revised May 15, 2003

IRON DEFICIENCY

Magnitude of the problem

 Iron deficiency is a leading cause of anemia, affecting over one-half billion people world wide. Blood loss nearly invariably is the culprit producing iron deficiency in adults. The high demand for iron created by neonatal and adolescent growth spurts occasionally produces iron deficiency in children. Nonetheless, blood loss is the most frequent cause of iron deficiency in this group as well. Body iron stores for women normally vary between one and two grams while men average three to four grams. The liver is the site of most storage iron. Depletion of iron stores precedes impaired production of iron-containing proteins, the most prominent of which is hemoglobin. The two key stages of iron deficiency, are (1) depletion of iron stores without anemia, and (2) depletion of iron stores with anemia. Iron replacement therapy cannot be comfortably undertaken until the cause of the iron deficit is ascertained.

Etiology

Abnormal iron uptake from the gastrointestinal tract

  1. Poor Bioavailability

  2.  All iron in the universe is produced in the cores of stars and by supernovae in a process called nucleosynthesis (Ferris, 1997). Although the element is the second most abundant metal in the earth's crust, iron's low solubility makes its acquisition for metabolic use a major challenge. Most environmental iron exists as insoluble salts. Gastric acidity assists conversion to absorbable forms, but the efficiency of this process is limited. Many plants produce powerful chelators, such as the phytates (organic polyphosphates) found in wheat products, that further impair iron absorption (Gillooly et al., 1984). The challenge of acquiring sufficient iron from the environment possibly the fueled the spread of the gene for hereditary hemochromatosis.
     
     
    Table 1. Factors that modifiy iron absorption
    Physical State (bioavailibility) heme > Fe2+> Fe3+
    High Gastric pH hemigastrectomy, vagotomy, pernicious anemia
    histamine H2 receptor blockers, calcium-based antacids
    Disruption of Intestinal Structure Crohn's disease, celiac disease (non-tropical sprue)
    Inhibitors phylates, tannins, soil clay, laundry starch, iron overload
    Competitors cobalt, lead, strontium
    Facilitators ascorbate, citrate, amino acids, iron deficiency

    High gastric pH reduces the solubility of inorganic iron, impeding absorption. Surgical interventions, such as vagotomy or hemigastrectomy for peptic ulcer disease, formerly were the major causes of impaired gastric acidification with secondary iron deficiency (Baird et al., 1974; Magnusson, et al., 1979). Calcium carbonate based antacid compounds are more likely to produce iron deficiency than are those that contain magnium hydroxide or aluminum hydroxide (Wienk, et al, 1996), (O'Neil-Cutting, et al, 1986). Large doses of histamine H-2 blockers can reduce iron absorption (Skine, et al., 1981). Rountine use of these agents produce iron deficiency only occasionaly, and then often with other confounding issues such as phytate ingetion with bran cereals. Despite markedly reducing gatric acidity, the more recently introduced acid pump blockers appear to cause iron deficiency infrequently (Koop, et al., 1992; Stewart, et al., 1998). These apparent paradoxes highlight the substantial  deficits that exist in our understanding of the mechanics of iron absorption.

    The impaired function of the gastric parietal cells associated with pernicious anemia not only reduces the production of intrinsic factor, but also lessens gastric acidity. Impaired iron absorption can result. In addition, the megaloblastic enterocytes absorb iron poorly. Frank iron deficiency can accompany the anemia produced by cobalamin deficiency. The serum iron level and transferrin saturation often are artifactually high in patients with pernicious anemia (Demiroglu, et al., 1997). Pernicious anemia slows hematopoiesis significantly. Since 80% of circulating iron is destined for the the erythron and red cell production,  the iron accumulates on plasma transferrin awaiting use. Correction of pernicious anemia dramatically increases hematopoiesis, with a consequent drop in plasma tranferrin iron levels. A recheck of plasma transferrin saturation should be performed within a week or two of the start of treatment for pernicious anemia to ensure that iron stores can support the higher rate of erythropoiesis.

     Hemin, the most readily absorbed form of iron, is taken up independently of gastric pH. The molecule is, of course, derived primarily from animal tissue. The scarcity of dietary meat for much of the world's population increases the risk of iron deficiency. The fact that cultivated grasses, such as rice, are dietary staples for many people exacerbates the problem since these plants are very poor sources of iron. The consequences of iron deficiency can be quite severe.
     

  3.  Inhibition of Iron Absorption

  4.  A number of environmental factors can produce dietary iron deficiency, including metals that share the iron absorption machinery, such as lead, cobalt, and strontium (Table 1). Of these only lead is a significant problem. For children, the threat is particularly marked. Iron deficiency increases the rate of uptake both of iron and lead from the gastrointestinal tract. Iron deficiency and lead intoxication, consequently, are common companions (Nicholls and McLachlan, 1990).
     
  5.  Disruption of the Enteric Mucosa

  6.  Some disorders disrupt the integrity of the enteric mucosa, thereby hampering iron absorption. Inflammatory bowel disease, particularly Crohn's disease, can injure extensive segments of the small intestine, occasionally extenting to the jejunum and even the duodenum. Invasion of the submucosa by inflammatory cells and disruption of the tissue architecture of the intestine impair iron absorption and uptake of dietary nutrients (Beeken, 1973). Occult gastrointestinal bleeding can accompany these condtions and exacerbate the problem of iron balance. The resulting iron deficiency anemia is further complicated by the anemia of chronic inflammation. Also, Crohn's disease frequently involves the terminal ileum, producing concurrent cobalamin deficiency.

      Sprue, both of the tropical and non-tropical variety (celiac disease), can also interfere with iron absorption. Degeneration of the intestinal lining cells along with chronic inflammation causes profound malabsorption. The anemia due to chronic inflammation and iron deficiency often is complicated further by nutritional deficiency. Celiac disease frequently improves dramatically with a gluten-free diet. Some patients with deranged iron absorption lack gross or even histologic changes in the structure of the bowel mucosa. The disease can be mild to the point that it produces few or no symptoms (Corazza et al., 1995). A gluten-free diet improves bowel function in many such patients, with secondary correction of the anemia.
     

  7.  Loss of Functional Bowel

  8.  Substantial segments of bowel are sometimes removed surgically, disrupting iron absorption. Intractable inflammatory bowel disease occasionally is treated by surgical excision. Traumatic abdominal injury, as occurs with motor vehicle accidents, also requires extensive bowel resection at times. Structural complications, such as intestinal volvulus or intusseception, sometimes necessitate removal of significant stretches of bowel in children. Iron deficiency usually develops slowly, and may not become evident for several years after the surgical procedure.

      Dysfunction of the gastrointestinal absorption machinery is a very rare cause of iron deficiency. The prototype of this genre of problem is the mk/mk mouse. These animals fail both to absorb iron from the gastrointestinal tract and to incorporate transferrin-bound iron into developing normoblasts in the bone marrow. The result is animals with a severe iron-deficiency anemia. Deficient synthesis of the recently cloned iron transport protein, NRAMP2 (now designated, DMT1), causes the problem (Fleming, et al., 1997). Impaired gastrointestinal iron absorption and defective erythrocyte production in at least one family appears to result from a deficit in the human analogue of NRAMP2.

Blood loss

 Blood loss is the world's leading cause of iron deficiency. The gastrointestinal tract is both the site of iron uptake and the most common site of blood loss. The gastrointestinal tract is unrivalled as a potential site of occult blood loss.
 
  1. Gastrointestinal Blood Loss
  2. Other sources of blood loss

Consequences of iron deficiency

 Although anemia is most prominently linked to iron deficiency, the condition produces a wide range of abnormalities, depending on its severity and duration. Some of these abnormalities, such as cognitive dysfunction in young children, have been recognized only recently.

Anemia

 Since most iron is directed to hemoglobin synthesis, erythrocyte production is among the first casualties of iron deficiency:
Figure 1. Photomicrograph of blood cells from a patient with iron deficiency anemia.
Photomicrograph of iron deficient red cells
 The microcytic, hypochromic anemia impairs tissue oxygen delivery, producing weakness, fatigue, palpitations, and light-headedness (Figure 1). Thalassemia trait is sometimes confused with iron deficiency, since the condition is characterized by microcytosis and sometimes mild anemia. Iron deficiency alters red cell size unevenly as shown in Figure 1. Electronic blood analysers determine the mean red cell volume (MCV) as well as the range of variation in red cell size (expressed as the RDW or red cell distribution width). The RDW (determined with every electronically processed complete blood count) is normal in patients with thalassemia trait but is high with iron deficiency (Lin et al., 1992). Other common features of thalassemia trait are basophilic stippling and target cells. These characteristics are not sufficiently unique to distinguish between thalassemia trait and iron deficiency, however.

  The plasma membranes of iron deficient red cells are abnormally rigid (Tillman, et al., 1980). This rigidity could contribute to poikilocytic changes, seen particularly with severe iron deficiency. These small, stiff, misshapen cells are cleared by the reticuloendothelial system, contributing to the low-grade hemolysis that often accompanies iron deficiency. The cause of this alteration in erythrocyte membrane fluidity is unknown.

Growth and developmental retardation

 Iron deficiency, with or without concomitant anemia, commonly impairs growth and intellectual development in children. Studies of cognitive development in the setting of iron deficiency produced disparate results for a time. In some investigations, dietary iron supplementation for infants reversed cognitive dysfunction, while others failed to show improvement. Some of the disparities may have resulted from differences in the instruments used in the analyses, and differences in the populations examined.

  The Bayles Scales of Infant Development, uncovers abnormalities in children as young as 9 to 12 months of age. Developmental abnormalities occur with or without anemia. In one study, iron replacement increased the Mental Development Index scores substantially in only seven days. Iron deficiency does not impair motor development even in infants with low scores on the tests of cognitive development. Concomitant lead exposure can further hamper the psychological development of these children.

  Information on the long-term effects of iron deficiency during infancy highlights the importance of early intervention. One group of children with iron deficiency anemia (hemoglobin less than 10 g/dl) was treated during infancy and tested for cognitive development at five years of age (Lozoff et al., 1991). This cohort scored lower in tests of mental and motor functioning than did their counterparts, despite correction of the deficiency during infancy. The disparity persisted despite adjustments for differences in socioeconomic background. The investigators soberly concluded that children who are iron deficient during infancy are at risk of long-lasting developmental disadvantage relative to peers with better iron status. Other investigations concur. Correction of iron deficiency during infancy clearly improves scores in short-term tests, such as the Bayles Scales of Infant Development, without preventing longer-term, and possibly more serious, impairment in cognitive function. Health care providers, therefore, must strive to prevent iron deficiency during infancy (Oski, 1993).

  The mechanism by which iron deficiency impairs neurologic function is unknown. Many enzymes in neural tissue require iron for normal function. The cytochromes involved in energy production, for example, predominantly are heme proteins. In rats, weanlings maintained on iron deficient diets develop severe behavioural anomalies, motor incoordination, and seizures. The abnormalities seen in iron deficient children and adults are much less pronounced, but are a source of serious concern nonetheless.

  The effect of iron deficiency on childhood growth is often difficult to separate from overall nutritional deficiency. The high prevalence of childhood iron deficiency among less affluent people has yoked deficiencies of iron and general nutrients. When the two factors have been separated, correction of iron deficiency improves growth independently of nutritional status.

Epithelial changes

 Iron deficiency produces significant gastrointestinal tract abnormalities, reflecting the enormous proliferative capacity of this organ. Some patients develop angular stomatitis and glossitis with painful swelling of the tongue. The flattened, atrophic lingual papillae makes the tongue smooth and shiny. A rare complication of iron deficiency is the Plummer-Vinson syndrome with the formation of a postcrycoid oesophageal web. Long-standing, severe iron deficiency affects the cells that generate the finger nails producing koilonychia. The nail substance is soft, so that ordinary pressure on the fingertips, as occurs with writing for instance, produces a concave deformity. The "spoon-shaped" changes featured in many text books are rare. Koilonychia usually is much more subtle. Often, a transverse depression or depressions across the thumb nail is the only manifestation. The alteration most commonly affects the right thumb nail, reflecting the right-handedness of most people.

Miscellaneous

 Pica occurs variably in patients with iron deficiency. The precise pathophysiology of the syndrome is unknown. Patients consume unusual items, such as laundry starch, ice, and soil clay. Both clay and starch can bind iron in the gastrointestinal tract, exacerbating the deficiency (Roselle, 1970). A dramatic example of the problems produced with clay consumption occurred in the 1960s with reports of iron deficiency in children along the border between Iran and Turkey (Say, et al., 1969). These youngsters had other, peculiar abnormalities including massive hepatosplenomegaly, poor wound-healing, and a bleeding diathesis. Presumably, the children initially had simple iron deficiency associated with pica, including geophagia. The soil contained compounds that bound both iron and zinc. The secondary zinc deficiency caused the hepatomegaly and other unusual abnormalities.

  Unexplained thrombocytosis occurs frequently with platelet counts in the range of 500,000 to 700,000 cells /fl. Megakaryocytes and normoblasts are derived from a common committed progenitor cell, the CFU-GEMM (colony-forming unit, granulocytic, erythroid, myelomonocytic). Thrombopoietin, the molecule that stimulates the growth of megakaryocytes and the production of platelets, is structurally similar to erythropoietin; the molecule that promotes red cell development. Some investigators speculated that the elevated levels of erythropoietin in patients with iron deficiency anemia might modestly increase platelet production by cross-reacting with the thrombopoietic receptor. Investigations with recombinant human erythropoietin and thrombopoietin indicate that cross-reactivity does not occur. The etiology of the thrombocytosis in iron deficiency remains a mystery.

Diagnosis of iron deficiency anemia

The Effect of Iron Deficiency on Serum Levels of Transferrin, Iron and Ferritin
Changes in serum levels of transferrin, ironandferritin produced by iron deficiency
Figure 2. Changes in Serum Levels of Transferrin, Iron, and Ferritin Produced by Iron Deficiency. The panel on the left shows the normal serum levels of transferrin, iron, and ferritin, in arbitrary units. The panel on the right shows the effect of iron deficiency on these values. The increase in serum transferrin level along with a fall in serum iron level lowers the transferrin saturation (serum iron level divided by the serum transferrin level.) The best laboratory indicator of body iron stores is the serum ferritin level, which drops substantially in people with iron deficiency.
 Iron deficiency anemia lowers the number of circulating red cells (a feature of all anemias). The red cells are microcytic (usually less than 80 fl in size) and hypochromic. The quantity of the iron-carrying protein, transferrin, in the circulation increases over baseline by 50% to 100%. The quantity of iron on transferrin can fall by as much as 90%. Consequently the transferrin saturation frequently declines from its usual 30% to under 10%. Figure 2 schematically illustrates these changes.

  The most useful single laboratory value for the diagnosis of iron deficiency may be the plasma ferritin. Ferritin is the cellular storage protein for iron. Plasma ferritin differs from its cellular counterpart in several respects, and appears to be a secreted protein of a different origin (Arosio, et al., 1977). The plasma ferritin value often falls to under 10% of its baseline level with significant iron deficiency.

  Other phenomena, such as chronic inflammation with rheumatoid arthritis, perturb the plasma values of iron, transferrin, and ferritin. Establishing the diagnosis of iron deficiency in these patients can be difficult. The situation is further complicated by the fact that chronic inflammation per se can produce anemia (Sears, 1992). Newer tests, such as assay of circulating transferring receptors, can help with the diagnosis of iron deficiency in many instances (Shih, et al., 1993). If all else fails, a bone marrow biopsy with Prussian blue staining for iron is the touchstone for the diagnosis.

Treatment of iron deficiency

 The most important steps in the evaluation and treatment of iron deficiency are determining the cause of the deficiency, and correcting the abnormality. Malignancy of the gastrointestinal is the haunting spectre in adults with iron deficiency. In children, iron deficiency due to growth spurts, poor dietary patterns, or benign gastrointestinal bleeding sources are much more common. After the cause of the iron deficiency has been ascertained, oral iron supplementation replaces stores most efficiently.

Oral iron supplementation

 Oral iron supplementation is the ideal way to replace iron stores as it uses the body's normal mechanisms. The shortcoming is the gastrointestinal tract's limited capacity for iron absorption. Only about 2 to 3 mg of elemental iron are absorbed, even when 50 or 100 mg are presented to the gut lumen. Most orally consumed iron flows untouched through the alimentary tract. Replenishing a 2,000 mg iron deficit can take most of a year. With ongoing blood loss, replacement of stores with oral iron becomes a herculean task. Many, if not most, patients fail to comply with such a prolonged medical regimen. Therapeutic failures are common with oral iron replacement.
 
 
Table 2. Poor Response to Oral Iron
Non Compliance
On-going Blood Loss
Insufficient Duration of Therapy
High Gastric pH
  • antacids
  • histamine H-2 blockers
  • vagotomy
Inhibitors of Iron Absorption/Utilization
  • lead
  • aluminum intoxication (hemodialysis patients)
  • chronic inflamation
  • neoplasia
Incorrect Diagnosis
  • thalssemia
  • sideroblastic anemia

 Although ferrous sulphate is often recommended to treat iron deficiency, frequent problems with the drug including gastrointestinal discomfort, bloating and other distress, make it unacceptable to many patients (Table 2). Ferrous gluconate, which is roughly equivalent in cost, produces fewer problems, and is preferable as the initial treatment of iron deficiency. Ascorbic acid supplementation enhances iron absorption. Combination tablets containing iron salts and ascorbic acid are significantly more expensive than separate tablets for each, however.

  Polysaccharide-iron complex, a replacement form of iron that differs from the iron salts, is a more recent option. The polar oxygen groups in the polysaccharide form coordination complexes with the iron atoms. The well-hydrated microspheres of polysaccharide iron remain in solution over a wide pH range. Most patients tolerate this form of iron better than the iron salts, even though the 150 mg of elemental iron per tablet is substantially greater than that provided by iron salts (50 to 70 mg per tablet). No study exists comparing iron uptake from polysaccharide-iron complex and ferric salts.

Parenteral iron replacement

 Parentaral iron is available either as iron dextran or iron saccharide (commonly ferric polymaltose). Only iron dextran has been licensed for general use in the U.S. A ferric polymaltose compound, Ferrlicit®, has been licensed for use in patients on hemodialysis who need parenteral iron replacement. Similar drugs have been used in Europe and the rest of the world for decades. The incidence of side effects, particularly the dreaded though uncommon anaphylaxis, is far less  with the iron saccharide compounds. Current investigations are underway aimed at broadening the FDA-approved indications. Ferrelicit has been used safely in patients who have had anaphylactic reactions to iron dextran.

  Parenteral iron is indicated when i) oral iron is poorly tolerated, ii) rapid replacement of iron stores is needed, or iii) gastrointestinal iron absorption is compromised. Iron-dextran can be administered via intramuscular or intravenous injection. Intramuscular injection of iron-dextran can be painful, and leakage into the subcutaneous tissue produces long-standing skin discoloration. A "Z-track" injection into the muscle minimizes the chance of subcutaneous leak. Suboptimal muscle mass frequently associated with nutritional deficiency further complicates this mode of replacement. Intravenous infusion of iron-dextran circumvents these problems altogether (Auerbach et al., 1988). Commonly, patients are given total dose infusion replacement with iron dextran, with entails the adminstration of up to 3,000 mg at a single sitting (Ahsan, 1998). This is the fastest way to replete iron stores. With either route of administration, a test dose of 10 mg of iron should be given and the patient observed by a physician for 30 minutes to rule out an anaphylactic reaction to the medication (such reactions are infrequent).

  About 10% to 15% of patients experience transient mild to moderate arthralgias the day after intramuscular or intravenous administration of iron-dextran. Acetaminophen usually effectively relieves the discomfort. Administration of methylprednisolone at a dose of 1 mg/kg after the test dose and before the repletion dose virtually eliminates this reaction. Premedication with steroids is particularly helpful for patients with autoimmune inflammatory disorders such as rheumatoid arthritis. The treatment strategy largely suppresses the pain flairs of disease that these patients can otherwise experience. The symptoms possible result from release of inflammatory cytokines such as interleukin-1 and tumour necrosis factor. The iron dextran is cleared from the circulation by fixed tissue macrophages, which probably are activated to release these proinflammatory peptides.

  Iron saccharide complexes work at least as well as iron-dextran when administered intravenously. Anaphylaxis occurs extremely infrequently with these drugs. The other side effects seen with iron dextran, such as fevers, dyspnea, and myalgias are also very uncommon with the iron saccharides. They are the preferred formulation in countries where it is available. Total dose infusion cannot be used with iron saccharide complexes because significant side-effects occur with doses that exceed about 250 mg. Patients wiht large iron deficits therefore require multiple treatments with these medications.

  In uncomplicated cases of iron deficiency, IV replacement produces subjective improvement in a few days. Peak reticulocytosis occurs after about 10 days, and complete correction of the anemia can take 3 to 4 weeks. The hematocrit rises sufficiently in a week or two to provide symptomatic relief for most patients.

Functional iron deficiency

 Human erythropoietin was one of the first agents of widespread clinical utility produced by recombinant DNA technology (rHepo) (Adamson, et al., 1990). Used to correct the anemia of end-stage renal disease (ESRD), this hormone has provided new insight into the kinetic relationship between iron and erythropoietin in red cell production. Erythropoietin treatment of anemia in patients with ESRD has also underscored the variable nature of storage iron. The shifting states of storage iron contribute to the inconsistency with which erythropoietin corrects the anemia of renal failure.

  With steady-state erythropoiesis, iron and erythropoietin flow to the bone marrow at constant, low rates. In patients with ESRD, recombinant human erythropoietin (rHepo) is administered in intermittent surges, most commonly as intravenous boluses. The resulting kinetics of erythropoiesis are markedly unphysiological and strain the production process (Madore, et al., 1997). Erythropoietin, the accelerator of erythroid proliferation is not coordinated with the supply of iron, the fuel for erythroid proliferation. This imbalance almost never occurs naturally. The rHepo jars previously quiescent cells to proliferate and produce hemoglobin. The requirement for iron jumps dramatically, and outstrips its rate of delivery by transferrin (Adamson, 1994).

Interplay between iron and erythropoietin

 Erythropoietin stimulates proliferation and differentiation of erythroid precursors, with an upsurge in heme synthesis. Iron is taken into the cells from ferrotransferrin by cell surface transferrin receptors, is transported to the mitochondria, and inserted into the protoporphyrin IX ring by ferrochelatase (Ponka, 1997). The newly synthesized heme modulates globin synthesis in part through its effect on the translational factor, eIF-2. Primitive erythroid cells have relatively few transferrin receptors. The number increases with differentiation, peaking at over 106 per cell in the late pronormoblasts. The number subsequently declines, to the point that mature erythroid cells lack transferrin receptors altogether. This variable expression of transferrin receptors means that iron delivery must be synchronized both with proliferation and stage of erythroid development. Late normoblasts, for instance, cannot compensate for iron that was not delivered during the basophilic normoblast stage. These cells have fewer transferrin receptors, and those receptors are busy supplying iron for currently produced heme molecules.

  Transferrin-bound iron bound is the only important source of the element for erythroid precursors. Even with normal body iron stores and normal transferrin saturation, robust proliferation of erythroid precursors can create a demand that outstrips the capacity of the iron delivery system (Brugnara et al., 1994). Transferrin iron saturation falls as the available iron on plasma transferrin is stripped off by voracious erythroid precursors. Plasma iron turnover (PIT) rises, as does erythron iron turnover (EIT) and erythron transferrin uptake (ETU) (Hotta, et al, 1991). The late arrival of newly mobilized storage iron fails to prevent production of hypochromic cells. This is "functional iron deficiency" or "iron-erythropoietin kinetic imbalance". The ESRD patients who initially received erythropoietin had substantial iron stores. More importantly, supraphysiological quantities of iron were bound to their circulating transferrin (i.e., transferrin saturations of 70 to 90%). As a result, even the bursts of heme production induced by pharmacological levels of exogenously administered rHepo could be matched by the available transferrin-bound iron. In these patients, the rate limiting factor in erythrocyte synthesis was the proliferative capacity of the erythroid precursors. The number of red cells produced was determined by the number of precursor cells and the quantity of erythropoietin they encountered.

 When patients with "ordinary" iron stores were begun on erythropoietin, the picture was quite different. Here, the cells were jolted into accelerated proliferation while transferrin saturations ranged only between 30% and 50%. Iron was rapidly pulled into the developing erythroid cells. In some instances, transferrin-bound iron could sustain maximal synthesis of hemoglobin. In other cases, however, iron availability was suboptimal, producing mild functional iron deficiency. Even when the hemoglobin concentration increased substantially, the transferrin saturation fell, reflecting the strain on the supply system (Rutherford et al., 1994). The quantity of iron in storage was more than adequate to meet the demands of hemoglobin synthesis, but could not be mobilized with sufficient speed to satisfy the developing normoblasts. The kinetic mismatch in circulating rHepo and circulating ferrotransferrin is the key to functional iron deficiency.


References