Iron Deficiency State Research Articles

A Hematologic “Gold Standard” for Iron-deficient States?1

In this issue of the Journal, Thomas and Thomas (1) provide a novel approach to the diagnosis of iron-deficient states. Traditionally, iron deficiency studies have used various biochemical indicators of iron metabolism to establish the absence or presence of biochemical iron deficiency and to assess the performance of a potentially novel biochemical or hematologic marker. Biochemical indicators have included serum iron, serum transferrin, transferrin saturation, serum ferritin, and serum circulating transferrin receptor (TfR) as well as various ratios of these variables. Hematologic indices that have been used to establish the presence of iron deficiency anemia have included, in addition to hemoglobin (Hb) or hematocrit (Hct), abnormal erythrocytes or reticulocyte indices that identify the presence of hypochromic and microcytic cells. Additional variables that have been investigated include zinc protoporphyrin and erythrocyte ferritin. Studies on the diagnosis of iron-deficient states have been complicated by the absence of a clear reference method to detect biochemical iron deficiency. Iron staining of bone marrow biopsy is widely quoted as a gold standard, but the invasive nature of this procedure severely limits its use. A less invasive standard for iron deficiency is based on the hematologic response to iron replacement therapy: an increase of the reticulocyte count or reticulocyte index after oral or intravenous iron replacement therapy reveals the presence of iron deficiency. The use of biochemical markers to diagnose iron-deficient states is problematic in clinical conditions such as the anemia of chronic disease (ACD). When a disturbed iron metabolism is associated with acute or chronic phase inflammatory responses, serum ferritin and transferrin concentrations are affected and not informative. Biochemical markers are also less effective in diagnosing functional iron deficiency, a transient discrepancy between iron supply and utilization that is mostly seen in individuals treated with recombinant human erythropoietin (r-HuEPO). In their report, Thomas and Thomas …

Anaemia, rHuEPO resistance, and cardiovascular disease in end-stage renal failure; links to inflammation and oxidative stress.

End-stage renal disease (ESRD) is characterized by a high mortality rate, derived largely from cardiovascular disease (CVD). In patients with ESRD, high levels of pro-inflammatory cytokines and increased oxidative stress are common features that may contribute to malnutrition, anaemia, recombinant human erythropoietin (rHuEPO) resistance, and atherosclerosis. Inflammation predicts poor outcome in ESRD. It is multifactorial in cause and, while it may reflect the underlying CVD, the acute-phase response may also contribute to both oxidative stress and progressive vascular injury. In patients with ESRD, the acute-phase response may be influenced by a number of factors unrelated to dialysis and perhaps by the dialysis procedure itself. Inflammation and the acute-phase response interact with the haematopoietic system at several levels resulting in reduced erythropoiesis, accelerated destruction of erythrocytes, and blunting of the reactive increase in erythropoietin in response to reduced haemoglobin levels. In patients with ESRD, rHuEPO resistance has been linked with inflammation, the latter of which is often associated with a state of functional iron deficiency. Patients with ESRD are thought to have a reduced capacity to handle oxidative stress. There is recent evidence that a relationship may exist between inflammation and oxidative stress and treatment of anaemia with rHuEPO. However, iron may also generate oxidative stress. Controlled trials are needed before evidence-based recommendations for the management of inflammation-induced anaemia and resistance to rHuEPO can be defined.

Iron metabolism and requirements in early childhood: do we know enough?: a commentary by the ESPGHAN Committee on Nutrition.

*University of Lancashire, Lancashire, United Kingdom; †University of Milano, Milano, Italy; ‡University of Lund, Malmo, Sweden; §Hopital des Enfants Malades, Paris, France; Hopital Necker Enfants-Malades, Paris, France, ¶Umea University, Umea, Sweden; #University of Munich, Munich, Germany; **Free University of Amsterdam, Amsterdam, The Netherlands; ††Royal Veterinary and Agricultural University, Frederiksberg, Denmark; ‡‡CHUV University Hospital, Lausanne, Switzerland; §§University of Liege, Liege, Belgium; Medical University of Warsaw, Warsaw, Poland; and ¶¶University of Glasgow, Glasgow, United Kingdom

Iron Cytotoxicity in Chronic Hepatitis C.

Iron-induced cytotoxicity in chronic hepatitis C was described in detail based on effectual iron reduction therapy. Without treatment, chronic hepatitis C may progress to cirrhosis and develop hepatocellular carcinoma. The first choice treatment for the disease is interferon. However, the cost-benefit of interferon treatments is so poor that options other than interferon are needed for poor responders. A minimum requirement for managing these patients is good control of disease activity shown by maintaining a low serum level of alanine aminotransferase activity. An index accepted widely is 80 IU/l or less of the enzyme activity. When patients were treated with repeated phlebotomy to remove body iron stores, the mean serum levels of enzyme activities changed from 128 to 63 IU/l. Maintaining patients at an iron deficient state for 5 years or more did not change the staging of liver histology, in contrast with the advanced liver histology in the control patients. Iron generates hydroxyl radicals that mediate peroxidation of membrane lipids. After the proposal of our empirical treatment based on the ultrastructural study of hepatic iron stores, evidence has been accumulated that oxidative stress via lipid peroxides plays an important role in the pathogenesis of chronic hepatitis C. The treatment is not yet authorized in Japan, but is approved in the United States as the first of the options to interferon. Considering the therapeutic effects of iron removal on both biochemical and histological parameters, the safety-proved economical procedure might be recommended for patients as an option to interferon.

Long term effects of phlebotomy on biochemical and histological parameters of chronic hepatitis C

OBJECTIVES: There is considerable evidence that iron is a risk factor for liver injury in chronic hepatitis C. Known as iron reduction therapy, phlebotomy reduces serum ALT activity. This effect might continue with maintenance phlebotomy and result in slower progression of liver fibrosis. METHODS: We examined the biochemical parameters and liver histology of patients with chronic hepatitis C treated by maintenance phlebotomy. For biochemical evaluation, 25 patients were treated by initial phlebotomy to reduce serum ferritin levels to 10 ng/ml or less and then observed for 5 yr with maintenance phlebotomy to maintain the iron-deficient state. For histological evaluation, liver biopsies were performed before and after the study period in 13 of the patients. Thirteen patients who were virological nonresponders to interferon alone and had undergone second liver biopsies after more than 3 yr served as histological controls. RESULTS: Serum aminotransferase levels were decreased significantly by initial phlebotomy and remained at the same levels during the study period ( p < 0.05). The grading scores were improved significantly in the study group ( p < 0.05) and unchanged in the controls. The staging scores remained unchanged in the study group but were increased in the controls ( p < 0.005). Disease progression was significantly different between the two groups ( p < 0.05). CONCLUSIONS: These results suggest that phlebotomy with maintenance lowers serum aminotransferase levels, improves liver inflammation, and suppresses the progression of liver fibrosis in chronic hepatitis C.

The Intracellular Location of Iron Regulatory Proteins Is Altered as a Function of Iron Status in Cell Cultures and Rat Brain

Iron regulatory proteins (IRPs) are proteins involved in the regulation of intracellular iron homeostasis that bind to specific mRNA structures termed iron responsive elements (IREs). Because the target mRNAs for the IRPs are both cytosolic and membrane associated, we hypothesize that movement of IRPs between the cytosolic and the membrane associated subcellular fractions occurs in response to intracellular iron changes. We tested this hypothesis in a cell culture model, using mouse fibroblast cells (NIH 3T3) and macrophage cells (J774), and in a rat model of early iron deficiency and excess. This presented the first opportunity to examine IRP binding activity in rat brain during states of dietary iron deficiency and excess. Binding activity for IRPs was demonstrated in both membrane and cytosolic fractions in the cell lines and the rat brain homogenates. Although IRP binding activity is predominantly located in the cytosol (90%), there was increased IRP/IRE binding activity in both cytosolic and membrane fractions when the cells were treated with deferoxamine, and decreased binding activity after treatment with iron. In the rat study, brain cortex, hippocampus and striatum homogenates had more IRP binding activity in iron-deficient rats and less in iron-supplemented rats in a region- and time-specific manner. The intracellular distribution of IRPs also changed between the cytosolic and membrane fractions of the brain homogenates in conjunction with changes in iron. These in vivo studies are consistent with the cell culture analyses showing intracellular redistribution of IRPs as a function of iron status. The results of these experiments extend our understanding of cytoplasmic mRNA binding protein activity and raise questions regarding the mechanism by which mRNA binding proteins can locate their target mRNAs within cells. The elucidation of this mechanism will have a significant impact on our understanding of eukaryotic gene regulation.

Prevalence of Undernutrition and Iron Deficiency in Pre-school Children from Different Socioeconomic Regions in the City of Oaxaca, Oaxaca, Mexico.

Lack of adequate food and in particular high quality protein, is one of the causes of malnutrition in children which could result in retarded growth. Iron deficiency is common in populations where protein sources are of vegetable origin; however in northern Mexico where the bean consumption is high, anemia is not a problem. The primary staples in the Mexican diet are corn tortillas and beans. The objective of this study was to evaluate the anthropometric condition of children 4-6 years old living in the city of Oaxaca, Oaxaca, Mexico and to determine the prevalence of anemia. Mean Z scores for children 4-6 years old living in poor conditions in the city of Oaxaca, Oaxaca, Mexico showed significant differences between socioeconomic groups (p<0.002) for height/age (H/A) and weight/age (W/A) (p<0.001) after adjusting for age and sex. Weight/height (W/H) was not different (p=0.30). By using the Waterlow classification system, 28.8% of the pre-school children of this study were stunted and only 0.9% were classified as wasted. There were no children that presented both stunting and wasting. Iron deficiency was very prevalent in both boys and girls, ranging from 56-79% depending on the indicator used. When classified by the combination of serum ferritin, % transferrin saturation and hemoglobin values, 23.7% of the children were classified as anemic, 11.9% in a state of iron deficiency and 13.6% with low iron reserves.

Site-directed Mutagenesis of Human Ceruloplasmin: PRODUCTION OF A PROTEOLYTICALLY STABLE PROTEIN AND STRUCTURE-ACTIVITY RELATIONSHIPS OF TYPE 1 SITES

A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis. The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains. The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity. This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction. During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene. This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake.

Relationships among serum triacylglycerol, fat pad weight, and lipolysis in iron-deficient rats

We studied the relationships among serum triacylglycerol (TG), fat pad weight, and lipolytic response to norepinephrine (NE) in iron-deficient rats. We used male Sprague-Dawley International Golden Standard rats. The rats were randomly divided into four groups: two iron-adequate groups for 1 week (1A) and 5 weeks (5A), and two iron-deficient groups for 1 week (1D) and 5 weeks (5D), based on the AIN-93G diet. Iron-deficient treatment caused a significant decrease in hemoglobin (Hb) and hematocrit (Hct) values and an increase in relative heart weight in 1D and 5D rats. Although serum TG was not affected by the 1-week iron-deficient treatment, it was significantly increased by 5-week iron-deficient treatment. The 1-week iron-deficient treatment significantly decreased the relative weight of the retroperitoneal fat pads, but not that of the epididymal fat pads. On the other hand, the 5-week iron-deficient treatment significantly decreased the relative weight of both fat pads; the degree of decrease was 41% and 32% for retroperitoneal and epididymal fat pads, respectively. Basal lipolysis significantly decreased in the epididymal adipocytes from 1D rats, whereas lipolytic response to NE markedly increased. No effect due to the 5-week treatment on basal lipolysis was observed in either retroperitoneal or epididymal adipocytes. In addition, lipolytic response to NE significantly increased in the retroperitoneal, but not the epididymal adipocytes. These results demonstrate that the effects of an iron-deficient diet on fat pad weight are different, depending on the duration of the treatment and the location of fat pads. In addition, iron deficiency-caused hypertriacylglycerolmia may be predominantly related to the increase in lipolysis in retroperitoneal rather than in epididymal adipocytes. The data further show that the increase in lipolysis of epididymal adipocytes occurs in the earlier stage prior to a severe iron-deficient state.

Iron stores and haemoglobin iron deficits in menstruating women. Calculations based on variations in iron requirements and bioavailability of dietary iron.

Iron stores and haemoglobin iron deficits in menstruating women can be calculated from body iron losses and absorption of dietary iron using recently developed methods. To examine iron balance (iron status) expressed as body iron stores or haemoglobin iron deficits in menstruating women from amounts of iron lost (iron requirements) and amounts of dietary iron absorbed. Calculations are made both of stationary states and of the rate of changes in iron stores (iron status) when any of the two main factors determining iron balance are changed. The study is based on (1) previous and new equations describing relationships between iron absorption, iron requirements (losses), iron stores and/or haemoglobin deficits and (2) published data on iron requirements and their variation in menstruating adult women. Both iron stores and haemoglobin iron deficits are strongly related to iron requirements and absorption of dietary iron and follow the same equations during states of iron repletion and iron deficiency. When, for example, increasing or decreasing the bioavailability of the dietary iron, about 90% of the change in iron stores will occur within 1 y. There are strong relationships between iron requirements, bioavailability of dietary iron and amounts of stored iron. The observations that a reduction in iron stores and a calculated decrease of haemoglobin iron had the same increasing effect on iron absorption suggest that the control of iron absorption is mediated from a common cell, that may register both size of iron stores and hemoglobin iron deficit, eg the hepatocyte. European Journal of Clinical Nutrition (2000) 54, 650-657.

Anemia in pregnancy.

Anemia is one of the most frequent complications related to pregnancy. Normal physiologic changes in pregnancy affect the hemoglobin (Hb), and there is a relative or absolute reduction in Hb concentration. The most common true anemias during pregnancy are iron deficiency anemia (approximately 75%) and folate deficiency megaloblastic anemia, which are more common in women who have inadequate diets and who are not receiving prenatal iron and folate supplements. Severe anemia may have adverse effects on the mother and the fetus. Anemia with hemoglobin levels less than 6 gr/dl is associated with poor pregnancy outcome. Prematurity, spontaneous abortions, low birth weight, and fetal deaths are complications of severe maternal anemia. Nevertheless, a mild to moderate iron deficiency does not appear to cause a significant effect on fetal hemoglobin concentration. An Hb level of 11 gr/dl in the late first trimester and also of 10 gr/dl in the second and third trimesters are suggested as lower limits for Hb concentration. In an iron-deficient state, iron supplementation must be given and follow-up is indicated to diagnose iron-unresponsive anemias.

Insight into the pathophysiology of restless legs syndrome

Restless Legs Syndrome (RLS) is a disorder of sensation with a prevalence of around 2-5% of the population. Relevant to understanding the possible pathophysiological mechanism is the fact that RLS is extremely responsive to dopaminergic agents. A second issue is that iron deficiency states may precipitate RLS in as much as 25-30% of people with iron deficiency. Studies looking at basal ganglia dopaminergic function using PET and SPECT techniques have shown a decrease in binding potential for the dopamine receptor and transporter. Similar phenomena occurs in iron-deficient animals. Using MRI techniques and CSF analysis of iron-related protein, studies have suggested a reduction in brain iron concentration occurs in RLS patients. The relevance of CNS iron metabolism to the pathophysiology of RLS is discussed.

The importance of the proportion of heme/nonheme iron in the diet to minimize the interference with calcium, phosphorus, and magnesium metabolism on recovery from nutritional ferropenic anemia.

The digestive utilization of Fe and its nutritive interaction with Ca, P, and Mg were studied in rats with nutritional ferropenic anemia. The diet contained 80% ferric citrate and 20% heme iron (80/20 diet). The weight gain, digestive utilization of Fe, and regeneration efficiency of hemoglobin and seric Fe were higher in iron-deficient rats (ID) fed the 80/20 diet than in iron-deficient rats fed the 50/50 diet (Campos et al., 1996). The phospho-calcic metabolism, which is adversely affected in ferropenic anemia, returned to normal values when iron was added to the diet. The digestive utilization of Mg, which fell with the 50/50 diet (Campos et al., 1996), returned to normal values when the ferropenic anemia was reversed with the 80/20 diet. In a state of iron deficiency, certain parameters related to the glucose and lipid metabolism are affected; the glucose and triglycerides values return to a normal range with the 80/20 diet.

鉄欠乏によるerythron transferrin uptakeの亢進とineffective erythropoiesis

鉄欠乏によるerythron transferrin uptakeの亢進とineffective erythropoiesis

Effect of Dietary Cadmium on Iron Metabolism in Growing Rats

Little is known regarding the interactions between iron and cadmium during postnatal development. This study examined the effect of altered levels of dietary iron and cadmium loading on the distribution of cadmium and iron in developing rats ages 15, 21, and 63 days. The uptake of iron, transferrin, and cadmium into various organs was also examined using59Fe, [125I]transferrin, and109Cd. Dietary cadmium loading reduced packed cell volume and plasma iron and nonheme iron levels in the liver and kidneys, evidence of the inducement of an iron deficient state. Dietary iron loading was able to reverse these effects, suggesting that they were the result of impaired intestinal absorption of iron. Cadmium loading resulted in cadmium concentrations in the liver and kidneys up to 20 μg/g in rats age 63 days, while cadmium levels in the brain reached only 0.16 μg/g, indicating that the blood–brain barrier restricts the entry of cadmium into the brain. Iron loading had little effect on cadmium levels in the organs and cadmium feeding did not lower tissue iron levels in iron loaded animals. These results suggest that cadmium inhibits iron absorption only at low to normal levels of dietary iron and that at high levels of intake iron and cadmium are largely absorbed by other, noncompetitive mechanisms. It was shown that109Cd is removed from the plasma extremely quickly irrespective of iron status and deposits mainly in the liver. One of the most striking effects of cadmium loading on iron metabolism was increased uptake of [125I]transferrin by the heart, possibly by disrupting the process of receptor-mediated endocytosis and recycling of transferrin by heart muscle.

Absorption of the oral iron preparation ferrous acetyl transferrin in achlorhydric patients: a pilot study

Gastric acid secretion is an important factor in iron absorption in humans. Chronic corporal atrophic gastritis (CAG) is a pathologic condition characterized by oxyntic mucosal atrophy and hypochlorhydria that may cause unexplained iron-deficiency states that are difficult to treat with standard oral iron therapy. The molecule ferrous acetyl transferrin (FAT) displays a high degree of homology with human transferrin and may thus use the same transferrin receptor-mediated iron absorption system on ileal and duodenal crypts. We tested this oral iron preparation in nine achlorhydric CAG patients to evaluate its absorption in the total absence of gastric acid secretion. FAT 40 mg was administered twice daily by mouth for 7 days. Blood samples for determination of serum iron, ferritin, and transferrin levels were collected and calculation of the transferrin saturation index was performed before drug administration at 8 am on the first day of the study (when patients had been fasting), on day 8 (13 hours after the last dose of FAT), and on day 15 (7.5 days after the last dose of FAT). Serum iron levels on day 15 (median, 65 μg/dL) were significantly increased over pretreatment levels (median, 32.5 μg/dL) and over the levels observed on day 8 (median, 47.0 μg/dL). A similar increase in median transferrin saturation index was observed (17.52% on day 15 vs 7.33% before treatment and 11.53% on day 8). Serum transferrin levels decreased significantly from day 8 (median, 281.5 mg/dL) to day 15 (median, 264.5 mg/dL). The study shows that in CAG patients with achlorhydria, iron in the form of FAT administered by mouth can be absorbed, resulting in increases in serum iron level and transferrin saturation index, during short-term treatment.

Pattern of Iron Storage in the Rat Heart Following Iron Overloading with Trimethylhexanoyl-Ferrocene

Female Wistar rats with slight iron deficiency anemia were kept on a diet containing 0.5% trimethylhexanoyl (TMH)-ferrocene for up to 79 weeks. In the state of iron deficiency, the heart was free of light-microscopically detectable iron. After 7 weeks of the TMH-ferrocene diet, the first iron-positive granules appeared in perivascular macrophages. Further oral administration caused a progression of iron deposition in these cells, visible in the form of a granular staining but also as a diffuse iron staining of the cytoplasm. Accordingly, at the electron-microscopical level, the iron was stored partly as free ferritin molecules in the cytosol, and partly in lysosomes in the form of ferritin and/or hemosiderin. After 11 weeks, further iron-positive cells with relatively small dark-blue granules were found in the vicinity of capillaries, which could be identified as fibrocytes by means of electron microscopy. In addition, slight iron deposition occurred in the endothelial cells of the cardiac capillaries, likewise mainly in the form of small, uniform siderosomes. The myocytes showed no product of Perls' Prussian blue reaction during the whole period of investigation. From the 11th week onwards, discrete ferritin molecules were detected electron microscopically within lysosomes of these cells. Their amount increased slowly with progression of the TMH-ferrocene feeding period. Free ferritin molecules could be observed in the cytosol of fibrocytes, endothelial cells and myocytes in only very slight concentrations, whilst they were more plentiful in macrophages. In hereditary hemochromatosis and posttransfusional siderosis, the iron is found predominantly in myocytes and appears to cause cell damage, whilst this is not the case in experimental iron overload in rats.

The relationship between iron deficiency and anemia in Venezuelan children

The iron status of 3228 subjects from the nutrition survey Proyecto Venezuela was studied. The sample included children from 1 to 16 y of age grouped by age and sex. Values for three indicators of iron status were compared: hemoglobin concentration, serum ferritin concentration, and percentage saturation of serum transferrin. In all groups there was a strong overlap in the hemoglobin concentration distribution curves for non-iron-deficient and iron-deficient subjects classified as such according to the other two indexes. The prevalence of iron deficiency ranged from 35% in 1-3 y olds to 10% in adolescent males, the values being almost identical in the nonanemic group compared with the total population. In the different groups, 80-97% of the subjects with abnormal values of at least one of these two indexes were not anemic. The difficulties involved in establishing a state of iron deficiency according to these indexes are discussed.

Iron Status of Female Runners

This study was designed to observe iron status and prevalence of iron deficient conditions in adult female habitual runners (n = 111) and inactive females of comparable age (n = 65). The runners were significantly lower (p < .05) than the reference group in mean serum ferritin (SF), total iron binding capacity, and red blood cell count, but significantly higher (p < .05) in mean corpuscular hemoglobin. The groups did not differ significantly in hemoglobin concentration, hematocrit, serum iron, percent saturation of transferrin, or red cell protoporphyrin. Chi square analysis indicated that iron depletion (SF < 20 ng.ml-1) was significantly more prevalent (p < .005) in the runners than in the controls. Anemia was extremely rare in both groups. A multiple regression analysis revealed significant negative associations between serum ferritin and coffee/tea intake (p < .001) and running activity (p < .05). These results indicate that habitual runners, as compared with inactive women, are at increased risk for iron deficient states but that full-blown anemia is a rare consequence of this deficient iron status.

Prolonged Clinical Use of a Heme Oxygenase Inhibitor: Hematological Evidence for an Inducible but Reversible Iron-Deficiency State

The heme oxygenase inhibitor tin (Sn4+)-mesoporphyrin, administered to two 17-year-old Crigler-Najjar type I patients during a 400-day study to lower plasma bilirubin levels, also produced changes, beginning approximately 50 days after initiation of treatment, in hematological and iron metabolism indices consistent with the development of iron deficiency anemia. These indices were responsive to iron supplementation and reverted to normal after termination of inhibitor treatment. Tin-mesoporphyrin enhances biliary heme excretion and inhibits intestinal heme oxygenase when administered orally or parenterally; the changes in blood indices could thus reflect, in part, blockade of heme catabolism and therefore of uptake of heme-derived iron, by intestinal epithelium. This action of the inhibitor suggests that such agents may facilitate studies involving aberrant metabolism of heme-derived iron in humans and that they merit further investigation with respect to their potential value in enhancing iron disposal in certain disorders such as those related, for example, to transfusion-induced iron overload states.