Anaemias
The megaloblastic anaemias are disorders in which there are distinctive morphological abnormalities in the bone marrow. Deficiency of vitamin B12 or folate typically causes megaloblastic anaemia but it may also be due to abnormal metabolism of these vitamins. In addition, a comparable haematological picture may result from therapy with antifolate drugs such as methotrexate, and nitrous oxide inactivates cobalamin and megaloblastic haemopoiesis may ensue after protracted exposure to this anaesthetic agent. The bone marrow morphological abnormalities can also occur independently of cobalamin and folate deficiency as a result of treatment with drugs interfering with synthesis of DNA (eg. hydroxyurea, cytosine arabinoside, 6-mercaptopurine), and in haematological malignancy (AML and myelodysplasia); in these cases there is no response to cobalamin or folate therapy. Some additional, rare causes of a megaloblastic picture refractory to cobalamin and folate are orotic aciduria, Lesch-Nyhan syndrome, the autosomal recessive disorder thiamine-responsive megaloblastic anaemia (Rogers syndrome, characterised by anaemia, diabetes mellitus and sensorineural deafness).syn Pathogenesis Deficiencies of cobalamin and folate affect all rapidly growing tissues through interference with DNA synthesis. The blood pancytopenia is due to impaired synthesis of DNA because of a reduced supply of one or more of its four immediate precursors, the purines dATP and dGTP, and the pyrimidines dTTP and dCTP. Folate deficiency principally impairs synthesis of dTTP since it is needed as a coenzyme in thymidilate synthesis, but is also required in many other reactions. Cobalamin is required in two reactions, methylmalonyl coA isomerisation and methylation of homocysteine to methionine, which also requires folate. In addition to haemopoietic elements, tissues affected include the epithelium of the gastrointestinal tract, especially that of the oral cavity, stomach and small intestine, and the epithelial linings of the urinary and respiratory tracts and of the female genital tract. The gonads are also affected, and fetal tissues. Cells of involved tissues are macrocytic, with prominent multinucleate and dying cells. Folate and cobalamin deficiencies may also affect the central nervous system and result in psychiatric disturbance, and cobalamin deficiency is well recognised to cause peripheral neuropathy, degeneration of the posterior and pyramidal tracts of the spinal cord, and optic atrophy. Clinical Picture The clinical picture in folate and cobalamin deficiencies can be explained by the pathology described above. There is typically symptomatic anaemia, with weakness, tiredness, dyspnoea and even angina and cardiac failure. Unconjugated hyperbilirubinaemia due to death of megaloblasts (ineffective erythropoiesis) may be sufficient to result in clinical jaundice. Reversible melanin pigmentation is also an occasional feature and reversible greying of scalp hair has been noted. Thrombocytopenia of sufficient severity to cause bruising and mucosal bleeding occasionally occurs and neutropenia and impaired phagocyte function predispose to infection. Anorexia is a frequent feature, often with weight loss, glossitis, constipation or, rarely, diarrhoea. Impotence is not uncommon and infertility usual, in both sexes. The most common and usually the first symptom of nervous system involvement is symmetrical paraesthesiae of fingers and toes. Weakness, spasticity, unsteadiness, visual impairment, difficulty with micturition, postural hypotension, mood disturbance, impaired memory and psychotic symptoms may be present. Haematological consequences The haematological findings range from a raised MCV and the presence of a few oval macrocytes and occasional hypersegmented neutrophil polymorphs on the blood film to severe macrocytic anaemia with pancytopenia. Sometimes there is a leucoerythroblastic blood film with obvious megaloblasts. Bone marrow is typically hypercellular with an accumulation of primitive cells due to selective death of more mature forms. The erythroblasts show characteristic dissociation between nuclear and cytoplasmic development, the nucleus appearing primitive despite haemoglobinisation of the cytoplasm. Mitotic cells are prominent. Giant and abnormally shaped metamyelocytes and enlarged polyploid megakaryocytes are typical. There is usually increased marrow iron. In the early stages the marrow changes may be subtle and are also easily overlooked when the amaemia is predominantly due to another condition such as iron deficiency or haemolysis. Diagnostic difficulty may also arise when megaloblastosis arises in a subject with iron deficiency or thalassaemia in which case the MCV may not be raised and the blood film has dimorphic features. Also there are many causes of raised MCV, other than cobalamin and folate deficiencies. These include alcohol abuse, haemolysis, hypothyroidism, bone marrow failure, myelodysplasia and drug therapy, for example with hydroxyurea. Neural tube defects, homocysteine and vascular disease There has been a recent reawakening of interest in cobalamin and folate due to observations on the role of folate supplementation before and during pregnancy in the prevention of neural tube defects in the fetus, and on the ability of folate and cobalamin supplementation to lower plasma homocysteine concentration, even in subjects who are not obviously deficient in either vitamin. Higher serum homocysteine concentrations have been linked to increased risk of arterial and venous thromboembolic events, although whether supplementation with cobalamin or folate has a protective effect remains unknown. It is recognised that the concentration of homocysteine is determined in part by genotype; in particular a common polymorphism (677 G-T)of the gene coding for 5,10-methylene-tetrahydrofolate reductase (MTHFR) which results in a thermolabile enzyme variant of reduced enzyme activity. The increased serum homocysteine in subjects with the thermolabile enzyme is thought to result from a reduced availability of 5-methyl-tetrahydrofolate, the product of MTHFR. 5-methyl-THF and cobalamin are required for the methylation of homocysteine to methionine. Cobalamin deficiency Cobalamin exists in a number of chemical forms. These have a planar group (corrin ring) and a nucleotide. 5-deoxyadenosylcobalamin is the principal form in tissues. Cobalamin is synthesised solely by microorganisms. The only source for humans is food of animal origin especially offal and muscle, with small amounts in eggs, cheese and milk. Unless vegetables, fruits or other non-animal dietary components are contaminated by bacteria they do not contribute to dietary cobalamin. A mixed diet contains 5-30 microg. of cobalamin and daily losses in urine and faeces are around only 0.1% of the body stores of 3 mg. These stores are therefore sufficient to last for 3 to 4 years even if supplies are completely cut off. The principal mechanism for absorption of cobalamin is active and efficient and is mediated by gastric intrinsic factor (IF), a glycoprotein synthesised by the parietal cells of the body and fundus of the stomach. Cobalamin released from food binds first to a glycoprotein R binder in the stomach, but the binder is digested by pancreatic trypsin allowing cobalamin to bind to IF, the complex being resistant to enzyme digestion. The cobalamin-IF binds to the the brush border surface of ileal mucosal cells through specific IF receptors (cubilin). Cobalamin enters the ileal cell and after around 6 hours appears in portal blood attached to Transcobalamin II (TCII). After presentation of IF-cobalamin complex, ileal cells become temporarily refractory to further cobalamin absorption. There is some enterohepatic circulation of cobalamin. TC II, is synthesised by liver, ileal cells and possibly other tissues, and readily gives up cobalamin to marrow and placenta. In contrast, a second main cobalamin transport protein, TCI, does not enhance cobalamin uptake into tissues. It is closely related to the R protein in gastric juice and to TCIII, another binding protein in plasma. TCI and TCIII are derived from neutrophil specific granules and plasma levels are raised in myeloproliferative diseases, some reactive neutrophilias and hypereosinophilia, as well as hepatoma, this accounting for the high serum B12 levels seen in these conditions. TCII may be increased in liver disease and autoimmune diseases but is not necessarily accompanied by raised B12 levels. Congenital absence of TCII results in megaloblastic anaemia from a few weeks of age with normal serum cobalamin concentration in plasma. In contrast, absence of TCI causes a low serum cobalamin with no clinical abnormality. Cobalamin deficiency is principally due to malabsorption, the only other cause being inadequate dietary intake in vegans. Although subnormal cobalamin levels have been found in up to 50% of young adult Indian vegans, most do not develop cobalamin deficiency sufficient to cause anaemia or neurological damage. Several explanations have been proposed, including underestimation of cobalamin stores by serum cobalamin level, protection through the efficiency of the enterohepatic circulation, and reduced daily losses in the presence of limited body stores. Clinically important deficiency occasionally occurs in infants born of severely cobalamin deficient vegan mothers. In addition, a degree of cobalamin malabsorption, usually without clinical consequence, may occur in simple atrophic gastritis, severe chronic pancreatitis, intestinal graft-versus-host disease, folate deficiency, coeliac disease and Zollinger-Ellison syndrome. Low serum cobalamin, with normal stores, occurs in normal pregnancy, due to haemodilution and preferential uptake by placenta in the third trimester. Pernicious anaemia is a severe lack of intrinsic factor due to gastric atrophy. It is a common disease in Northern Europe but it occurs in all ethnic groups. It is rare under the age of 40 years. There is a familial tendency and associations with blood group A, organ-specific autoimmune diseases, premature greying of hair, vitiligo and blue eyes. It seems that autoimmune mechanisms play a major role in pathogenesis. Around half of affected individuals have so-called blocking antibodies which prevent IF binding to cobalamin, and around one third have so-called precipitating antibodies which interfere with IF binding to ileal mucosa. These autoantibodies are fairly specific for PA but may occasionally also be found in serum from subjects with other autoimmune diseases. Parietal cell antibody is detectable in around 90% of cases of pernicious anaemia but is also present in 16% of healthy females over 60 years of age. The detection of intrinsic factor antibody, but not of parietal cell antibody only, in a subject with the features of cobalamin deficiency, is generally regarded as acceptable evidence of a diagnosis of pernicious anaemia as the cause of the deficiency. Other investigations used in the diagnosis of cobalamin deficiency are the Schilling test and the deoxyuridine suppression test. The former tests for absorption of cobalamin, rather than deficiency, but as most causes of the deficiency, apart from nutritional, are due to malabsorption, a normal Schilling test in a subject on a good diet excludes cobalamin deficiency. The accuracy of the test is dependent on complete urine correction and for this reason the use of plasma sampling at 8 to 10 hours is probably preferable. There is controversy over the claim that megaloblastic anaemia due to cobalamin deficiency may occur with a normal Schilling test due to an inability to release cobalamin from binders in food. This is detectable only when protein bound cobalamin is used in the absorption test, but this type of test has not been routinely introduced. The deoxyuridine suppression test measures the synthesis of thymidine and its incorporation into DNA by bone marrow cells. It is impaired from an early stage in all subjects with megaloblastic anaemia due to cobalamin or folate deficiency but the test is technically demanding and not in routine use. Folate deficiency Folic acid (pteroylglutamic acid) is the parent compound of the folate family. It consists of pteridine, para-aminobenzoate and L-glutamic acid. Food folates are reduced di- or tetrahydrofolate derivatives with methyl or formyl groups and typically 4, 5 or 6 glutamate residues. Folate is present in most foods with highest levels in green vegetables, nuts, yeast and liver. Unlike cobalamin, it is rapidly degraded during cooking of foods. Around 100 mcg. is required daily. Some losses occur in sweat and shed skin, and especially in urine. The body stores of about 10 mg. are equivalent to 4 months’ requirement. In contrast to cobalamin, folate is absorbed in the upper small intestine, probably by an active process. Around 50% of food folates are absorbed, monoglutamates being absorbed more efficiently than polyglutamates. Food folates are converted to 5-methyltetrahydrofolate before entering portal plasma. In all body fluids folate exists as the monoglutamate form of 5-methyltetrahydrofolate. The causes of folate deficiency are numerous. Unlike cobalamin deficiency, dietary inadequacy is a major cause of folate deficiency. Folate requirements are doubled in pregnancy due to increased catabolism and the demands of the fetus. Megaloblastic anaemia is common if supplements are not given, especially in the third trimester and early in the postpartum phase. Twin pregnancies are at increased risk. Diagnosis of folate and cobalamin deficiency The diagnosis of significant deficiency of cobalamin or folate depends upon recognition of the typical clinical picture, assay of the vitamin levels and demonstration of a complete response to replacement. Cobalamin is measured by radioisotope dilution or enzyme linked immunosorbant assay. The lower limit of normal is 160 to 200 ng/l and megaloblastic anaemia due to cobalamin deficiency is usually accompanied by a level of less than 100 ng/l. Lesser reductions may be found in mild states of cobalamin malabsorption such as atrophic gastritis, in pregnancy and in folate deficiency. Folate is measured in serum and red cells by radioassay or enzyme-linked immunoassay. Serum folate is easily influenced by recent diet and red cell folate gives a more accurate representation of body folate stores. Normal adults have more than 160 microg./l of packed red cells and low values are found in megaloblastic anaemia due to folate deficiency, but also in that due to cobalamin deficiency. Although the diagnosis of megaloblastic anaemia due to cobalamin or folate deficiency is generally straightforward, it has recently been suggested that a large number of subjects have subclinical or mild deficiency which is undetected by conventional approaches. This is based on observations of raised concentrations of methylmalonic acid or homocysteine in many subjects with normal levels of cobalamin and folate. The assumption that these findings really represent important deficiency is by no means universally accepted, however. Treatment of folate and cobalamin deficiency Treatment of megaloblastic anaemia is generally straightforward. It is preferable to treat with the vitamin which has been demonstrated to be deficient, but in the urgent situation it is acceptable to start treatment with cobalamin and folate having obtained serum for testing. Transfusion should be avoided as there is a risk of death from cardiac failure due to volume overload. If it is unavoidable the minimum volume (often one unit) of packed red cells should be given, with diuretics. The use of calcium supplements has been advocated as the initial haematological response in severely anaemic subjects may cause hypokalaemia. In most cases of malabsorptive cobalamin deficiency the cause is irreversible and lifelong cobalamin therapy is needed. Oral therapy is inadequate except in dietary deficiency. Six intramuscular doses of 1000 microg. of hydroxocobalamin given over 3 day intervals replaces the body stores. An injection of 1000 microg. every three months is entirely adequate for maintenance. In contrast, there is no requirement for parenteral folate therapy. If 5 to 15 mg is administered orally, daily, sufficient is absorbed for a complete response, even in severe malabsorption. Duration of folate therapy is determined by the nature of any underlying disease and its response to treatment. It is essential that large doses of folate are not given to a subject with uncorrected cobalamin deficiency as cobalamin neuropathy may be precipitated. It is important to ensure a complete haematological response to treatment, for confirmation of the diagnosis and to allow identification of the reasons for failure to respond. These include wrong diagnosis, untreated additional deficiency (eg. of iron), underlying haematological disease (eg. haemolysis), renal failure and hypothyroidism. Reticulocytosis commences on the third day of treatment and peaks at around 7 days. The leucocyte and platelet counts should be normal at seven days. In conclusion, although the causes and pathophysiology of megaloblastic anaemias are well recognised, there are recent observations linking folate and cobalamin to metabolic derangements which could be of much broader significance than previously recognised. Further reading Chanarin I, Metz J. (1997) Diagnosis of cobalamin deficiency: The old and the new. Brit. J. Haematol. 97, 695-700 Frosst P, Blom HJ, Milos R et al (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylene terahydrofolate reductase. Nature (Genetics) 10, 111-3 Mills JL, McPartlin JM, Kirke PN et al (1995) Homccysteine metabolism in pregnancies complicated by neural tube defects. Lancet 345, 149-51 Toh B-H, Van Driel IR, Gleeson PA (1997) Pernicious anaemia. New. Eng. J. Med. 337, 1441-8