Diagnosis of megaloblastic anaemias

Blood Reviews (2006) 20, 299–318

www.elsevierhealth.com/journals/blre

REVIEW

Diagnosis of megaloblastic anaemias S.N. Wickramasinghe Department of Haematology, Faculty of Medicine, Imperial College, St Mary’s Campus, Praed Street, London W2 1PG, UK

KEYWORDS

Summary There are a large number of causes of megaloblastic anaemia. The most frequent are disorders resulting in vitamin B12 or folate deficiency. The diagnostic process often consists first of establishing the presence of B12 or folate deficiency and then of determining the cause of deficiency. The blood count, blood film, serum B12 assay, and red cell and serum folate assays are the primary investigations. Other useful investigations include serum/plasma methylmalonic acid (MMA), plasma total homocysteine (tHCYS) and serum holo-transcobalamin II assays. All currently used tests have limitations regarding specificity or sensitivity or both and the metabolite assays are not widely available. An understanding of these limitations is essential in formulating any diagnostic strategy. The wide use of serum B12 and metabolite assays has resulted in the increasingly early diagnosis of B12 deficiency, often in patients without B12-related symptoms (subclinical deficiency). Food cobalamin malabsorption is the most frequent cause of a low serum B12. At least 25% of low serum B12 levels are not associated with elevated metabolite levels and may not indicate B12 deficiency. Some of these are caused by partial deficiency of transcobalamin I. c 2006 Elsevier Ltd. All rights reserved.

Megaloblastic anaemia; Diagnosis; B12 deficiency; Folate deficiency; Inborn errors of metabolism



Introduction The megaloblastic anaemias are a subgroup of macrocytic anaemias in which the bone marrow shows distinctive morphological abnormalities of red cell precursors, namely, megaloblastic erythropoiesis. In the other subgroup, the bone marrow shows normoblastic erythropoiesis. Hence, it was customary in the past to examine the marrow of patients with macrocytic anaemia to determine which subgroup a patient fell into. On the basis of data from serum vitamin B12 (B12, cobalamin) and red cell and serum E-mail address: [email protected].



folate assays, serum intrinsic factor antibody assays and tests of B12-and folate-dependent metabolic functions, many patients with megaloblastic anaemia can now be diagnosed without marrow aspiration. The causes of macrocytic anaemia with megaloblastic erythropoiesis are listed in Table 1 and with normoblastic erythropoiesis in Table 2. The most common causes of megaloblastic anaemia are vitamin B12 and folate deficiency. In mild or subclinical deficiency of these vitamins, there may be macrocytosis without anaemia or neither macrocytosis nor anaemia. In these cases, the marrow shows little or no megaloblastic change. In investigating a patient with macrocytosis, the diagnoses

0268-960X/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.blre.2006.02.002

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Table 1

Causes of megaloblastic anaemia (macrocytic anaemia with megaloblastic erythropoiesis).

Vitamin B12-related 1. Dietary deficiency 2. Impaired release of B12 from food - food cobalamin malabsorption 3. Inadequate secretion of intrinsic factor Pernicious anaemia Total or partial gastrectomy Congenital intrinsic factor deficiency Congenitally abnormal intrinsic factor 4. Impaired release of B12 from B12–R binder complex Pancreatic insufficiency Zollinger-Ellison syndrome 5. Diversion of dietary B12 Abnormal intestinal bacterial flora – jejunal diverticula, small intestinal strictures, stagnant intestinal loops (blind loop syndrome) Fish tapeworm infestation 6. Malabsorption due to abnormalities in the terminal ileum Ileal resection Crohn’s disease Chronic tropical sprue Other acquired diseases Imerslund-Gra ¨sbeck syndrome (selective malabsorption with proteinuria) 7. Drug-induced malabsorption (aminosalicylic acid, neomycin, colchicine, slow-release potassium chloride, metformin, phenformin, biguanides, cholestyramine) 8. Complex or uncertain mechanism – HIV infection 9. Inactivation of methylcobalamin by nitrous oxide 10. Inherited abnormalities of B12 transport or intracellular B12 metabolism Deficiency or abnormality of transcobalamin II Defective methylcobalamin synthesis (hyperhomocysteinaemia) Defective synthesis of both methylcobalamin and adenosylcobalamin (hyperhomocysteinaemia and methylmalonic acidaemia) Folate-related 1. Dietary deficiency 2. Malabsorption due to abnormalities of the jejunum Coeliac disease Jejunal resection Tropical sprue 3. Drug-induced malabsorption Sulfasalazine 4. Increased requirement Pregnancy Prematurity Chronic haemolytic anaemias Malignant diseases Chronic inflammatory disease 5. Increased loss Some skin diseases Long-term dialysis 6. Acquired abnormality of folate metabolism: dihydrofolate reductase inhibitors 7. Complex or uncertain mechanism Ethanol abuse (some cases) Anticonvulsant drugs (some cases) Oral contraceptive drugs, glutethimide, cycloserine 8. Inherited abnormalities of folate absorption and metabolism Hereditary folate malabsorption Glutamate formiminotransferase-cyclodeaminase deficiency Vitamin B12- and folate-independent

1. Acquired abnormalities of nucleic acid synthesis Purine analogues (e.g. azathioprine, 6-mercaptopurine, 6-thioguanine, acyclovir) Pyrimidine analogues (e.g. 5-fluorouracil, cytarabine, 5-azacytidine, zidovudine) Other drugs (hydroxycarbamide, cyclophosphamide, procarbazine) Arsenic poisoning

Diagnosis of megaloblastic anaemias Table 1

301

(continued)

2. Inherited abnormalities of nucleic acid synthesis Orotic aciduria Lesch-Nyhan syndrome 3. Complex or unknown mechanism Anticonvulsant therapy (some cases) Chronic alcohol abuse (some cases) Protein-energy malnutrition Myelodysplastic syndromes (some cases) and erythroleukaemia Congenital dyserythropoietic anaemia, types I and III Thiamine-responsive megaloblastic anaemia syndrome

test on a biopsy sample to detect Helicobacter pylori, serology for Helicobacter pylori infection and radiological imaging of the small intestine. In some inherited disorders, a proportion of cases may be diagnosed by mutational analysis of the disease genes.

Table 2 Causes of macrocytosis with normoblastic erythropoiesis. Chronic alcohol abusea Myelodysplastic syndromesa Hypothyroidism Chronic lung disease with hypoxia Heavy smoking Chronic liver diseasea Chronic haemolytic anaemia Therapy with certain anticonvulsant drugsa Myeloma (some cases) Hypoplastic and aplastic anaemia Physiological Normal neonates Normal pregnancy (some cases)

B12-dependent and folate-dependent reactions Two reactions are known to require B12 in man. The first is the methylcobalamin-dependent methylation of homocysteine to methionine by methionine synthase (homocysteine methyltransferase), which is also dependent on 5-methyltetrahydrofolate.

a Some patients show B12- and folate-independent megaloblastic erythropoiesis.

in both Tables 1 and 2 must be considered. The commonest cause of macrocytosis with normoblastic erythropoiesis is chronic alcohol abuse. In most cases, the diagnosis of megaloblastic anaemia is made in two stages: (1) establishing that the patient is B12 or folate deficient and (2) determining the cause of deficiency. Determining the cause of megaloblastic anaemia is considerably helped by a carefully taken clinical history and the clinical examination. The history often immediately reveals which of the many causes listed in these Tables should be investigated (Table 3). Gastrointestinal investigations are required for diagnosis in some cases. These include the Schilling test and food cobalamin absorption test, serological tests for coeliac disease (anti-endomysial, anti-reticulin and anti-gliadin antibodies), oesophagogastroduodenoscopy and biopsy of the gastric and duodenal mucosa for histology, CLO (Campylobacter-like organism)

Deoxyuridylate (dUMP)

Homocysteine

Methionine synthase Methylcobalamin

5-Methyltetrahydrofolate

Methionine

Tetrahydrofolate

The second is the adenosylcobalamin-dependent conversion of methylmalonyl CoA to succinyl CoA by the enzyme methylmalonyl CoA mutase. Adenosylcobalamin Methylmalonyl CoA Succinyl CoA Methylmalonyl CoA mutase

Impairment of these two reactions in B12 deficiency leads to increased plasma homocysteine and serum/plasma methylmalonic levels. Folate coenzymes transfer single carbon units during aminoacid metabolism and purine and pyrimidine synthesis. 5,10-methylenetetrahydrofolate is required for the thymidylate synthasedependent methylation of deoxyuridylate to thymidylate, a rate-limiting step in DNA synthesis.

Thymidylate synthase

5,10-Methylenetetrahydrofolate

Thymidylate (dTMP) Dihydrofolate

dTTP

DNA

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Table 3

Possible diagnoses suggested from a detailed history.

History

Details

Diagnostic possibilities

Diet

Strictly vegetarian without milk, cheese or eggs Ovo-lactovegetarian (some cases, especially in pregnancy) Generally inadequate, excessive cooking of vegetables Chronic alcohol abuse

B12 deficiency

Eating (or handling) raw fish

Neurological symptoms

Symptoms of malabsorption Associated diseases

Previous surgery Family history Drug history, exposure to chemicals

Age at presentation

a

Infants fed goat’s milk Paraesthesiae, altered gait, impaired vision due to optic neuritis or atrophy, psychiatric symptoms Diarrhoea, weight loss, steatorrhoea Autoimmune disorders: autoimmune thyroid diseasea, vitiligo, hypofunction of adrenal gland, hypoparathyroidism Zollinger-Ellison syndrome Crohn’s disease Partial or total gastrectomy, ileal resection Pernicious anaemia or other autoimmune disorders Nitrous oxide, use and abuse Metformin, aminosalicylates and others Dihydrofolate reductase inhibitors Anticonvulsants Antipurines, antipyrimidines, hydroxycarbamide, cyclophosphamide, procarbazine, arsenic Premature infant, 4–6 wks Infancy - vegan mother Infancy or childhood or life-long history of macrocytic/megaloblastic anaemia

Folate deficiency Usually folate-independent macrocytosis, occasionally folate deficiency B12 deficiency due to infestation with Diphylobothrium latum Folate deficiency Primarily in B12 deficiency, rarely in folate deficiency Folate deficiency or B12 deficiency or both Pernicious anaemia

B12 deficiency B12 deficiency B12 deficiency Pernicious anaemia Inactivation of intracellular methyl-B12 B12 deficiency Folate deficiency Usually folate-independent macrocytosis, occasionally folate deficiency B12- and folate-independent macrocytosis and megaloblastic change Folate deficiency B12 deficiency Congenital disorders of B12 transport or metabolism, folate absorption or metabolism and nucleic acid synthesis, some congenital dyserythropoietic anaemias, thiamine-responsive anaemia

Some patients with hypothyroidism have B12-and folate-independent macrocytosis.

In folate deficiency, and indirectly in B12 deficiency, this reaction is impaired leading to disturbed DNA synthesis and megaloblastic haemopoiesis.

Biochemical tests for assessing B12 status Serum B12 The assay of total serum B12 remains the first-line investigation in the assessment of B12 status. A number of rapid fully-automated B12 assays are

now used widely.1 However, unlike the microbiological and non-automated competitive protein binding assays such as the Quantaphase II radioassay (Bio-Rad) that were subjected to several thorough investigations some of the new fullyautomated assays have not been studied so well. Consequently, their sensitivity in detecting B12 deficiency has to be accepted largely on the basis of data provided by the manufacturers. With the well-studied older assays, about 3–5% of B12responsive patients had serum B12 levels within the reference range2 (usually in the lower end of

Diagnosis of megaloblastic anaemias this range). With the fully-automated assays, this percentage is likely to be at least as high so that the the sensitivity of the B12 assay would be expected to be 95–97%. Thus, the finding of a normal serum B12 level does not completely exclude the possibility of B12 deficiency. Normal or high serum B12 levels due to an increase in the concentration of transcobalamin Ibound B12 (holotranscobalamin I) may be found when B12 deficiency develops in chronic myeloid leukaemia, other chronic myeloproliferative disorders and liver disease. Normal serum B12 levels are also found in some cases of B12 deficiency due to prolonged exposure to nitrous oxide, in congenital transcobalamin II deficiency and, usually, in inborn errors of intracellular B12 metabolism. The most common causes of a high serum B12 are parenteral B12 therapy and renal failure. Studies of elderly populations have shown increases of the B12-related metabolites, serum methylmalonic acid (MMA) and/or plasma total homocysteine (tHCYS), in a substantial proportion of cases with low-normal and even high-normal B12 levels. For example, in one study 35% of subjects with low-normal B12 levels, i.e. 140–258 ng/l, and 24% of subjects with high-normal levels, i.e. >258 ng/l, had increased levels of MMA and/or tHCYS; the corresponding figures for increased MMA only were 12 and 11%.3 Therefore, some investigators have recommended that B12 status should be fully investigated below a serum B12 level that is considerably higher than the lower 95% reference limit for this vitamin. However, this approach would lead to the investigation of an unmanageably large number of individuals, most of whom would turn out not to have clinical B12 deficiency and some to have subclinical deficiency.4 As with folate assays, the reference ranges with different non-automated and fully-automated assays differ widely and the ranges quoted by the manufacturers have sometimes been found to be different from those determined in diagnostic laboratories.1 Method-specific reference ranges should therefore be established in individual diagnostic laboratories. When a low serum B12 level is found, it should ideally be confirmed by assay of a second sample before further investigations and treatment are undertaken. Low serum B12 levels are not specific for B12 deficiency but may also be found, in the absence of B12-related metabolic abnormalities, in folate deficiency, pregnancy, HIV infection, myeloma, mild or severe transcobalamin I deficiency and in patients receiving anticonvulsant drugs. When serum B12 levels are assayed, folate levels must also be assayed at the same time in order to explore the pos-

303 sibility that the primary deficiency may be of folate rather than B12. Many low serum B12 levels remain unexplained after thorough investigation; only about half are due to known causes such as food cobalamin malabsorption, pernicious anaemia and dietary deficiency. About 25% of low B12 levels in subjects over the age of 60 yrs are not associated with increased levels of serum MMA and/or plasma tHCYS, and either do not represent deficiency3 or represent the early stages of negative B12 balance (see below). 15% of all low serum B12 levels and 15% of low levels in patients without B12 malabsorption or metabolite abnormalities seem to be due to mild and, occasionally, severe TC I deficiency.5 Diagnosis of TC I deficiency is by RIA.

Serum/plasma methylmalonic acid (MMA) and plasma total homocysteine (tHCYS) Both MMA and tHCYS increase early during the development of B12 deficiency. The MMA assay is expensive, not widely available and has a long turnaround time. Provided impaired renal function is excluded elevated MMA is highly specific for B12 deficiency, being more specific than a low serum B12. Its sensitivity in detecting B12 deficiency is similar to that of a low serum B12. About 98% of clinically confirmed cases of B12 deficiency and less than 2% of confirmed cases of folate deficiency (without renal failure) have elevated MMA levels.6 tHCYS assays are cheaper and more widely available than MMA assays but have the disadvantage of poor specificity. tHCYS is increased in 96% of B12 responsive cases and 91% of folate-deficient cases6 as well as in renal failure, alcohol abuse, vitamin B6 deficiency, hypothyroidism, patients receiving certain drugs (e.g. isoniazid) and inborn errors of homocysteine metabolism. The increased MMA and tHCYS levels in B12 deficiency return to normal after treatment with B12 but not with folate.7 The proportion of elderly subjects with elevated metabolite levels is higher than that with low serum B12 levels. In one study of subjects over the age of 60 years, 12% had low serum B12, 17% had high MMA and 26% had high tHCYS levels; 35% had increases in MMA and/or tHCYS levels.3

Holotranscobalamin II (holo-TC II) Only 6–20% of the B12 in serum is bound to transcobalamin II (TC II), the transport protein involved in delivering B12 to cells; the remainder is bound to transcobalamin I (haptocorrin) whose function is

304 uncertain. Measurement of TC II-bound B12 (holoTC II or holo-TC) would be expected to provide a more reliable measure of B12 availability to tissues than total serum B12. With the initial assay methods, holo-TC II appeared to be influenced by factors other than B12 status and its specificity for B12 deficiency was low.8 Studies with the new radioimmunoassays have shown that holo-TC II is slightly more sensitive than serum B12 in detecting individuals with high MMA and tHCYS levels.9 However, they have provided conflicting data on specificity and further studies are required before the value of holo-TC II in determining B12 status is established;10–13 in one study the specificity was found to be 89%.10 In a recent investigation, Chen et al14 found that deficiency of B12 was much more important than impaired B12 absorption in determining holo-TC II levels. There was a considerable overlap between serum holo-TC II levels of treated patients with pernicious anaemia and control subjects indicating that contrary to previous suggestions holo-TC II levels cannot be used as a surrogate for the Schilling test.

Deoxyuridine suppression test (DUST) This test gives a measure of the efficiency with which marrow cells methylate deoxyuridylate to thymidylate and is abnormal in both B12 and folate deficiency.15–17 Worldwide, it is only performed in a very few laboratories. The DUST is more sensitive in detecting B12 deficiency than MMA or tHCYS levels18 but has the disadvantage that it has to be done on bone marrow cells.

Biochemical tests for assessing folate status Serum and red cell folate Serum or red cell folate assays or both are the initial tests used for the assessment of folate status. Most of the folate in serum is in the form of 5methyltetrahydrofolate monoglutamate and that in red cells in the form of various polyglutamates. The red cell folate level is a measure of folate status over the preceding three months (i.e. over one red cell lifespan). It is generally considered that the serum folate level mainly reflects current and recent folate intake and, consequently, is more useful in detecting acute folate deficiency than long-term deficiency.19,20 On the basis of highly significant statistical correlations between serum folate and red cell folate measured by microbiolog-

S.N. Wickramasinghe ical and fully-automated methods,21,22 some have suggested that the serum and red cell folate assays give similar information. Though statistically significant, the correlations are weak and the regression coefficients of 0.55 found by Jaffe & Schilling21 and 0.49 found by Phekoo et al22 indicate that only 30% and 24% respectively of the variation of the serum folate can be accounted for by variations of red cell folate. The accuracy of folate assays and particularly of fully-automated red cell folate assays is questionable. The results obtained depend on the assay method - microbiological, radioassay, fully-automated or GC-MS.1,23,24 This may arise from differences in the method of preparation of the haemolysate and in the completeness of the deconjugation of folate polyglutamates to monoglutamates. There are also difficulties in assay design resulting from the fact that the red cell-derived monoglutamates do not consist of one species of folate molecule but a mixture of folates.1 The specificity of a low red cell folate in the diagnosis of folate deficiency is poor largely because upto 60% of B12-deficient patients have low red cell folate levels.19 By contrast, serum folate levels are increased in 20% and low in only 10% of such patients. In practice, folate deficiency is assumed if a low folate level is found together with a normal B12 level. If the B12 level is also low, it is important to consider the possibility that the primary deficiency is of B12 rather than folate by considering the clinical setting, measuring MMA levels (if possible) or by performing a Schilling test. Not all patients with a low red cell folate level have metabolic evidence of folate or/and B12 deficiency. In one study with a radioassay, low red cell folate levels were found in 8 of 45 macrocytic patients who gave normal DUST results.25

Plasma total homocysteine (tHCYS) and serum/plasma methylmalonic acid (MMA) As mentioned earlier, tHCYS is elevated in both folate and B12 deficiency. The increased tHCYS levels in folate deficiency return to normal after treatment with folate but not with B12.7 MMA levels are normal in folate deficiency.

Thrapeutic trial as an investigational tool An optimal response to therapeutic doses of B12 confirms the diagnosis of B12 deficiency. A suboptimal response may indicate that the initial diagnosis

Diagnosis of megaloblastic anaemias was wrong but is more often due to co-existing iron deficiency, infection, chronic inflammatory disorder or renal failure or the intake of drugs such as cotrimoxazole. It is important to note that treatment with 5 mg or more of folic acid daily may cause an optimal haematological response in megaloblastic anaemia due to B12 deficiency but that neuropathy or haematological relapse or both develop after about 6–30 months of therapy.19,26 Thus, a response to therapeutic doses of folate cannot be taken as definitive evidence of folate deficiency. However, B12 therapy does not cause an optimal response in folate deficiency. A response to small parenteral doses of B12 (e.g. 2lg i.m. daily) or small daily parenteral doses of folic acid provides evidence of B12 or folate deficiency, respectively: the criteria of response must be carefully defined. An initial response to one vitamin followed by a second response to the other demonstrates a double deficiency. This is a lengthy approach and is now rarely undertaken.

Haematological changes in B12 and folate deficiency The availability of fully-automated machines for blood counts and serum B12 assays, allows B12 deficiency to be diagnosed much earlier than previously. In today’s practice, 25–40% of patients with cobalamin-responsive disorders have normal MCVs and a similar percentage are not anaemic.27–29 In 15–25% both the Hb and MCV are within the reference range. Therefore, B12 deficiency should not be excluded on the basis of a normal blood count or, as discussed earlier, a low-normal serum B12 level in a clinical setting suggestive of deficiency, particularly in patients with symptoms consistent with B12 neuropathy (see next section). Evidently, in cases of B12 deficiency that are diagnosed very early, both the Hb and MCV are within the reference range and somewhat later the Hb remains within the reference range but the MCV is increased. As the extent of deficiency increases, there is a progressive reduction in Hb and increase in MCV and the blood film shows increasing macrocytosis (usually with oval macrocytes) and anisopoikilocytosis. The poikilocytes consist of tear-drop-shaped and irregularly-shaped cells as well as small red cell fragments. The blood film usually contains hypersegmented neutrophil granulocytes (i.e. more than 3% of neutrophils with five or more nuclear segments) and, occasionally, also contains macropolycytes (i.e. very large neutrophils with upto 8 or more nuclear segments). Hypersegmentation of neutrophils may be seen

305 early, at a stage when both the Hb and MCV are within the reference range. In some severely anaemic patients, usually with Hb less than 7 g/dl, there are many red cell fragments resulting in an unusually mild degree of macrocytosis for the extent of anaemia. The coexistence of iron deficiency, a chronic disorder or a thalassaemia syndrome may also lead to an unexpectedly low MCV. In severe vitamin B12 deficiency there may be neutropenia, thrombocytopenia or pancytopenia. The haematological changes in folate deficiency evolve in a manner similar to that described above for B12 deficiency. In both B12 and folate deficiency, the bone marrow shows megaloblastic erythropoiesis and is hypercellular. The severity of these changes is proportional to the degree of anaemia so that nonanaemic cases may show normoblastic erythropoiesis or only mild megaloblastic changes. The M/E ratio is usually reduced but may be increased. The marrow contains giant metamyelocytes. Occasionally, some megakaryocyte nuclei are hypersegmented. Abnormal sideroblasts are present and in occasional cases there may be a few ring sideroblasts.

Clinical manifestations of B12 and folate deficiency The important difference between B12 and folate deficiency is that symptoms and signs due to peripheral neuropathy, subacute combined degeneration of the cord or focal demyelinization of the white matter of the brain are found in some cases of B12 deficiency but not usually in folate deficiency. These include paraesthesiae in the extremities, difficulty in walking, muscle weakness and impairment of memory. Less common features are poor vision due to optic neuritis or optic atrophy, psychiatric disorders (depression, cognitive impairment, mania, psychotic symptoms and obsessive compulsive disorder), stiffness of the limbs, impotence and impairment of bladder and rectal control. Neurological signs include symmetrical sensory loss in the extremities (particularly loss of position and vibration sense), ataxia, positive Romberg sign and, rarely, the features of spastic paraplegia. In B12 neuropathy, there is an inverse correlation between the severity of the neurological abnormality and the haematocrit30 so that some of the most affected patients may have a normal blood count. In a study of 141 patients with B12 neuropathy,

306 the haematocrit was within the reference range in 24%, the MCV in 18% and either the haematocrit or MCV in 28%.31 Although rare, peripheral neuropathy, optic neuropathy and subacute combined degeneration of the cord have also been reported in folate deficiency.32–36 The early case reports were published at a time when metabolite assays were not available in order to fully exclude B12 deficiency. In some of the later cases, the folate deficiency was associated with alcohol abuse or anticonvulsant drug therapy. Nevertheless, folate-responsive neuropathy appears to be a definite entity. A number of studies have reported an association between low folate levels and depression.37,38 There are several non-neurological manifestations of B12 and folate deficiency, reflecting the requirement of both these vitamins for the division of all types of cell. Manifestations include glossitis, symptoms of anaemia, gastrointestinal disturbances, loss of weight and subfertility. Ongoing work also suggests that there may be an increased risk of vascular occlusive lesions secondary to hyperhomocysteinaemia.

Vitamin B12: requirements, absorption and development of deficiency B12 is found only in bacteria and foods of animal origin. None is found in vegetables and fruit other than by virtue of bacterial contamination. The average mixed Western diet contains about 5 lg/ day. The estimated average requirement for B12 in adults, including non-pregnant women, is 2.4 lg/day and recent data suggest that it should be at least 3 lg/day in pregnant women.39 Most of the B12 in food is protein-bound and is released when the protein is subjected to acid peptic digestion in the stomach. The released B12 rapidly attaches to a B12 binding protein, Rbinder, present in salivary and gastric juice. The R binder in the B12-R binder complex is broken down in the alkaline environment of the jejunum by pancreatic trypsin and the released B12 binds to intrinsic factor produced by the parietal cells of the body and fundus of the stomach. The B12intrinsic factor complexes, which are resistant to proteolysis, travel down to the terminal ileum where they attach to specific epithelial cell receptors mediating B12 absorption: this attachment is calcium-dependent, the calcium being provided by the pancreas. B12 is absorbed very inefficiently by passive diffusion in the absence of intrinsic factor.

S.N. Wickramasinghe The total amount of B12 in the body is 3–5 mg, most of which is in liver stores. B12 is lost at a rate of 0.05–0.1% of the body content per day, mainly in the urine and faeces (in desquamated cells) and in the bile. There is an enterohepatic circulation of B12, with 3–6 lg/day excreted in bile and all but about 1lg of this reabsorbed in the terminal ileum.

Development of B12 deficiency When an individual enters a state of negative B12 balance, i.e. when the quantity of B12 absorbed daily is less than the amount lost, the B12 stores become progressively depleted. From the rate of loss of B12 mentioned above, it can be calculated that following an abrupt cessation of absorption, such as occurs after total gastrectomy, it takes several years for the B12 stores to become severely depleted and for megaloblastic anaemia or B12 neuropathy to develop. This gradual process has been divided into overlapping stages.40–42 In the early stages of negative B12 balance, the serum holotranscobalamin II (holo-TC II) concentration and % TC II saturation are reduced but the serum B12 level is normal. Later, when the B12 stores become more depleted, the serum B12 levels fall but there are no metabolic or clinical abnormalities. At the next stage, one or more metabolic abnormalities (abnormal DUST result, increases in the levels of serum MMA and/or plasma tHCYS) develop but there are still no subclinical or clinical abnormalities. This is followed by a stage in which subclinical abnormalities (mild electrophysiological neurological changes or mild haematological abnormalities without anaemia) are also present. Finally, clinical symptoms and signs resulting from mild to severe haematological or neurological abnormalities, or both, appear. Although patients with pernicious anaemia would be expected to progress through all these stages, not all patients in negative B12 balance will progress to the stage with clinical manifestations. Some may regain B12 balance and remain in any of the earlier stages. In others, a temporary phase of negative B12 balance may be followed by positive balance and restoration of normal B12 stores.

Folate: requirements, absorption and development of deficiency Folates are found in foods of both animal and vegetable origin. The average mixed Western diet contains about 400 lg folates/day. Folates are

Diagnosis of megaloblastic anaemias destroyed by heat and 30–90% may be lost during cooking. The estimated average requirement for folates in adults is 100–200 lg/day, which is two orders of magnitude greater than that for B12. The main site of absorption is the jejunum. Dietary folate polyglutamates are first hydrolysed to monoglutamates by the enzyme folate hydrolase (folate conjugase) located in the jejunal brushborder surface. The monoglutamates are absorbed via a brush-border receptor (reduced folate carrier) and converted to 5-methyltetrahydrofolate within the enterocytes before transfer to portal blood. The adult liver stores 8–20 mg folate. Folate is lost in desquamated cells, sweat and urine at a rate of about 1–2% of the total hepatic store per day; this is 10–20 times greater than the rate of loss of B12. In the steady state, this rate of loss is balanced by the daily absorption of 100–200 lg folate. Because of the larger daily requirement and higher turnover rate, folate stores may become depleted much more rapidly than B12 stores; macrocytosis due to folate deficiency may develop within 5 months of taking a a severely folate-deficient diet.

Development of folate deficiency The progression from a folate replete state to megaloblastic anaemia due to folate deficiency involves the intermediate stages of negative folate balance (low serum folate and normal red cell folate), depletion of folate stores (low red cell folate, no folate-related metabolic abnormalities, no clinical or haematological abnormalities) and development of folate-related metabolic abnormalities (high plasma tHCYS levels and abnormal DUST results) without either clinical manifestations or haematological changes other than some macrocytes and hypersegmentation of neutrophils.40

Vitamin B12-related megaloblastic anaemia Causes of B12 deficiency These are listed in Table 1. The most common causes are food cobalamin malabsorption, pernicious anaemia and dietary deficiency. Dietary deficiency The dietary history over a number of years is important in investigating the cause of megaloblastic anaemia. Strict vegetarians (vegans) who have not consumed meat, fish, eggs, cheese or milk for

307 several years frequently have low serum B12 levels. However, most vegans have normal blood counts (including MCVs) and seem to be in good health despite their reduced B12 intake and low B12 stores presumably because they have achieved a new B12 balance within these constraints, with the enterohepatic circulation of B12 efficiently conserving the losses in the bile. Nevertheless, a very high proportion of such individuals have increased serum MMA and plasma tHCYS levels and low serum holo-TC II levels.43 A lower proportion of lactovegetarians and ovo-lactovegetarians also have these abnormalities. Some vegans develop macrocytosis or megaloblastic anaemia that responds to oral or parenteral B12 and a few develop B12 neuropathy. Megaloblastic anaemia and neurological impairment including epilepsy and severe encephalopathy may develop during the first months of life in breast-fed infants of vegan mothers or of mothers with undiagnosed subclinical pernicious anaemia.44,45 People on a predominantly vegetarian diet who consume a small amount of dairy products or meat may develop low serum B12 levels when they also have a condition causing a mild abnormality of B12 absorption (e.g. food cobalamin malabsorption). Recent studies indicate that some ovo-lactovegetarians develop low serum B12 and high tHCYS levels during pregnancy.39

Impaired release of B12 from food – food cobalamin malabsorption (FCM) FCM results from a failure of adequate amounts of B12 to be released from its protein-bound state in food due to impaired acid peptic digestion in the stomach. There are considerably more patients with FCM than with pernicious anaemia. The accurate diagnosis of FCM requires that five criteria are met: a low seum B12 level, an adequate dietary B12 intake, high serum MMA and/or plasma tHCYS levels, a normal result with part I of the standard Schilling test and an abnormal result with a modification of the test employing protein-bound instead of free radiolabelled vitamin B12. 46 In the modified test, the vitamin is bound in vitro to egg yolk or chicken serum or foods such as salmon or trout. Unfortunately, the modified Schilling test (protein-bound B12 absorption test) is unavailable in most laboratories and the diagnosis has to be made without this key criterion. Assays of MMA and tHCYS are also not readily available and most haematologists have to rely only on the serum B12, dietary history and Schilling test. This leads to the overdiagnosis of FCM as about 15% of low serum B12 levels are due to partial TC I deficiency5 and

308 some are of unknown aetiology. Nevertheless, treatment with 50–100 lg cyanocobalamin orally once daily, which is effective in FCM, is unlikely to be harmful to those who are incorrectly diagnosed as having FCM by the above three criteria. About 30–40% of patients with low serum B12 levels and normal results with the conventional Schilling test have FCM.47–49 In studies employing all five of the diagnostic criteria, including the protein-bound B12 absorption test, the majority of cases of FCM had subclinical B12 deficiency, i.e. did not have clinical symptoms or signs attributable to deficiency but had metabolic evidence of deficiency. Mild elevations of MMA or tHCYS or both were usually found and most cases gave abnormal results with the DUST. In a few cases, mild B12-responsive haematological abnormalities such as slight increases in the MCV or hypersegmented neutrophils were present and occasional cases had megaloblastic anaemia.47,50–52 Neuro-electrophysiological abnormalities were found in some asymptomatic patients53 and in several patients with dementia who had FCM.54 In the 47 patients with FCM studied by Carmel et al.,47 the average age was 53 years. The B12 status in FCM usually remains more-or-less stable or deteriorates slowly. However, in some patients the gastric dysfunction eventually affects intrinsic factor secretion and pernicious anaemia develops.47,50,51,55,56 In a recent study of 92 patients over the age of 65 years (median, 76 years) in whom the diagnosis of FCM was based on a low serum B12 level, the lack of dietary deficiency and a normal conventional Schilling test result (food cobalamin absorption tests were not performed), clinical abnormalities were much more common than in previous reports on younger patients.57 Mild sensory polyneuropathy, confusion or impaired mental function and physical asthenia were found in 44.6, 22.8 and 20.7% of cases, respectively. B12-responsive haematological abnormalities were also common with anaemia, leukopenia, thrombocytopenia and pancytopenia being found in 21, 10.9, 8.7 and 6.5%, respectively. Amongst 172 elderly cases of B12 deficiency, 57% had food cobalamin malabsorption and 33% had pernicious anaemia.57 FCM is often caused by atrophic gastritis and hypochlorhydria or achlorhydria. In some patients, it is associated with and presumably caused by various other conditions, namely, chronic gastritis due to Helicobacter pylori, alcohol abuse or bacterial overgrowth in the stomach; gastric surgery (partial gastrectomy, vagotomy, gastric bypass surgery for treatment of obesity); and the long-term use of acid-suppressing drugs such as H2-receptor antago-

S.N. Wickramasinghe nists, proton pump inhibitors and gelusil. A history of one of these conditions supports the diagnosis.

Inadequate secretion of intrinsic factor Pernicious anaemia. The most common cause of impaired B12 absorption leading to clinical manifestations is pernicious anaemia (PA), which probably accounts for about 10–30% of low serum B12 levels in the elderly; the higher value was found in the very old.57,58 In PA there is an inherited tendency to develop immunologically-mediated severe atrophic gastritis or gastric atrophy leading to loss of parietal cells and, consequently, a marked reduction of intrinsic factor (IF) secretion. The normal basal secretion of IF is around 3000 units per hour and increases 3–5-fold following stimulation by histamine or gastrin. These rates provide far more than the 2000–3000 units required to absorb the daily requirement of 2–3lg B12. In PA, IF secretion is reduced to 0–250 units per hour and is unaffected by stimulants. PA is uncommon before the age of 30 and its prevalence increases with advancing age; most patients are 50–70 years old. The male:female ratio is about 1:1.5. Early studies indicated that it affects about 1 per 1000 of the population in the United Kingdom, and nearly 1% after the age of 70 years. A more recent study from Los Angeles showed that the prevalence of mild cobalamin deficiency due to undiagnosed PA in people aged 60 years or over was at least 1.9% (2.7% in women and 1.4% in men).59 About 25% of patients give a family history of pernicious anaemia and 10% have clinical or subclinical autoimmune thyroid disease. There is an increased prevalence of autoimmune diseases (thyroid diseases, vitiligo, hypoparathyroidism, and hypofunction of the adrenal glands) in both patients and their relatives and, probably, of type I diabetes in patients.19 PA may occur as part of the polyendocrinopathy syndrome and then presents in the second decade. Occasional cases of PA have hypogammaglobulinaemia or pure red cell aplasia. As assays of IF in gastric juice are no longer performed, the diagnosis of PA is based on indirect evidence. The usual way of establishing the diagnosis is by doing part I of the Schilling test, which assesses the absorption of free B12 by quantitating the appearance of radioactivity in the urine over 24 h after a small oral dose (usually 1 lg) of radiolabelled B12. The patient is given 1mg of non-radioactive B12 intramuscularly immediately before the test to saturate plasma transcobalamins and thereby increase the amount of radiolabelled B12 appearing in the urine. In PA, the urinary excretion of radiolabelled B12 is reduced indicating reduced

Diagnosis of megaloblastic anaemias absorption. When the test is repeated giving both radiolabelled B12 and IF by mouth (part II of the Schilling test), absorption is usually improved, indicating that the impaired absorption is due to IF deficiency, the most common cause of which is PA. However in some patients with PA, the absorption is not improved with a standard dose of IF because of the presence of high titres of IF antibodies in the gastric juice. The absence of improvement with IF may also indicate that the diagnosis is not PA but, for example, a condition that causes malabsorption of B12 in the terminal ileum or diversion of dietary B12 (see below). If the clinical and laboratory features are consistent with PA and the patient has no other relevant diseases, it is unnecessary to perform part II of the Schilling test. Even part I is usually required only in patients who do not have IF antibodies (see below). It is important that patients having Schilling tests are not taking oral B12 supplements or abusing alcohol at the time of the test as this will result in misleading low results. Recently, the Schilling test was unavailable for about 2 years due to a manufacturing problem. Because of this as well as the cost of the test, the failure of some patients to provide a full 24-hour urine collection and the large number of patients who are now being discovered to have low serum B12 levels, alternative approaches for diagnosing PA were revisited. Measurement of serum levels of pepsinogen (PG) I, gastrin and IF antibodies are all informative: of these IF antibodies are the most specific and only IF antibody tests are widely available. All patients in whom the diagnosis of PA is considered should have serum IF antibodies measured. IF antibodies are present in 50–70% of cases of PA and their presence is highly specific for this disorder. IF antibodies in the absence of evidence of PA are rare in control subjects (around 0.3%) but have been found in some patients with Graves disease and insulin-dependent diabetes and, rarely, in hypothyroidism, atrophic gastritis and relatives of patients with PA.19 There are two types of IF antibodies: type I antibodies that block the B12-binding site of the IF molecule and type II antibodies that block the site involved in the attachment of the B12-IF complex to the ileal receptor. IF antibodies can be measured by RIA methods that detect type I antibodies or by ELISA methods that detect both type I and type II antibodies. It is important that when RIA methods are used, samples are not taken within a few days of a B12 injection as sera with B12 levels greater than 3,500–5,000 ng/l may give false positive results. When IF antibodies are unequivocally present, the diagnosis of PA can be made with

309 a high degree of certainty and a Schilling test is usually unnecessary. Low serum PG I levels (<30 lg/l) are found in about 90% of cases of PA but are less specific than IF antibodies, with a specificity of 92–95%;60,61 low levels may be seen in moderate or severe atrophic gastritis without PA, carcinoma of the stomach and after Roux-en-y gastric bypass surgery. High serum gastrin levels (> 200 ng/l) are seen in about 90% of patients with PA but are less specific than low serum PG I levels;60 moderate elevations may be found in patients with gastric ulcer, chronic renal failure, rheumatoid arthritis, vitiligo, hyperparathyroidism, pyloric obstruction, carcinoma of stomach, vagotomy without gastric resection, gastric surgery with retained antrum and ZollingerEllison syndrome. The absence of a low PG I or high gastrin level suggests that the diagnosis may not be PA. Gastric parietal cell antibodies have been reported in 50–90% of patients with PA. This wide range may result from the small numbers of cases studied and in differences in assay methods. The prevalence in the largest series (324 cases) was 55%.62 Parietal cell antibodies are unhelpful in establishing the diagnosis of PA as they are found in the absence of IF deficiency in 20–40% of other groups of individuals, namely, patients with autoimmune thyroid disease, relatives of cases of PA and thyroid disease, patients with Addison’s disease of the adrenal gland and cases of iron deficiency anaemia as well as in 16% of people over the age of 60 years. Total and partial gastrectomy. All patients subjected to total gastrectomy develop megaloblastic anaemia due to B12 deficiency 2–10 years after surgery, this being the time taken for normal B12 stores to become exhausted after B12 absorption ceases after surgery:19 serum B12 levels begin to fall during the first year. Neuropathy develops in some cases. After partial gastrectomy, B12 deficiency develops in 5% of cases usually 5 years or more after surgery.19 Deficiency is often primarily due to FCM resulting from a reduction of gastric acid and pepsin.46 It is sometimes due to a lack of intrinsic factor as a consequence of the removal of a substantial amount of intrinsic-factor-secreting mucosa at surgery and the subsequent atrophy of the remainder of the gastric mucosa. In a few cases, and especially when the surgery creates blind loops (e.g. during a Polya gastrectomy), the B12 deficiency is a consequence of the development of an abnormal intestinal bacterial flora. Congenital intrinsic factor deficiency. This rare disorder with autosomal recessive inheritance is

310 characterized by megaloblastic anaemia and B12 neuropathy due to absence of IF production, the presence of hydrochloric acid and pepsin in gastric juice, a histologically-normal gastric mucosa and the absence of IF antibodies. The age at presentation varies from infancy to adulthood suggesting that its expression is affected by factors that remain to be identified. The Schilling test gives results similar to those in pernicious anaemia and the diagnosis is based on examination of gastric juice and the demonstration of the absence of IF by radioimmunoassay.63,64 In a recent study of 5 cases, a genetic polymorphism in the coding region of the gastric intrinsic factor gene (GIF) was associated with the disease65 and in another study homozygous nonsense and missense mutations in the GIF gene were found in 7 families.66 Congenitally abnormal intrinsic factor. There is a report of a 13-year-old child with megaloblastic anaemia due to a structurally abnormal IF that had a markedly decreased affinity for ileal IF-B12 receptors.67 In another family, three siblings presented in their second year with megaloblastic anaemia due to the production of an abnormal IF.68 Impaired release of B12 from B12-R binder complex Disorders affecting exocrine pancreatic function. A proportion of patients with chronic pancreatitis or after pancreatectomy give abnormal Schilling test results and some have low serum B12 levels. Absorption of B12 is decreased mainly because the impaired secretion of pancreatic trypsin leads to reduced degradation of vitamin B12-R binder complexes and, consequently, reduced availability of B12 for binding to IF. Rarely, megaloblastic anaemia develops. Zollinger-Ellison syndrome (gastrinoma of pancreas/duodenum). The impaired absorption of B12 that may occasionally occur in this syndrome is often caused by long-term therapy with proton pump inhibitors leading to achlorhydria and FCM. In untreated patients, the excessively produced acid may inactivate pancreatic trypsin leading to impaired release of the vitamin from B12-R binder complexes. Diversion of dietary B12 Dietary B12 may be converted into inactive cobamides by abnormal bacterial flora that develop in the proximal part of the small intestine when there is intestinal stasis. B12 deficiency by this mechanism may be seen in patients with multiple jejunal diverticula, small-intestinal strictures, systemic sclerosis affecting the intestine, and stagnant

S.N. Wickramasinghe intestinal loops resulting either from gastrointestinal surgery or from fistulae complicating regional iletis or tuberculosis. In affected patients, Part I of the standard Schilling test gives an abnormal result and this is not improved in Part II of the test. The diagnosis is confirmed by considerable improvement of B12 absorption (in Part I of the test) after a course of broad-spectrum antibiotics. The fish tapeworm (Diphyllobothrium latum)may cause B12 deficiency by extracting B12 from food.19 This infestation is contracted by eating raw or undercooked fresh water fish (containing a larval form) from the lakes of Finland, the Baltic States, Northern Italy, Switzerland, Germany, Rumania, the Soviet Union, Japan and North America. Only a few percent of infested subjects, almost exclusively from Finland, the Baltic States and the Soviet Union, developed B12 deficiency. There are no recent case reports of B12 deficiency due to this tapeworm. However, several cases of diphyllobothriasis are still detected each year in Finland, Sweden and the French and Italian speaking areas of the subalpine lakes and there have been sporadic cases in Austria, Spain, Greece, Rumania, Poland and Norway.69 Diphyllobothriasis has also been reported in previously unaffected areas such as Korea, Brazil and Argentina.

Malabsorption due to abnormalities of the terminal ileum Crohn’s disease, chronic tropical sprue, ileal resection and other acquired diseases. Since B12 is absorbed in the terminal ileum, deficiency may develop in diseases affecting this region of the ileum such as Crohn’s disease and chronic tropical sprue or after resections of more than about 60 cm of the terminal ileum. The extent of impairment of B12 absorption is proportional to the extent of pathological involvement or resection of the terminal ileum. Between 60 and 90% of patients with chronic tropical sprue have megaloblastic anaemia; about 90% malabsorb B12 and many also malabsorb folate. In all of the above situations, the Schilling test shows reduced absorption of B12 and no correction with intrinsic factor. In tropical sprue, the absorption of B12 in Part I of the Schilling test frequently returns to normal after a course of broadspectrum antibiotics. Impaired B12 absorption has been found in about 30% of patients with coeliac disease, after total body irradiation or ileal irradiation (e.g. during radiotherapy to the cervix), in small intestinal lymphomas, graft-versus-host disease, giardiasis, folate deficiency and Whipples disease (intestinal lipodystrophy), and when the terminal ileum is

Diagnosis of megaloblastic anaemias affected by amyloidosis, tuberculosis or scleroderma. B12 deficiency itself may cause some impairment of B12 absorption as evidenced by an improvement in absorption after B12 therapy. Selective malabsorption of B12 with proteinuria ¨sbeck syndrome). This autosomal (Imerslund-Gra recessive disorder is characterised by megaloblastic anaemia resulting from malabsorption of B12 due to a defect in the receptor for IF-B12 complexes and, in over 95% of cases, proteinuria. Patients usually present between the ages of 1 and 5 years but some have presented between 5 and 16 years; a high proportion of the reported cases are from Finland, Norway and the Middle East. The IF content of gastric juice is normal as is the histology of the stomach and terminal ileum. The Schilling test shows markedly reduced absorption and no improvement with IF. The disease usually results from mutations in one of two genes that encode components of the receptor for the IF-B12 complex, namely, cubulin (CUBN) and amnionless (AMN).70,71 In some families, the disease is not linked to either of these genes.71 The anaemia responds to parenteral B12 but the proteinuria persists. In children with megaloblastic anaemia, the diagnosis of Imerslund-Gra ¨sbeck syndrome should be considered by testing the urine for protein. Mutational analysis of the CUBN and AMN genes can be used to confirm the diagnosis in many cases. Drug-induced malabsorption Aminosalicylates, neomycin, colchicine, slow-release potassium chloride, metformin, phenformin, biguanides and cholestyramine may impair B12 absorption.19 Rarely, patients who have received prolonged treatment with aminosalicylates or metformin have developed megaloblastic anaemia. Complex or uncertain mechanism HIV infection. Low serum B12 levels occur in upto one-third of all HIV-positive cases and may be found even at the early stages. Some HIV-positive patients have increased tHCYS or MMA levels, give abnormal results in the DUST or show B12 malabsorption with the Schilling test but these abnormalities correlate poorly with a low B12 level.72–75 The low B12 levels in HIV-positive patients appear to have a multifactorial basis, including a decrease in the level of TC I,75 a reduction in the output of gastric acid (leading to FCM) or of acid and IF, and HIV-related enteropathy affecting the terminal ileum.76 Inactivation of vitamin B12 by nitrous oxide Nitrous oxide (N2O) irreversibly inactivates methylcobalamin-dependent methionine synthase by

311 oxidising the enzyme-bound cob(I)alamin form of methylcobalamin to the inactive cob(II)alamin form with the generation of damaging OH radicals and nitrogen.77 Chronic abuse of N2O by healthcare workers (mainly dentists and anaesthetists) and others (e.g. those with access to whipped cream dispensers) and prolonged intermittent exposure for analgesia may lead to B12 neuropathy and megaloblastic anaemia.78–81 The serum B12 level is usually low but may be normal and the Schilling test gives a normal result. Patients with pre-existing mild B12 deficiency may be especially prone to rapidly develop N2O-induced neuropathy.

Inherited abnormalities of B12 transport or intracellular B12 metabolism Congenital transcobalamin II (TC II) deficiency. In this rare autosomal recessive disorder, patients usually present within the first few weeks or months of birth with severe megaloblastic anaemia, leucopenia, thrombocytopenia, failure to thrive and vomiting.82–84 Some cases also have recurrent infections due to hypogammaglobulinaemia, leucopenia and granulocyte dysfunction, bizarre red cell morphology or erythroid hypoplasia. Neurological symptoms (impaired cognitive development, epilepsy, abnormalities of gait) are generally seen only when treatment with B12 is inadequate or delayed. As most of the B12 in serum is bound to TC I, serum B12 levels are usually normal and the diagnosis requires measurement of total serum TC II levels. The unsaturated B12-binding capacity of the serum, which is normally largely dependent on the presence of apotranscobalamin II, is greatly reduced. The Schilling test shows decreased absorption of B12 that is not corrected by IF, indicating that TC II is involved in B12 absorption. Some patients show a complete absence of TC II and a few produce a functionally abnormal TC II or are doubly heterozygous for the absence of TC II and an abnormal TC II molecule.85–87 In a few cases, the disorder is caused by homozygosity for various mutations in the TC II gene.88 Congenital abnormalities of intracellular B12 metabolism. Infants and children affected by some of these disorders do not present with megaloblastic anaemia but with metabolic acidosis, feeding difficulties, failure to thrive or various neurological or psychiatric symptoms. Megaloblastic anaemia is only seen in patients with defective methylcobalamin synthesis. The important biochemical abnormalities that point to an inborn error of intracellular B12 metabolism are markedly increased plasma tHCYS or serum MMA levels or both. The serum B12 level is usually normal. Using

312 cultured fibroblasts from patients and the technique of complementation analysis, a number of complementation groups have been identified, and of these cblC-cblG commonly have megaloblastic anaemia. (1) Defective methylcobalamin synthesis (hyperhomocysteinaemia). This results from impaired methylation of cob(I)alamin on its apoenzyme due to mutations in methionine synthase (cblG) or from mutations in methionine synthase reductase which is required for the reduction of cobalamin to cob(I)alamin prior to its methylation (cblE).4,64,89 Most patients present before the age of 2 years with megaloblastic anaemia and various neurological abnormalities but the diagnosis has sometimes been made in adults. They have normal serum B12 and folate levels, hyperhomocysteinaemia, homocysteinuria, hypomethioninaemia and no methylmalonic acidaemia. (2) Defective synthesis of both methylcobalamin and adenosylcobalamin. Complementation analysis has shown three groups of patients with different primary defects (cblC, cblD and cblF) who have both hyperhomocysteinaemia due to a failure to synthesise methylcobalamin as well as methylmalonic acidaemia due to a failure to synthesise adenosylcobalamin, the coenzyme for methylmalonyl CoA mutase responsible for converting methylmalonyl CoA to succinyl CoA.4,64,89 Most cases belong to the cblC group and the majority of these cases present in infancy with lethargy, feeding difficulties and failure to thrive. Others present in childhood or adolescence with neurological symptoms such as spasticity, psychosis or a retinopathy with perimacular pigmentation. Megaloblastic anaemia is frequently present and the DUST reveals impaired methylation of deoxyuridylate irrespective of whether erythropoiesis is megaloblastic or normoblastic.

Folate-related megaloblastic anaemia Causes of folate deficiency These are listed in Table 1. In many countries, the most common cause is dietary deficiency. Dietary deficiency Megaloblastic anaemia due to dietary folate deficiency may be seen in those who chronically abuse

S.N. Wickramasinghe alcohol, in the poor, the neglected elderly and the mentally abnormal and in some cases of proteinenergy malnutrition. In the USA and Canada, the introduction in 1998 of mandatory fortification of breads, cereals, flours, corn meals, pastas, rice, and other grain products with folate has resulted in a marked reduction of the prevalence of folate deficiency, and B12 deficiency is now the main determinant of nutritional hyperhomocysteinaemia in these countries.90 As goat’s milk only contains 12% of the folate in cow’s milk, an important cause of folate deficiency anaemia in infants is the almost exclusive use of goats milk instead of cow’s milk; this condition has been described as ‘‘goat’s milk anaemia’’. Reduced intake of folate contributes to the folate deficiency that may develop following gastric surgery, during prolonged illnesses and in some epileptics on anticonvulsant drugs. Malabsorption Because folate is absorbed mainly in the jejunum, coeliac disease and tropical sprue, both of which predominantly affect the upper part of the small intestine, often cause folate deficiency. Impaired folate absorption may also be seen following partial gastrectomy or jejunal resection, when the upper small intestine is affected by Crohn’s disease and in patients taking sulfasalazine (a non-competitive inhibitor of the reduced folate carrier) or abusing alcohol (see below). Increased requirement or loss There is an increased requirement for folate during pregnancy (because of the needs of the growing fetus), as well as in a number of other situations. The increased requirement may lead to folate deficiency especially in individuals with a borderline dietary folate intake. Megaloblastic anaemia due to folate deficiency usually develops after the thirty-sixth week of pregnancy, around the time of delivery or early in the postpartum period. Because of folate supplementation before conception and during pregnancy, the prevalence of megaloblastic anaemia associated with pregnancy in the developed world is low. There is an increased prevalence in twin pregnancies. In poor countries, where dietary intake is inadequate and folate supplements are not taken during pregnancy, the prevalence of megaloblastic haemopoiesis may be upto 50%. The newborn requires 10 times the amount of folate per day (per unit weight) when compared with an adult. Premature babies have an even higher folate requirement because of the rapid

Diagnosis of megaloblastic anaemias growth during the first 2–3 months and may develop megaloblastic anaemia at 4–6 weeks of age. Other situations in which there is an increased folate requirement because of increased cell proliferation and in which folate deficiency may develop include chronic haemolytic anaemias, idiopathic myelofibrosis, malignant diseases (e.g. leukaemia, lymphoma, myeloma and carcinoma) and chronic inflammatory disorders such as rheumatoid arthritis. Increased loss of folate from the body may cause folate deficiency or, more likely, contribute to its development. Excessive loss may result from the increased desquamation of cells in psoriasis and exfoliative dermatitis. Because folates are only loosely bound to plasma proteins, there is some loss of folate during long-term haemodialysis or peritoneal dialysis. Acquired abnormality of folate metabolism: treatment with dihydrofolate reductase inhibitors Megaloblastic anaemia or megaloblastic haemopoiesis may develop in patients receiving dihydrofolate reductase inhibitors such as methotrexate, pyrimethamine and triamterene.16,91 The megaloblastic change results from an impairment of the regeneration of 5,10-methylenetetrahydrofolate from dihydrofolate and, consequently, a reduction of the rate of methylation of deoxyuridylate to thymidylate. Trimethoprim is a weak inhibitor of dihydrofolate reductase. With conventional doses it causes megaloblastic haemopoiesis only in patients with a pre-existing mild B12 or folate deficiency. Complex or uncertain mechanism Anticonvulsant therapy and ethanol abuse. The macrocytosis associated with anticonvulsant therapy16,92 or chronic alcohol abuse16,19 is not usually due to folate deficiency. However some patients develop folate deficiency, mainly as a consequence of a poor diet. Malabsorption of folate and the induction of enzymes involved in folate catabolism may also play a role. Alcohol probably inhibits brush-border folate hydrolase. Oral contraceptive drugs, glutethimide and cycloserine. Cases of megaloblastic anaemia associated with long-term oral contraceptive therapy in which other causes of megaloblastic change appear to have been fully excluded are rare. There is some evidence that oral contraceptives may precipitate folate deficiency in individuals whose folate intake is borderline or who have other conditions causing negative folate balance but no

313 good evidence that they have a significant affect on folate replete subjects.93,94 By contrast, oral contraceptives may decrease serum B12 levels without associated metabolic disturbances by reducing TC I.95 Glutethimide and cycloserine have also been reported to cause folate deficiency. Congenital disorders of folate absorption and metabolism There are three well-documented inborn errors of folate absorption and metabolism - hereditary folate malabsorption, severe methylenetetrahydrofolate reductase (MTHFR) deficiency (the most common of the three) and glutamate formiminotransferase-cyclodeaminase deficiency.4,64,96 Megaloblastic anaemia develops in hereditary folate malabsorption and the severe phenotype of glutamate formiminotransferase deficiency but not in severe MTHFR deficiency in which there is no shortage of methylenetetrahydrofolate (MTHF), the methyl donor for the thymidylate synthasedependent methylation of deoxyuridylate to thymidylate. Severe MTHFR deficiency is an autosomal recessive disorder due to mutations in the MTHFR gene. MTHFR catalyses the conversion of MTHF to methyltetrahydrofolate required for the methylcobalamin-dependent methylation of homocysteine to methionine. Patients usually present early in infancy with severe developmental delay, marked hypotonia, seizures, breathing disorders and coma, and sometimes present in childhood with abnormalities of gait. Arterial and venous thrombosis may occur. The diagnosis has occasionally been made in adults. Despite the absence of megaloblastic anaemia, they have low serum, red cell and CSF folate levels. Their serum B12 levels are normal. As in cblE and cblG, there is hyperhomocysteinaemia, homocysteinuria and reduced plasma methionine levels but the presence of megaloblastic anaemia in cblE and cblG distinguishes them from severe MTHFR deficiency. Hereditary folate malabsorption is a disorder of intestinal folate absorption and of folate transport across the blood-brain barrier. Patients present in the first few months of life. Clinical features have varied in different cases and include megaloblastic anaemia, pancytopenia, mental retardation, convulsions, ataxia, athetosis, peripheral neuropathy, vomiting, diarrhoea, mouth ulcers, infections and failure to thrive. Serum, red cell and CSF folate are very low. The diagnosis can be confirmed by folate absorption studies. Glutamate formiminotransferase-cyclodeaminase (FTCD) deficiency is the second most common inherited disorder of folate metabolism. FTCD is a

314 bifunctional enzyme that channels 1-carbon units from formiminoglutamate to the folate pool. FTCD deficiency is an autosomal recessive disorder which presents either with a severe or a mild phenotype. The severe phenotype is characterised by increased formiminoglutamic acid (FIGLU) in blood and urine after histidine loading, megaloblastic anaemia and mental retardation. In the mild phenotype, there is high urinary excretion of FIGLU in the absence of histidine administration and mild developmental delay but no megaloblastic anaemia. In glutamate formiminotransferase deficiency the serum folate is high or normal. Three cases with the mild phenotype were compound heterozygotes for mutations in the FTCD gene.97

Vitamin B12-independent and folateindependent causes of megaloblastic erythropoiesis Abnormalities of nucleic acid synthesis Drug-induced impairment of DNA synthesis. A number of drugs cause macrocytosis with megaloblastic erythropoiesis by impairing DNA synthesis.16,91 These include the purine analogues mercaptopurine, thioguanine, azathioprine and acyclovir; the pyrimidine analogues 5-fluorouracil (which inhibits thymidylate synthase), 5-azacytidine, zidovudine and cytarabine; and an inhibitor of ribonucleotide reductase, hydroxycarbamide. Other drugs that cause megaloblastic changes by vitamin B12-independent and folate-independent interference with DNA synthesis include cyclophosphamide and procarbazine. Arsenic poisoning also causes megaloblastic changes. Hereditary orotic aciduria (uridine monophosphate synthase deficiency). The features of this rare autosomal recessive disorder of pyrimidine metabolism are severe megaloblastic anaemia, failure to thrive, the excretion of large quantities (0.5–1.5 g/d) of orotic acid in the urine and, in some cases, impaired cellular immunity.98,99 Patients present between the ages of 3 months and 7 years. They have greatly reduced activity of two enzymes catalyzing the last two steps of de novo pyrimidine biosynthesis, namely orotate phosphoribosyltransferase (OPRT) and orotidine-50 monophosphate decarboxylase (ODC), resulting in impaired conversion of orotic acid to uridine monophosphate, a precursor of the pyrimidine bases of DNA. This reduced enzyme activity has been shown in one Japanese case to be caused by mutations in the uridine monophosphate synthase gene which codes for a bifunctional enzyme with OPRT and ODC activity in different domains of the mole-

S.N. Wickramasinghe cule.100 The serum B12 and red cell folate levels are normal and the diagnosis requires measurement of orotic acid in the urine.101 There is no response to vitamin B12 or folate therapy but the anaemia and failure of growth respond to the daily administration of uridine. Lesch-Nyhan syndrome. The severe form of this X-linked syndrome, Lesch-Nyhan disease, is characterised by mental retardation, choreoathetosis, spastic cerebral palsy, self-mutilation (especially biting of the lips and fingers), hyperuricaemia and gout. The hyperuricaemia leads to renal damage and renal failure.102,103 Some cases have megaloblastic anaemia that responds to adenine.104 There may also be an increased susceptibility to infection due to defective function of B-lymphocytes. LeschNyhan disease is caused by a virtually complete deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) involved in purine salvage leading to hyperactivity of the de novo purine synthetic pathway with overproduction of purine and, consequently, uric acid.103 Over 300 mutations in the HPRT gene have been described that cause both the disease and its less severe variant in which there is only a partial HPRT deficiency.103,105

Complex or unknown mechanism Anticonvulsant therapy. Anticonvulsant drugs (phenytoin, phenobarbital, primidone) may cause megaloblastic changes by a folate-independent impairment of DNA synthesis and erythroblast proliferation.16,19 Chronic alcoholism. Patients who chronically abuse alcohol often have macrocytosis and mild megaloblastic changes without evidence of folate deficiency. In such patients the serum, red cell and hepatic folate levels are normal and there is no abnormality in the methylation of deoxyuridylate as judged by the DUST.16,19 It is possible that erythroblasts are damaged by acetaldehyde generated from ethanol by bone marrow macrophages106 or by acetaldehyde in circulating acetaldehydealbumin complexes.107,108 Protein-energy malnutrition (PEM). In some cases of PEM with normal red cell folate and serum B12 levels, megaloblasts and giant metamyelocytes are found in the marrow and the DUST indicates an impairment of the methylation of deoxyuridylate that is not related to folate or B12 deficiency. The bone marrow and DUST abnormalities may be a consequence of the protein deficiency.17 Myelodysplastic syndromes and erythroleukaemia. In some patients with these disorders, folate-independent megaloblastic changes are a

Diagnosis of megaloblastic anaemias feature of the neoplastic erythroblasts.16 However, because of an increased requirement for folate, secondary folate deficiency may develop. Congenital dyserythropoietic anaemias. Patients with congenital dyserythropoietic anaemia types I and III usually have a macrocytic anaemia. Their bone marrow smears show the dysplastic features that define these two conditions and display megaloblastic erythropoiesis.109 Thiamine-responsive megaloblastic anaemia syndrome. The features of this rare autosomal recessive disorder are megaloblastic anaemia, progressive bilateral sensorineural deafness and diabetes mellitus; the megaloblastic anaemia does not respond to vitamin B12, folate or pyridoxine. Some cases also have sideroblastic erythropoiesis.110 The anaemia, and sometimes the diabetes and deafness, respond to pharmacological doses of thiamine given orally. The disease gene, SLC19A2, has been localised to chromosome 1q23.3 and mutations were found in this gene in a few affected families. The gene product may be a thiamine transporter protein.111–113

Practice points  Although several tests are available for assessing B12 and folate status, each of them has its limitations. A full blood count, blood film and assay of serum B12 and red cell (or serum) folate remain the most useful initial investigations.  About 30% of patients with B12-responsive disorders have normal MCVs, 30% are not anaemic and 20% are neither anaemic nor macrocytic.  3–5% of cases with confirmed B12 deficiency have serum B12 levels within the reference range.  Neither a low serum B12 level nor a low red cell folate level are specific for B12 or folate deficiency, respectively.  30–40% of low serum B12 levels are caused by food cobalamin malabsorption (FCM); the percentage may be higher in the elderly.  Most cases of FCM have more-or-less stable subclinical B12 deficiency but some have clinical manifestations. Some cases with subclinical deficiency progress to develop pernicious anaemia.  High serum/plasma methylmalonic acid (MMA) levels are found in B12 but not folate deficiency and high plasma total homocyste-

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ine (tHCYS) levels are found in both B12 and folate deficiency. In the absence of renal failure, high MMA levels are highly specific for B12 deficiency but high tHCYS levels have poor specificity for either deficiency. About 25% of low serum B12 levels are unassociated with elevations of MMA or tHCYS or abnormal DUST results and either do not indicate deficiency or indicate early negative B12 balance. 15% of low serum B12 levels are due to partial deficiency of transcobalamin I. Assay of MMA and tHCYS are useful in defining subclinical deficiency and are essential in the investigation of megaloblastic anaemia due to some inborn errors of metabolism. Only between 10 and 30% (depending on age) of low serum B12 levels are associated with clinical manifestations of B12 deficiency, often caused by pernicious anaemia (PA). In B12 neuropathy, there is an inverse correlation between the haemoglobin level and the severity of neurological damage. Some patients with neuropathy have a normal blood count. Serum intrinsic factor antibodies are highly specific for PA but are only found in 50–70% of cases. Serum holo-transcobalamin II levels cannot be used as a surrogate for the Schilling test. In infants and children, megaloblastic anaemia is usually caused by nutritional deficiency. It may rarely be caused by some inherited abnormalities of B12 transport, B12 or folate absorption or B12, folate or nucleic acid metabolism.

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