 3.3 yrs; mean +/- SD) on selected indices of iron and copper status. The regimen involved low-zinc diets based on egg albumin and soy protein with added phytate and calcium such that mean [phytate]/[Zn] and [phytate] X [Ca]/[Zn] molar ratios were 209 and 4116, respectively, for 1 wk, followed by 70 and 2000, respectively, for 6 wks. Subjects were then repleted with 30 mg Zn/d for 2 wks. Plasma copper, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity in plasma and red blood cells (RBC), hemoglobin, hematocrit, and serum ferritin were determined weekly on fasting blood samples. Significant reductions (p less than 0.05) after 7 wks in RBC Cu,Zn-superoxide dismutase (49.5 +/- 7.2 vs 33.6 +/- 6.3 U/mg Hb) and serum ferritin (69.2 +/- 38.7 vs 53.8 +/- 33.7 micrograms/L) occurred; no comparable decline was noted for plasma Cu, hemoglobin, or hematocrit. Significant (p less than 0.05) but less consistent changes were also observed in plasma superoxide dismutase activity. None of the changes were associated with the decreases in plasma, urinary and hair zinc concentrations, and alkaline phosphatase activity in RBC membranes. Results indicate that the biochemical iron and copper status of the subjects was marginally impaired, probably from the dietary regimen that induced marginal zinc deficiency.     

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.