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Megaloblastic anaemia and miscellaneous deficiency anaemias 

Megaloblastic anaemia and miscellaneous deficiency anaemias

Chapter:
Megaloblastic anaemia and miscellaneous deficiency anaemias
Author(s):

A.V. Hoffbrand

and Drew Provan

DOI:
10.1093/med/9780199204854.003.220506_update_002

Update:

Discussion of new meta-analyses concerning effects of folate deficiency and folate supplementation on cognitive function/dementia, cardiovascular disease and cancer.

Updated on 29 May 2014. The previous version of this content can be found here.
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Essentials

Megaloblastic anaemias are characterized by red blood cell macrocytosis. They arise because of inhibition of DNA synthesis in the bone marrow, usually due to deficiency of one or other of vitamin B12 (cobalamin) or folate, but sometimes as a consequence of a drug or a congenital or acquired biochemical defect that disturbs their metabolism, or affects DNA synthesis independent of vitamin B12 or folate.

Biochemical and nutritional aspects of vitamin B12 and folate

Vitamin B12—synthesized by bacteria; in humans the daily requirement of 1 to 2 μ‎g is acquired from secondary animal sources including fish, eggs, milk, and meat. Processing within the body occurs as follows: (1) proteolysis of food releases dietary vitamin B12 for binding to a glycoprotein haptocorrin; (2) pancreatic trypsin degrades the glycoprotein, releasing vitamin B12 for attachment to intrinsic factor; (3) the B12–intrinsic factor complex binds to a specific receptor—cubilin amnion—expressed on the luminal brush border of the mucosal cells of the ileum, and is endocytosed; (4) after lysosomal degradation, vitamin B12 is complexed with transcobalamin (TC)II and secreted into the circulation; (5) the TCII–B12 complex is incorporated by cellular endocytosis in peripheral tissues and vitamin B12 released by digestion in the lysosomal compartment.

Folate—occurs principally in leaves and vegetables, but is destroyed by cooking. The daily requirement is about 100 μ‎g, with absorption occurring through a proton-coupled folate transporter in the proximal small intestine and duodenum. Attached glutamate residues are cleaved, releasing methyl tetrahydrofolate into the portal plasma.

Biochemical basis of megaloblastic anaemia—(1) Folate deficiency reduces the availability of 5,10-methylene tetrahydrofolate, thus inhibiting synthesis of thymidylate, which is the rate-limiting precursor for DNA synthesis. (2) Vitamin B12 deficiency impairs DNA synthesis indirectly because it is an acceptor for single-carbon moieties required for conversion of methyltetrahydrofolate to tetrahydrofolate, the source of active folate co-enzymes (folate polyglutamates). It also appears to be critical for function of the enzyme methionine synthase, inhibition of which appears to be the principal cause of the neuropathy and spinal cord disease characteristic of severe vitamin B12 deficiency

Causes of megaloblastic anaemia

Vitamin B12 deficiency—(1) malabsorption—including (a) gastric causes—e.g. acquired (addisonian) pernicious anaemia, gastrectomy; (b) intestinal causes—e.g. bacterial overgrowth, ileal resection, Crohn’s disease; (2) nutritional—e.g. vegans.

Folate deficiency—(1) poor diet—e.g. poverty, alcoholism; (2) malabsorption—e.g. gluten-induced enteropathy, tropical sprue; (3) excessive requirements—e.g. pregnancy, haemolytic anaemia; (4) excess excretion—e.g. chronic haemodialysis; (5) drugs—e.g. anticonvulsants; (6) liver disease.

Not due to vitamin B12 or folate deficiency—(1) abnormalities of vitamin B12 or folate metabolism—including (a) congenital—e.g. TCII deficiency; (b) acquired—e.g. dihydrofolate reductase inhibitors; (2) independent of vitamin B12 or folate—including (a) congenital—e.g. orotic aciduria; (b) acquired—e.g. various myeloid leukaemias; (c) drugs—e.g. antimetabolites.

Laboratory investigation

This consists of three stages: (1) Recognition that megaloblastic anaemia is present—the mean corpuscle volume (MCV) is raised to 100 to 140 fl, and the peripheral blood shows hypersegmented neutrophils, with the leucocyte count often moderately reduced. The bone marrow is hypercellular, with megaloblastic erythroblasts and giant metamyelocytes. (2) Distinction between vitamin B12 or folate deficiency (or rarely some other factor) as the cause of the anaemia—usually achieved by assay of serum vitamin B12 and serum folate. (3) Diagnosis of the underlying disease causing the deficiency—depends on taking a dietary history, measurement of parietal-cell and intrinsic factor antibodies and serum gastrin (see below), transglutaminase antibody and pursuing clinical clues to other possible causes.

Acquired (addisonian) pernicious anaemia

Antibodies in serum and gastric juice directed against parietal cells (85–90% of cases) and intrinsic factor (50%), and raised serum gastrin are associated with gastritis and failure of absorption of vitamin B12.

Clinical features—anaemia usually develops gradually, and symptoms may not occur until it is severe. Aside from pallor, other manifestations can include (1) mild jaundice; (2) mild pyrexia; (3) psychiatric disturbance; (4) glossitis and angular cheilosis; (5) features of an associated disorder—e.g. vitiligo, thyroid disease. Complications include (1) peripheral sensorimotor neuropathy; (2) subacute combined degeneration of the spinal cord—manifest as loss of proprioception and pyramidal weakness; (3) psychiatric disturbance.

Treatment and prevention of megaloblastic anaemia

Vitamin B12 deficiency—may be treated with intramuscular hydroxocobalamin (1-mg doses, given frequently at initiation of treatment, then every 3 months) or (provided malabsorption is not responsible) oral hydroxocobalamin. Neurological complications are irreversible unless treated early.

Folate deficiency—high-dose oral folic acid (5 mg daily) overcomes folate malabsorption, but this should not be given alone where vitamin B12 deficiency coexists because neurological disease may be precipitated or exacerbated (although the haematological abnormalities improve). Where folate metabolism is disturbed, oral or parenteral folinic acid may restore pyrimidine synthesis.

Prevention—the role of dietary folate supplementation is an accepted and highly effective public health measure in many countries for preventing neural tube birth defects.

Introduction

The megaloblastic anaemias are a group of disorders characterized by a macrocytic anaemia and distinctive morphological abnormalities of the developing haemopoietic cells in the bone marrow. In severe cases, the anaemia may be associated with leucopenia and thrombocytopenia. Megaloblastic anaemia arises because of inhibition of DNA synthesis in the bone marrow, usually due to deficiency of one or other of two water-soluble B vitamins: vitamin B12 (B12, cobalamin) or folate. Vitamin B12 deficiency may also cause a severe neuropathy. In a minority of cases, megaloblastic anaemia arises because of a disturbance of DNA synthesis due to a drug or a congenital or acquired biochemical defect that causes a disturbance of vitamin B12 or folate metabolism or affects DNA synthesis independent of vitamin B12 or folate. Vitamin B12 and folate are discussed first and the other rare megaloblastic anaemias are mentioned later in this chapter.

Folic acid supplements and food fortification with folic acid are aimed at preventing neural tube defects. Possible relations between folate and vitamin B12, and cardiovascular or malignant diseases and cognitive defects in older people are also discussed.

Biochemical and nutritional aspects of vitamin B12 and folate

Vitamin B12

Biochemistry

Four major forms of the vitamin exist in humans, all with the same cobalamin nucleus, which consists of a planar corrin ring (hence the term ‘corrinoids’ for vitamin B12 compounds) attached at right angles to a nucleotide portion, 5,6-dimethylbenzimidazole joined to ribose-phosphate (Fig. 22.5.6.1; Table 22.5.6.1). 5′‎-Deoxyadenosyclobalamin (adocobalamin) accounts for about 80% of vitamin B12 inside mammalian cells and is located mainly in mitochondria; methylcobalamin is a minor cellular component but the main form in plasma. Both are extremely light-sensitive and are rapidly photolysed to hydroxocobalamin by daylight; hydroxocobalamin is present in small amounts in tissues and plasma and is available commercially for therapeutic use. The fourth form, cyanocobalamin, is found only in trace amounts naturally, but is stable and used therapeutically. Hydroxo- and cyanocobalamins are converted to the two biochemically active forms. The fully reduced compounds are termed Cob(I)alamins, and the oxidized compounds Cob(III)alamins. Analogues of vitamin B12 (pseudo-vitamin B12s) exist in nature, endogenous production of which in humans is suggested by their presence in all sera (including fetal serum) and their fall in parallel with physiologically active vitamin B12 in vitamin B12 deficiency.

Fig. 22.5.6.1 The structure of cyanocobalamin.

Fig. 22.5.6.1
The structure of cyanocobalamin.

Table 22.5.6.1 Vitamin B12 and folate

Vitamin B12

Folate

Parent form

Cyanocobalamin (cyano-B12), mol. wt. 1355

Folic acid (pteroyglutamic acid), mol. wt. 441.4

Crystals

Dark-red needles

Yellow, spear-shaped

Natural forms

Deoxyadenosylcobalamin

Reduced (di- or tetrahydro-), methylated, formylated, other single carbon additions; mono- and polyglutamates

Methylcobalamin

Hydroxocobalamin

Foods

Animal produce (especially liver) only

All, especially liver, kidney, yeast, greens, nuts

Adult daily requirements

2 μ‎g

100 μ‎g

Adult body stores

2–5 mg

6–20 mg

Length of time to deficiency

2–4 years

4 months

Daily diet content

5–30 μ‎g

About 200–250 μ‎g

Cooking

Little effect

Easily destroyed

Absorption

Intrinsic factor (+ neutral pH + Ca2+) via ileum

Deconjugated, reduction, and methylation via duodenum and jejunum

Plasma transport

Tightly and specifically bound to transcobalamins

One-third loosely bound albumin, other proteins; ?specific protein

Enterohepatic circulation

3–9 μ‎g/day

60–90 μ‎g/day

Vitamin B12 is known to be involved in only three reactions in human tissues: as adocobalamin in the isomerization of methylmalonyl CoA to succinyl CoA and of α‎-leucine to β‎-leucine, and as methylcobalamin in the methylation of homocysteine to methionine, a reaction that also requires methyltetrahydrofolate (Fig. 22.5.6.2). In some bacteria, but not in humans, vitamin B12 has a direct role in DNA synthesis by virtue of its involvement in ribonucleotide reductase.

Fig. 22.5.6.2 Biochemical reactions of vitamin B12 (cobalamin) in human tissues.

Fig. 22.5.6.2
Biochemical reactions of vitamin B12 (cobalamin) in human tissues.

Nutrition

Vitamin B12 is synthesized by microorganisms; animals obtain it by consuming the flesh of other animals or their produce (milk, cheese, eggs, etc.)—or vegetable foods contaminated by bacteria. A healthy mixed diet contains between 5 and 30 µg daily. In some species, but not in humans, vitamin B12 is absorbed after synthesis by bacteria in the large intestine. The vitamin B12 content in humans is about 3 to 5 mg; it is found mainly in the liver (c.0.7–1.1 µg/g). Adult daily losses are related to body stores; to maintain normal body stores, daily requirements are of the order of 1–2 µg. It takes 3 to 4 years, on average, for deficiency to develop if supplies are totally cut off by malabsorption. There is an enterohepatic circulation for vitamin B12, variously estimated at 3 to 9 µg daily, which is intact in vegans, which may partly account for their tendency to maintain low body stores without incurring severe deficiency. The body is unable to degrade vitamin B12 and deficiency has not been shown to be due to excess utilization or loss.

Absorption

About 15% of dietary vitamin B12 is available for absorption. It is released from protein binding in food by proteolytic enzymes, heat, and acid, and combines one molecule to one molecule with a glycoprotein R vitamin B12-binding protein (also called haptocorrin) in gastric juice. The glycoprotein binds dietary forms of vitamin B12 but does not facilitate its absorption. Pancreatic trypsin degrades this protein and so releases vitamin B12 for attachment to intrinsic factor (IF) and subsequent absorption. IF is a glycoprotein produced mainly by the parietal cells (Table 22.5.6.2). The normal stomach produces a vast excess of IF, measured in units (1 unit binds 1 ng vitamin B12). Vitamin B12 in bile is also attached to IF and reabsorbed through the ileum. At neutral pH, in the presence of calcium ions, the vitamin B12–IF complex attaches passively to a complex specific IF receptor, cubilin amnion, on the brush border of the mucosal cells of the terminal ileum. Cubilin is a 640-kDa peripheral membrane protein present in the epithelium of intestine and kidney. Amnionless (AMN) (50 kDa) binds to cubilin and is essential for production of mature cubilin and its transport to the apical brush border. AMN directs sublocalization and endocytosis of cubilin and the IF–vitamin B12 complex. Mutations of cubilin or AMN underlie hereditary malabsorption of vitamin B12 (discussed later in this chapter).

Table 22.5.6.2 Vitamin B12-binding proteins

Intrinsic factor

Transcobalamin I and III1

Transcobalamin II

Present in

Gastric juice

Plasma

Plasma, cerebrospinal fluid

Source

Gastric parietal cell

Granulocytes? other organs

Macrophages, liver parenchyma, ileum

Molecular weight

45 000

60 000

45 500

Structure

Glycoprotein (15% sugar)

Gl ycoprotein

Polypeptide

Normal total binding capacity

30–110 μ‎g/l

700–800 ng/l

900–1000 ng/l

B12 content

No B12

300–400 ng/l B12

30–60 ng/l B12

Function

B12 absorption (not itself absorbed)

? storage of B12

  • B12 delivery to marrow, placenta, brain, and other tissues,

  • B12 absorption

? protection of B12

Binding of B12 analogues

1 Related ‘R’ binders (haptocorrins) occur in other tissues and secretions, e.g. milk, gastric juice, saliva, and tears.

After cubilin–AMN-mediated endocytosis, IF undergoes lysosomal degradation. After a delay of 3 to 5 h, vitamin B12 appears in portal blood, with a peak concentration 8 h after ingestion, complexed with transcobalamin II (TCII) secreted into the circulation from the basolateral side of the intestinal cells. Ileal absorption of vitamin B12 is limited by the number of cubilin receptors to a few micrograms daily, and although 80% of a single dose of 1 to 2 µg may be absorbed, the proportion diminishes steeply at higher doses. A small (<1%) trace of a large (≥1 mg) dose of vitamin B12 can be absorbed passively and rapidly through the buccal, gastric, and duodenal mucosae without the involvement of the IF pathway and thus forms the basis for treatming malabsorption of vitamin B12 with large oral doses.

Transport

Vitamin B12 in plasma is 70 to 90% attached to a glycoprotein, transcobalamin I (TCI), and 0–10% to transcobalamin III (TCIII) which does not enhance cell uptake of vitamin B12 (see Table 22.5.6.2). TCI and III belong to a group of glycoproteins, the R binders or haptocorrins (see above), that are present in many tissues and fluids; these molecules have the same amino acid composition but differ in the carbohydrate moiety. The haptocorrins may have the role of binding analogues of vitamin B12 derived from food or intestinal organisms and transporting them to the liver for excretion in the bile. Genetic mutations of the gene TCN1, which codes for TCI cause subnormal serum B12 levels.

The most important plasma vitamin B12-binding protein, TCII, is synthesized in macrophages, liver, the ileum and possibly endothelium. TCII is loaded with vitamin B12 from the ileum and by release of vitamin B12 from the liver and other organs. It is normally almost completely unsaturated because it actively enhances uptake of vitamin B12 by bone marrow, placenta, and other tissues of the body that contain TCII receptors. TCII–vitamin B12 is internalized by endocytotosis; vitamin B12 is released by proteolytic cleavage in lysosomes but TCII is not reutilized (Table 22.5.6.1). TCII has a 20% amino acid homology and greater than 50% nucleotide homology with human TCI and with rat IF. It shows at least five genetic variants. Serum TCII is normally higher in women than men and in black populations compared with white. The concentration of vitamin B12 in cerebrospinal fluid is low, with a mean of 10 ng/litre in normal subjects. Most of this is attached to TCII. There is virtually no vitamin B12 in normal urine.

Folate

Biochemistry

This vitamin exists in nature in over 100 forms, all of which are derivatives of folic acid (pteroylglutamic acid), which consists of a pteridine, a para-aminobenzoic acid moiety and l-glutamic acid (Fig. 22.5.6.3). Natural folates differ from folic acid by:

  • being reduced in the pteridine ring to di- or tetrahydo- forms

  • having a single carbon moiety attached at positions N5 or N10 (e.g. methyl, formyl, etc.)

  • having a chain of glutamate moieties attached by γ‎-peptide bonds to the l-glutamate moiety

Fig. 22.5.6.3 The structure of pteroylglutamic (folic) acid.

Fig. 22.5.6.3
The structure of pteroylglutamic (folic) acid.

In human and other mammalian cells, the number of glutamates is mainly four, five, or six. Polyglutamate forms of folate are the active coenzymes; these show increased affinity or lowered Km values for most of the enzymes of one-carbon metabolism. In body fluids however, folates are monoglutamate derivatives. In plasma, 5-methyltetrahydrofolate (methyl-THF) predominates.

The biochemical reactions of folates are shown in Table 22.5.6.3. In each there is transfer of a single carbon group, methyl (–CH3), formyl (–CHOH), methenyl (≡CH), methylene (=CH2), or formimino (=CHNH), from one compound to another. Three of the reactions are concerned with synthesis of DNA precursors (two purine and one pyrimidine). During thymidylate synthesis, oxidation of folate to the dihydro state occurs; the enzyme dihydrofolate reductase, the principal target for the antifolates methotrexate and pyrimethamine, returns folate to the active tetrahydro state (Fig. 22.5.6.4). During its reactions, folate is not completely reutilized, some degradation at the C9–N10 bond occurs to non-folate compounds. Thus, folate utilization is increased and folate deficiency likely when cell turnover and DNA synthesis are increased.

Table 22.5.6.3 Biochemical reactions of folates

Reaction

Enzyme

1.

Conjugation or deconjugation

Hydrolysis of poly- to monoglutamates

Folate ‘conjugase’ (α‎-glutamylcarboxypeptidase; pteroylpolyglutamate hydrolase)

Conjugation of monoglutamates to polyglutamates

Folate-polyglutamate synthetase

2.

Oxidation–reduction

Oxidized or dihydrofolates, converted to tetrahydrofolates

Dihydrofolate reductase

3.

Amino acid interconversions

(a) homocysteine → methionine1

++

+ +

5-Methyl THF methyltransferase

methyl THF →THF

(b) 5-formiminoglutamic acid → glutamic acid (Figlu)

Figlu transferase

+ +

THF → formimino THF

Serine–hydroxymethyltransferase

(c)serine → glycine

++

THF → 5,10-methylene THF

4.

DNA synthesis

Purine synthesis:

(a) GAR → formyl GAR

GAR transformylase

+ +

5,10 methenyl THF →THF

(b) AICAR → inosinic acid

AICAR transformylase

+ +

10-formyl THF →THF

Pyrimidine synthesis:

Deoxyuridine monophosphate (dUMP) → thymidine monophosphate (TMP)

Thymidylate synthetase

5,10-methylene THF → THF

5.

Formate fixation

Formic acid + ATP + THF → 10-formyl-THF + ADP

THF formylase

6.

? Methylation of biogenic amines

e.g. dopamine → epinine

? dopamine methyltransferase

++

methyl THF → THF

1 See Figs. 2 and 4.

THF, tetrahydrofolate; DHF, dihydrofolate; GAR, glycinamide ribotide; AICAR, 5-amino-4-imidazolecarboxamide ribotide.

Reaction (6) has been demonstrated only in vitro and may not take place in vivo.

Fig. 22.5.6.4 Suggested mechanisms by which vitamin B12 deficiency affects folate metabolism and interferes with DNA synthesis. Indirect involvement of vitamin B12, as methylcobalamin, in DNA synthesis is suggested by the ‘methylfolate’ trap (‘tetrahydrofolate starvation’) hypothesis. Methylcobalamin is involved in formation of intracellular THF from plasma methyl-THF. THF and/or its formyl derivative, but not methyl-THF, are the ‘ground substances’ from which all folate coenzymes are made by glutamate addition and single carbon unit transfer. 5,10-Methylene-THF polyglutamate is involved in thymidylate synthesis. A, adenine; C, cytosine; D, deoxyribose; DP, diphosphate;G, guanine; T, thymine; THF, tetrahydrofolate; TP, triphosphate; U, uridine.

Fig. 22.5.6.4
Suggested mechanisms by which vitamin B12 deficiency affects folate metabolism and interferes with DNA synthesis. Indirect involvement of vitamin B12, as methylcobalamin, in DNA synthesis is suggested by the ‘methylfolate’ trap (‘tetrahydrofolate starvation’) hypothesis. Methylcobalamin is involved in formation of intracellular THF from plasma methyl-THF. THF and/or its formyl derivative, but not methyl-THF, are the ‘ground substances’ from which all folate coenzymes are made by glutamate addition and single carbon unit transfer. 5,10-Methylene-THF polyglutamate is involved in thymidylate synthesis. A, adenine; C, cytosine; D, deoxyribose; DP, diphosphate;G, guanine; T, thymine; THF, tetrahydrofolate; TP, triphosphate; U, uridine.

Nutrition

Folate occurs in most foods, the highest concentrations (more than 30 µg/100 g wet weight) in liver (in which it is easily destroyed by cooking). Vitamin C protects folate from oxidative destruction. An average Western daily intake is about 250µg, with 50% or more in the polyglutamate form. Body stores are about 10 to 12 mg, with a mean liver concentration of about 7 µg/g. Primitive or rapidly growing tissues have higher folate concentrations than corresponding mature tissues. Daily adult requirements are about 100 µg.

Absorption

Folates are absorbed rapidly, mainly through the duodenum and jejunum. Polyglutamates are deconjugated in the intestinal lumen, at the brush border, and possibly in lysosomes of intestinal cells by an enzyme known as folate conjugase (γ‎-glutamylcarboxypeptidase, pteroylpolyglutamate hydrolase). They are reduced to the tetrahydro state and methylated at the N5 position so that methyl-THF enters portal plasma whatever food folate is ingested (Table 22.5.6.1). Folic (pteroylglutamic) acid itself, which is not present in food, but is used therapeutically, enters the portal blood largely unchanged at doses of more than 100 to 200 µg, as it is a poor substrate for reduction by dihydrofolate reductase. A proton-coupled folate transporter (PCFT) with high affinity and a low pH optimum is essential for absorption of reduced folates and folic acid. It is expressed particularly in the apical brush border of the enterocytes of the duodenum and jejunum. Various mutations have been found in this transporter in patients with a specific hereditary malabsorption of folate. The protein is expressed in other tissues and may be involved in intracellular transportation of folates from endocytic vesicles. As it is also active at neutral pH for methyl THF, it may play a role in delivering this folate to systemic cells, e.g. the liver. It may also transport antifolates, e.g. methotrexate and pemetrexed, into the acid interior of solid tumours. The small intestine has a large capacity to absorb folate; on average 50% of natural folate is absorbed whatever the dose. If excessive amounts are fed, the excess is largely excreted in urine as folates or their breakdown products after cleavage of the C9–N10 bond. There is a substantial enterohepatic circulation for folate, estimated at up to 90 µg folate daily; if this is interrupted, plasma folate concentrations decrease to about one-third within 24 h.

Transport

Folate is transported in plasma, two-thirds unbound and about one-third loosely bound to albumin and possible other proteins. There are two highly specific mammalian folate transporters. SLC19A1 is a facilitative transporter with the characteristics of an anion exchanger. The gene is located at chromosome 21q 22.2. The protein has 12 transmembrane domains and both N- and C-termini are directed to the cytoplasm. It is ubiquitously expressed on normal tissues and tumours. Its affinity for folic acid and methotrexate is one to two orders less than for reduced folates. The second is a group of high affinity binding proteins (FRs) encoded by three genes, designated α‎, β‎, and γ‎, localized to chromosome 11q 13.3-q 13.5. FRα‎ and FRβ‎ are both glycosylphosphatidylinositol (GPI) anchored proteins. The physiological role of the FRs is not clear. They are expressed in the apical brush border of the renal tubular epithelial cells so may have a role in renal reabsorption of folates.

Folate taken up by membrane-bound FRs is thought to enter endocytic vesicles. FRα‎ (but not FRβ‎) knockout mice show fatal morphological abnormalities, suggesting a critical role in mouse development. The FRs have enhanced expression on certain tumour cells and this has prompted studies aimed at developing tumour-specific antifolates or folate-conjugated radiopharmaceuticals or other molecules.

Plasma folate is filtered by the glomerulus and mostly reabsorbed unless the renal tubular maximum is exceeded. Normal urine folate is 0 to 13 µg in 24 h. Folate is secreted into cerebrospinal fluid (which has a mean concentration of 24 µg/litre) and is present in bile. Human milk has a folate concentration of 50 µg/litre. Prostate-specific membrane antigen is a folate hydrolase carboxypeptidase which can release glutamates in either α‎ or γ‎ linkages. The physiological significance of this is unknown.

Biochemical basis of megaloblastic anaemia

All known causes of megaloblastic anaemia, whether drugs, deficiencies, or inborn errors of metabolism, inhibit DNA synthesis by reducing the activity of one of the many enzymes concerned in purine or pyrimidine synthesis or by inhibiting DNA polymerization from its precursors. Folate deficiency, by reducing supply of the active coenzyme form, 5,10-methylene-THF, inhibits thymidylate synthesis, a rate-limiting reaction in DNA synthesis. Vitamin B12 does not have a direct role in this or any other reaction in mammalian DNA synthesis. Vitamin B12 deficiency inhibits DNA synthesis indirectly because of the requirement for methylcobalamin in the conversion of methyl-THF that has entered cells from the plasma to THF. Deficiency of vitamin B12 is considered to decrease the intracellular supply of THF, from which the natural folate coenzymes, folate polyglutamates, are made. Methyl-THF cannot act as a substrate for synthesis of folate polyglutamates in human cells. When vitamin B12 is deficient there is reduced activity of all reactions requiring folate coenzymes, including those involved in DNA synthesis (Fig. 22.5.6.4). Misincorporation of the base uracil, because of the accumulation of dUMP (Fig. 22.5.6.4). and hence of dUTP, has been proposed to contribute to the DNA abnormality.

Clinical features and causes of megaloblastic anaemia

Although pernicious anaemia (PA) is only one of the many causes of megaloblastic anaemia (Tables 22.5.6.422.5.6.6), it is convenient to describe the general clinical features of the anaemia under this heading; PA is the most frequent cause of megaloblastic anaemia in Western countries. The laboratory findings and treatment of PA and other megaloblastic anaemias are discussed later.

Table 22.5.6.4 Causes of B12 deficiency and malabsorption of B12

1. Causes of severe B12 deficiency

(a) Nutritional:

vegans

long-continued extremely poor diet (rarely)

(b) Malabsorption:

gastric causes

acquired (Addisonian) pernicious anaemia

congenital intrinsic-factor deficiency or abnormality

total and partial gastrectomy

destructive lesions of stomach

intestinal causes

gut flora associated with (jejunal diverticulosis, ileocolic, fistula, anatomical blind loop, stricture, Whipple’s disease, scleroderma, HIV disease)

ileal resection and Crohn’s disease

chronic tropical sprue

selective malabsorption with proteinuria

irradiation to cervix

HIV disease

fish tapeworm

transcobalamin II deficiency

2. Causes of malabsorption of B12 usually without severe B12 deficiency

Simple atrophic gastritis, gastric bypass, severe chronic pancreatitis

Zollinger–Ellison syndrome, adult gluten-induced enteropathy, giardiasis

Drugs:

PAS, colchicine, neomycin, slow K, ethanol, metformin, phenformin, anticonvulsants

Deficiencies of folate, B12, protein

Table 22.5.6.6 Megaloblastic anaemia not due to vitamin B12 or folate deficiency

1. Abnormalities of B12 or folate metabolism

Congenital

Transcobalamin II deficiency or functional abnormality

Inborn errors of folate metabolism e.g. methylfolate transferase deficiency

Homocystinuria and methylmalonic aciduria (some cases)

Acquired

Nitrous oxide

Dihydrofolate reductase inhibitors: methotrexate, pyrimethamine, trimethoprim, ?pentamidine, triamterene

2. Independent of B12 or folate

Congenital

Orotic aciduria, (responds to uridine)

Lesch–Nyhan syndrome, ? responds to adenine

Thiamine-responsive

Some cases of congenital dyserythropoietic anaemia

Acquired

AML FAB M6, other myeloid leukaemias (some cases)

Myelodysplasia

Drugs

Antimetabolites: 6-mercaptopurine, cytosine arabinoside, hydroxyurea, 5-fluorouracil, azathioprine, etc.

Acquired pernicious anaemia (Addisonian pernicious anaemia, Biermer’s anaemia, pernicious anaemia)

Definition

An autoimmune disease in which there is atrophy of the stomach with severely reduced or absent IF and acid secretion with consequent malabsorption of vitamin B12 and vitamin B12 deficiency. There is an autoimmune gastritis caused by pathological CD4 T cells reacting against gastric H/K-ATPase.

Aetiology

PA is a disease of older people: less than 10% of patients are under the age of 40 years. There is a female:male ratio in most (but not all) series of about 1.6:1. There is a slightly higher prevalence (c.44% vs 40%) of blood group A in patients with PA compared with controls in the United Kingdom. No overall association between PA and HLA type has been found, but those with an endocrine disease have a greater incidence of HLA B8, B12, and BW15. An association between autoimmune gastritis and HLA-DRB103 and 104 has been reported in Finnish and Italian populations. Contrary to previous opinions, PA occurs in all ethnic groups including African, Indian, Native American, and Chinese, as well as white Europeans. There is a higher incidence in close relatives, of either sex, of an affected person.

DNA sequence variants of a gene NLRP1, located at chromosome 17p13, encoding NACHT, a leucine-rich repeat protein which is a regulator of the innate immune response, have been associated with vitiligo and its associated diseases including PA.

About 55% of patients have serum thyroid antibodies and 33% with primary myxoedema have parietal-cell antibody. There is probably no association with diabetes mellitus. Other evidence for an immune aetiology of the gastritis of PA is the improvement in mucosal appearance and function with corticosteroid therapy, the presence of antibodies in serum and gastric juice directed against parietal cells and IF, and of cell-mediated immunity to IF (see Chapter 5.2). Parietal-cell antibody is present in the serum of 85 to 90% of patients. The autoantigens are the α‎- and β‎-subunit of the gastrin proton pump (H+,K+ ATPase). Two antibodies to IF exist in serum. Type I (‘blocking’) occurs in about 50% of patients and is directed against the vitamin B12-binding site. Type II (to the ileal binding site) occurs in 30 to 35% but only if type I antibody is also present. Antibodies to IF exist in gastric juice and here they may neutralize the action of remaining IF. The incidence of parietal-cell and IF antibodies in serum in PA may be different in different groups of patients, younger patients having a lower incidence of parietal-cell antibody while blacks and Hispanics may have a higher incidence of IF antibodies. The antibodies to IF are virtually specific for PA but parietal-cell antibody occurs in many subjects with atrophic gastritis without PA. An autoantibody to the gastrin receptor may also occur in serum in PA.

PA may be associated with hypgogammaglobulinaemia or with selective IgA deficiency when it tends to present at an early age. Serum gastrin concentrations are raised (>200 µg/litre) in 90% of patients with PA, and serum pepsinogen (PG) concentrations are less than 30 µg/litre in 92% of such patients with a low PGI/PGII ratio.

The relationship of PA, auto immune gastritis, and Helicobacter (H.) pylori infection is not clear. Young subjects (<40 years) with gastritis, hypergastrinaemia, and positive antiparietal cell antibody in serum will usually show iron deficiency anaemia whereas older (>60 years) with these features more frequently have macrocytic red cells and low serum vitamin B12 levels. H. pylori infection occurs in up to 40% of such subjects less than 20 years old but in only 10% of those older than 60 years. It has been proposed that H. pylori is an infective trigger to autoimmune gastritis by molecular mimicry.

Pathology

There is a gastritis in which all layers of the body and fundus of the stomach are atrophied with loss of normal gastric glands, mucosal architecture, and absence of parietal and chief cells, but mucous cells lining the gastric pits are well preserved. An infiltrate of plasma cells and lymphocytes with an excess of CD8 cells occurs and intestinal metaplasia may be present. The antral mucosa is well preserved except in hypogammaglobulinaemia, and, like the fundus, shows an increased number of gastrin-secreting cells.

Clinical features

The general features of megaloblastic anaemia are similar, whatever the underlying cause. Particular clinical features may point to the underlying disease, whether PA or some other cause. In PA, the anaemia usually develops gradually, perhaps over several years, and symptoms may not occur until it is severe. The most common complaints are due to the anaemia, but loss of mental and physical drive, numbness, or difficulty in walking suggest neurological complications. Psychiatric disturbances are common and range from mild neurosis to severe organic dementia. They may occur in the absence of anaemia or macrocytosis. Mild jaundice, loss of appetite and weight, indigestion, and episodic diarrhoea are frequent. An intercurrent infection may precipitate severe anaemia and thus symptoms. Older patients may present with congestive heart failure. In a few patients, bruising due to thrombocytopenia is marked. Many symptomless patients are diagnosed because a routine blood test is made.

Physical signs, if present, are those of anaemia, perhaps with mild jaundice, giving the patient a so-called lemon-yellow tint. A few patients with deficiency of either vitamin B12 or folate develop a widespread brown pigmentation, affecting nail beds and skin creases particularly, but not mucous membranes. This is reversible with the appropriate therapy. The tongue may be red, smooth, and shiny, occasionally with ulcers. A mild pyrexia up to 38°C is common in patients with moderate to severe anaemia. The liver may be enlarged while the cardiovascular system shows changes due to anaemia. Patients with PA may also have features of an associated disorder on presentation, most commonly myxoedema. Other thyroid disorders, vitiligo, carcinoma of the stomach (incidence three times controls), Addison’s disease, and hypoparathyroidism, may precede, occur simultaneously with, or follow the onset of the anaemia.

Neurological complications of vitamin B12 deficiency

Vitamin B12 deficiency may cause a symmetrical neuropathy affecting the lower limbs more than the upper (Chapter 24.13), which usually presents with paraesthesiae or with ataxia, particularly in the dark. In some cases, loss of cutaneous sensation, spastic paraparesis, muscle weakness, urinary or faecal incontinence, an optic neuropathy, or psychiatric disturbance dominates. The nervous system disease is due to severe deficiency judged by serum vitamin B12 levels or methylmalonic acid (MMA) excretion, but may occur with mild or no anaemia. A similar neurological syndrome with paraparesis has been described in dentists and others repeatedly exposed to nitrous oxide (N2O), which inactivates methionine synthase. The biochemical explanation for the neurological disease is not clear. A defect in fatty acid metabolism in myelin tissue has been suggested. Studies in N2O-treated monkeys have also suggested that the neuropathy results from accumulation of S-adenosyl homocysteine (caused by the block in conversion of homocysteine to methionine) with inhibition of transmethylation biogenic amines, proteins, phospholipids, and neurotransmitters in the spinal cord and brain. Methionine has been shown to prevent the neurotoxicity caused by N2O in experimental animals. There is a more rapid decline in cognitive function in subjects with low serum B12 levels, serum holotranscobalamin and raised serum MMA concentrations. Recent studies suggest that low serum folate levels are not associated with cognitive loss or depression in the elderly. High levels, have been associated with increased cognitive loss in subjects with low serum B12 levels, but some studies suggesting this have not been age adjusted. There are conflicting reports on whether administration of folic acid or vitamin B12 improve cognitive function in older people with or without low serum vitamin B12 or folate concentrations.

General tissue effects of vitamin B12 and folate deficiencies: the effects of folic acid supplementation

Both deficiencies cause macrocytosis and related cytopathic effects on proliferating epithelial cells throughout the body (e.g. bronchial, bladder, buccal, and uterine cervix), with glossitis and angular cheilosis, a mild malabsorption syndrome, and reduced regeneration of damaged liver cells. In both sexes, sterility (reversible with vitamin B12 or folate therapy) may result from effects on the gonads. It is possible that the deficiencies in children affect overall body growth. Nutritional vitamin B12 deficiency in infants long term causes failure to thrive and poor brain growth with poor intellectual outcome.

Generalized, reversible melanin pigmentation occurs in a few patients with vitamin B12 or folate deficiency, the cause of which is uncertain. Defective bactericidal activity of phagocytes due to impaired intracellular killing has been described in vitamin B12 deficiency but not in folate deficiency. Vitamin B12 deficiency reduces serum concentrations of the osteoblast-related proteins alkaline phosphatase and osteocalcin, but whether clinically important bone disease occurs is unknown.

Neural tube defects (NTD)

Folic acid supplements at the time of conception and in early (first weeks) of pregnancy reduce the incidence of NTD (anencephaly, encephalocele, and spina bifida) in the first pregnancy and in subsequent pregnancies where such a malformation has occurred previously.

Folic acid fortification of the diet has led to a substantial reduction of incidence of NTD, e.g. in the North America. The explanation for the effect of folic acid on NTD is not certain. Women carrying affected fetuses have lower serum folate and vitamin B12 concentrations and higher serum homocysteine levels than matched controls. There is a linear relationship when plotted on logarithmic scales between the birth incidence of NTD and maternal red-cell folate, indicating that an increase in red-cell folate even within normal range is associated with a constant, proportional decrease in the birth frequency of NTD. Folic acid prevention of NTD (and in some studies cleft lip and palate), despite apparently normal serum and red cell folate concentrations, suggests that folic acid is overcoming a metabolic abnormality in folate metabolism. Only one such defect, a mutated tetrahydofolate reductase, has been identified so far. Periconceptional use of vitamins or supplements containing folic acid is associated with a reduced incidence of birth defects associated with maternal diabetes mellitus.

Mutated 5,10 methylene tetrahydrofolate reductase (MHTFR), a common thermolabile variant (677C→T) (Ala225Val) of the enzyme MHTFR is associated with lower serum and red-cell folate concentrations and with higher plasma homocysteine than in control subjects in the general population. The prevalence of the homozygous state in the population is approximately 5% and in parents of fetuses with NTD the prevalence is approximately 13%. The presence of this mutation can therefore account for only a small proportion of NTDs. Mutations of other genes, e.g. VANGL1, not related to folate metabolism, have been found in NTD families. Serum B12 levels are also lower in sera of mothers with NTD infants than in controls. Also, transcobalamin II receptor polymorphisms are associated with increased risk for NTD. There are, however, no studies showing that B12 therapy or dietary fortification with B12 reduces the incidence of NTD.

Cardiovascular disease

McCully (1969) first implicated homocysteine as a cause of atherosclerosis. This was based on pathological studies of children or young adults with congenital homocystinuria, whether due to a defect of cystathionine synthase, methionine synthase, or MHTFR (Fig. 22.5.6.5). In these children, plasma homocysteine concentrations are raised to 10 to 100 times normal. It is now apparent that even mild rises in plasma homocysteine are associated with coronary or peripheral arterial disease, stroke, and deep vein thrombosis. Homocysteine can directly injure endothelial cells, activate platelets and leucocytes, stimulate vascular smooth muscle proliferation, oxidize low-density lipoprotein (LDL) and disturb collagen and extracellular matrix formation. Determinants of plasma homocysteine include age, sex, renal function, protein intake, vitamin B6, folate, and vitamin B12 status, the presence of the thermolabile variant MHTFR, smoking, and alcohol consumption, as well as intake of various drugs.

Fig. 22.5.6.5 The role of three enzymes (cystathione synthase, methionine synthase, and MHTFR) and three vitamins (vitamin B12, vitamin B6, and folate) in homocysteine metabolism.

Fig. 22.5.6.5
The role of three enzymes (cystathione synthase, methionine synthase, and MHTFR) and three vitamins (vitamin B12, vitamin B6, and folate) in homocysteine metabolism.

Megaloblastic anaemia and miscellaneous deficiency anaemiasFolate deficiency assessed by serum or red-cell folate or by dietary folate intake is also associated with coronary vascular disease, myocardial infarct, and peripheral vascular disease. Meta-analysis of prospective trials shows that a 25% lower starting homocysteine level is associated with 11% lower coronary heart disease risk (and 19% lower stroke risk). There is also an association of the MTHFR homozygous state TT, which is associated with a higher homocysteine level in serum than the wild type CC state, and ischaemic heart disease. Meta-analysis of 75 studies showed an increased risk of ischaemic heart disease in TT compared to CC homozygotes, odds ratio 1.16 (1.04–1.29). However, meta-analysis of 26 randomized control trials enrolling 58,804 participants showed that folic acid supplementation was not associated with a significant change in cardiovascular disease or all-cause mortality, although it was linked to a decreasing trend in stroke risk. This was more marked in populations without mandatory fortification of the diet with folic acid (Yang et al, 2012). Yi et al (2013) in a meta-analysis of 14 randomized trials with B vitamin supplementation including some of those included in the Yang 2012 study, found an overall reduction in stroke (RR 0.93, p=0.04). This was more marked in those followed for more than three years, and those without background fortification of breakfast cereals with folic acid.

Wald et al (2011) have suggested that in prevention of coronary disease aspirin may be negating or reducing the effect of lowering homocysteine. On this basis folic acid would have a role in primary prevention of ischaemic heart disease in those not taking aspirin.

It is relevant that a reduction in evidence of stroke occurred in the United States of America and Canada coinciding with the introduction of dietary folic acid fortification, whereas no reduction in incidence occurred over the same period (1998–2002) in England and Wales without fortification.

Malignancy

Positive and negative associations between the occurrence of various types if lymphoblastic or myeloblastic leukaemias in infancy and childhood and polymorphisms of folate-metabolizing enzymes including MHTFR have been reported. Folic acid prophylactically in pregnancy has been reported to reduce the incidence of a subsequent childhood acute lymphoblastic leukaemia and of brain tumours. In Canada, food fortification with folic acid has been associated with a 60% reduction in incidence of neuroblastoma.

Epidemiological studies show an inverse risk of colorectal cancer or adenoma and folate status and a less clear-cut relation exists with other gastrointestinal, lung, breast, ovary, and cervical carcinomas. Also small randomized and nonrandomised trials suggest a benefit of supplemental folic acid on incidence of colorectal cancer. A large randomized trial has shown no difference in overall incidence of colonic adenomas in women between controls and folic acid, vitamin B6 and vitamin B12 supplemented subjects.

It has also been suggested that an increased incidence of colorectal cancer in the United States of America and Canada is associated with the fortification of the diet with folic acid, but the temporal disassociation between fortification and risk in colorectal cancer incidence makes this unlikely—increased detection by screening endoscopy is a more likely explanation. There has been no increase in mortality rate from colorectal cancer in the United States or Canada since fortification started.

Megaloblastic anaemia and miscellaneous deficiency anaemiasSoon after its discovery, folic acid in large doses was found to promote the growth of existing cancers. Antifolate drugs were developed as a result of these observations. Most recent analysis has shown no significant effect of folic acid at large doses over prolonged periods on cancer incidence. Meta-analysis of 13 trials carried out before 2011, ten for cardiovascular disease prevention and three for colonic adenoma and cancer incidence, involving almost 50,000 subjects in the supplemented and control groups, and lasting for a mean of 5.2 years, did not show an effect on cancer incidence at doses of folic acid daily of 2–5mg. This was true for individual cancers of breast, prostate, lung or large bowel, as well as for rarer cancers. No overall increased incidence of cancer was found in the analysis of 14 trials of B vitamin supplementation (some included in the previous study).

Other effects

Megaloblastic anaemia and miscellaneous deficiency anaemiasComplications of pregnancy that have been ascribed to folic acid, including miscarriage and multiple pregnancy, have no sound basis.

Other causes of vitamin B12 deficiency

Juvenile PA

A few cases of PA with gastric atrophy, achlorhydria, and IF antibodies have occurred in children. They may show associated (‘autoimmune’) conditions, e.g. myxoedema, hypoparathyroidism, Addison’s disease, or chronic mucocutaneous candidiasis.

Congenital deficiency or structural abnormality of intrinsic factor

Fewer than 100 cases have been reported of a child being born with absent or nonfunctioning IF due to a mutation of the IF gene. There is an otherwise normal stomach on biopsy and normal secretion of acid. Inheritance is autosomal recessive. In different cases, IF may be present in the gastric juice but susceptible to acid degradation or cannot bind vitamin B12, or binds it but cannot attach it to ileal receptors. These children tend to present with irritability, vomiting, diarrhoea, and loss of weight, and are found to have megaloblastic anaemia. The usual age of diagnosis is about 2 years, although a few have been diagnosed as early as 4 months and others only in their teens.

Total gastrectomy

All patients who have this operation will develop vitamin B12 deficiency, which usually presents between 2 and 6 years postoperatively. They should be treated with prophylactic vitamin B12 injections from the time of the operation.

Partial gastrectomy

Iron deficiency usually accounts for the anaemia that occurs after this operation. About 6% develop megaloblastic anaemia due to vitamin B12 deficiency. In most of these patients, malabsorption of vitamin B12 is due to an abnormal jejunal flora. The exact incidence of vitamin B12 deficiency depends mainly on the size of the gastric remnant.

Small-intestinal lesions

Colonization of the upper small intestine with colonic bacteria, if sufficiently heavy as in the stagnant-loop syndrome, leads to malabsorption of vitamin B12. The most common causes are listed in Table 22.5.6.4. It appears that the bacteria destroy IF. Infestation with the fish tapeworm (Diphyllobothrium latum) has a similar effect but is now almost completely eradicated; infestation is only sufficiently marked in Finland and Russian lake regions to represent a likely cause of megaloblastic anaemia.

HIV infection

Serum vitamin B12 concentrations fall progressively in HIV-infected patients and subnormal serum values occur in 10 to 35% of individuals with AIDS. Increased concentrations of TCII are usual and malabsorption of vitamin B12, not corrected by IF, has been found in some of these patients. An abnormal small-intestinal flora is the most likely cause of vitamin B12 malabsorption. Megaloblastic anaemia due to vitamin B12 deficiency is, however, rare.

Resection of 1 m or more of terminal ileum

This causes severe malabsorption of vitamin B12. Other diseases that may affect ileal structure and function include: tropical sprue, in which severe vitamin B12 deficiency with anaemia or, rarely, neuropathy is a manifestation only in the chronic phase; gluten-induced enteropathy in which megaloblastic anaemia, if it occurs, is always due to folate deficiency (and vitamin B12 deficiency, if it occurs, is mild); in Crohn’s disease malabsorption of vitamin B12 is frequent but severe vitamin B12 deficiency is unusual unless an ileal resection, fistula, or stagnant loop occurs.

Selective malabsorption of vitamin B12 with proteinuria (Imerslund’s disease, Imerslund–Gräsbeck syndrome, recessive megaloblastic anaemia, MGA1) (OMIM 261100)

This congenital disorder with autosomal recessive inheritance is the most common cause of megaloblastic anaemia due to vitamin B12 deficiency in nonvegan children. The child secretes IF normally but is unable to transport vitamin B12 across the ileum to portal blood. Most Finnish patients with MGA1 carry the disease-specific mutation P1297L (FM1) in cublin. A second less frequent mutation (FM2) activates a cryptic splice site with insertion of multiple stop codons in the CUB6 domain. Other mutations in cublin have been described. In Norway at least six different mutations of the AMN gene have been reported in affected families. The proteinuria, present in over 90% of cases, is benign, non-specific, and persists after vitamin B12 therapy. The clinical presentation of the disease is identical to that of congenital IF deficiency.

Other causes of malabsorption of vitamin B12

Several other conditions and drugs may cause malabsorption of vitamin B12 but rarely cause deficiency of clinical severity. p-Aminosalicylate, colchicine, neomycin, ‘slow’ potassium tablets, metformin, phenformin and sunitinib, a tyrosine kinase inhibitor used to treat renal cell carcinoma, have all been reported to cause malabsorption of vitamin B12. In chronic pancreatitis and the Zollinger–Ellison syndrome, there is failure to release vitamin B12 from gastric haptocorrin due to absence or inactivation of pancreatic trypsin. Malabsorption of vitamin B12 also occurs in inherited TCII deficiency.

Malabsorption of vitamin B12 occurs temporarily after total-body irradiation before stem cell transplantation. In chronic graft vs host disease affecting the gut malabsorption of vitamin B12 is usual, due to the abnormal gut flora as well as any ileal defect. Irradiation to the ileum during radiotherapy treatment for carcinoma of the cervix has also been reported to cause vitamin B12 malabsorption.

Dietary vitamin B12 deficiency

This occurs most commonly in Hindus who omit all animal produce from their diet. The incidence of overt megaloblastic anaemia is much lower than the incidence of subclinical deficiency assessed by the serum vitamin B12 assay. These individuals have low vitamin B12 stores. Babies have been born vitamin B12 deficient with megaloblastic anaemia caused by severe vitamin B12 deficiency (due to poor diet or tropical sprue) in the mother. Breast milk may also be B12 deficient if the mother’s stores are low. Dietary deficiency of vitamin B12 also occurs in non-Hindu vegans, and rarely in nonvegetarian people living on inadequate diets because of poverty.

Folate deficiency

Clinical features

The main clinical features of megaloblastic anaemia due to folate deficiency are similar to those when the anaemia is due to vitamin B12 deficiency, except that a severe neuropathy does not occur and the underlying aetiology tends to be different. Cognitive changes and depression may be caused by the deficiency. Neurological abnormalities do occur with inborn errors of folate metabolism and may be precipitated by antifolate drugs. Folate deficiency may develop rapidly in a few months, and although many mildly deficient patients do not progress for months or years, in some patients the deficiency may lead to a severe pancytopenia (‘arrest of haemopoiesis’) over a short period, particularly if an infection supervenes.

Nutritional folate deficiency

Minor degrees of nutritional folate deficiency are frequent in most countries, but severe folate deficiency may account for about 17% of all cases of megaloblastic anaemia in the United Kingdom. It occurs mainly in people who are old, poor, and psychiatrically disturbed, living alone on an inadequate diet from which liver, fruit, and fresh vegetables are omitted; in some, barbiturates or consumption of spirits or cough mixtures or a physical abnormality such as rheumatoid arthritis, or tuberculosis may aggravate the effect of a poor diet. A few cases have developed because a special diet is taken, such as for phenylketonuria or for slimming. Scurvy is usually accompanied by severe folate deficiency. Goat’s milk anaemia is a nutritional folate deficiency due to the low (6 µg/litre) folate content of goat’s milk. In some countries (e.g. Burma, Malaysia, Africa, or India), nutritional folate deficiency is the main cause of megaloblastic anaemia, often presenting in pregnancy. Among Hindus, nutritional vitamin B12 deficiency is also common, however, and in many countries—e.g. Caribbean islands, Sri Lanka, and South-East Asia—tropical sprue (see Chapter 15.10.8) is an important cause of both deficiencies and is difficult to distinguish from ‘pure’ nutritional deficiency.

Malabsorption

(see Section 15)

Gluten-induced enteropathy

Folate deficiency due to malabsorption of folates occurs in virtually all untreated patients, the serum folate being subnormal in virtually 100% and red-cell folate subnormal in 80% or more. Anaemia occurs in about 90% of adult cases, due to folate deficiency alone in 30 to 50% and to mixed iron and folate deficiency in the remainder. Mild vitamin B12 deficiency may also occur, but it is not a cause of anaemia in uncomplicated cases. Spontaneous atrophy of the spleen occurs in most of the patients; in about 10 to 15% of cases; the blood film shows the presence of Howell–Jolly bodies, and other features of hyposplenism. A gluten-free diet produces a spontaneous rise in serum and red-cell folate in those patients who respond. In children with gluten-induced enteropathy, anaemia is most often due to combined iron and folate deficiency.

Patients with dermatitis herpetiformis almost all show some degree of gluten-induced duodenal and jejunal abnormality; the severity of folate malabsorption and deficiency correlates with the severity of the intestinal lesion.

Tropical sprue

(see Chapter 15.10.8)

Malabsorption of folate occurs in all severe, untreated patients in the acute phase and megaloblastic anaemia due to folate deficiency may develop within a few months. Not only does the anaemia respond to folate therapy but in many patients all the clinical features, and malabsorption of fat, vitamin B12, and other substances, improves on folate therapy alone. In the first year about 60% of patients appear to be cured by folic acid alone. Long-standing cases are more likely to be vitamin B12 deficient and thus to require vitamin B12 as well as folate and antibiotic therapy.

Congenital specific malabsorption of folate

This is a rare, autosomal recessive abnormality. Affected children show features of damage to the central nervous system (mental retardation, fits, athetotic movements) and present with megaloblastic anaemia responding to physiological doses of folic acid given parenterally but not orally. Folate levels in cerebrospinal fluid are low. It is due to inherited mutations of the proton-coupled folate transporter (PCFT) affecting protein stability or its membrane trafficking.

Other causes

Absorption of folate is impaired by systemic infections. Mild degrees of folate malabsorption have also been reported after jejunal resection or partial gastrectomy, with Crohn’s disease, and with lymphoma. In the intestinal stagnant-loop syndrome, folate levels tend to be high due to absorption of bacterially produced folate. Alcohol, anticonvulsants, oral contraceptives, antituberculous drugs, nitrofurantoin, and sulphasalazine have been suggested, on variable evidence, to cause malabsorption of folate in some subjects but none is definitely established except sulphasalazine.

Increased folate utilization

A general mechanism of increased folate utilization in conditions of increased cell turnover has emerged. This consists of partial degradation of folate at the C9–N10 bond rather than complete recycling of the folate coenzymes required in DNA synthesis.

Pregnancy

(see also Section 14)

This, associated with poor nutrition, is probably the most common cause of megaloblastic anaemia world-wide, unless folic acid supplements are taken. The frequency of the anaemia was about 0.5% in most Western cities and up to 50% in some areas of Asia and Africa until the introduction of prophylactic folic acid. The incidence increases with parity and is higher in twin pregnancies. Folate requirements in a normal pregnancy are increased to about 300 to 400 µg daily. Serum and red-cell folate tend to fall as pregnancy progresses, and to rise spontaneously about 6 weeks after delivery. Lactation may prove an additional cause of folate deficiency, however, which may precipitate megaloblastic anaemia postpartum.

The cause of the deficiency in pregnancy is increased degradation of folate. Folate transfer to the fetus may play a minor part; in a few, megaloblastic anaemia of pregnancy is the first sign of gluten induced enteropathy. The statistical association of iron and folate deficiencies in pregnancy is probably due to a poor quality of the diet in certain women. Lower folate levels and higher homocysteine levels (but not lower B12 levels) have been associated with increased risk of prematurity and preeclampsia.

Prophylactic folic acid should now be given routinely in pregnancy; 400 µg/day is recommended (see earlier) and intake in women who may become pregnant should be at least this amount daily from food or supplements. Larger doses (4–5 mg/day) should be used if there has been a previous infant with an NTD.

Prematurity

Newborn infants have higher serum and red-cell folate concentrations than adults. These fall to a lowest value at about 6 weeks of age. In premature infants, the decline is particularly steep and megaloblastic anaemia may develop, particularly if infections, feeding difficulties, or haemolytic disease with exchange transfusion have occurred. Prophylactic folic acid (e.g. 1 mg/week for the first 3–4 weeks of life) may be given, particularly to those babies weighing less than 1.5 to 1.8 kg at birth.

Malignant diseases

Mild folate deficiency is frequent in patients with cancer (Table 22.5.6.5). In general, the severity correlates with the extent and degree of dissemination of the underlying disease. Patients with megaloblastic anaemia due to folate deficiency are unusual and, as folic acid might ‘feed the tumour’, it should be withheld unless there is a real indication for its use, e.g. gross megaloblastosis causing severe anaemia, leucopenia, or thrombocytopenia. The potential effects of food fortification with folic acid on malignant disease were discussed earlier.

Table 22.5.6.5 Causes of folate deficiency

1. Poor diet

Especially poverty, psychiatric disturbance, alcoholism, dietary fads, scurvy, kwashiorkor, goat’s milk anaemia, partial gastrectomy, other gastrointestinal disease

2. Malabsorption

Gluten-induced enteropathy (child or adult or associated with dermatitis herpetiformis)

Tropical sprue

Congenital specific malabsorption

Minor factor: partial gastrectomy, jejunal resection, inflammatory bowel disease, lymphoma, systemic infections

Drugs: cholestyramine, sulphasalazine, methotrexate, ? others (see (5) below).

3. Excessive requirements

Physiological

Pregnancy

Prematurity and infancy

Pathological:

(a) Malignancies—leukaemia, carcinoma, lymphoma, myeloma, sarcoma, etc.

(b) Blood disorders—haemolytic anaemia (especially sickle-cell anaemia, thalassaemia major), chronic myelofibrosis

(c) Inflammatory—tuberculosis, malaria, Crohn’s diseases, psoriasis, exfoliative dermatitis, rheumatoid arthritis, etc.

(d) Metabolic—homocystinuria (some cases)

4. Excess urinary excretion

Congestive heart failure, acute liver damage, chronic dialysis

5. Drugs

Mechanism uncertain

Anticonvulsants (diphenylhydantoin, primidone, barbiturates)

? nitrofurantoin

? alcohol

Also drugs causing malabsorption of folate (see (2) above)

6. Liver disease

Mixed causes above, and poor storage

Blood disorders

Chronic haemolytic anaemia

Requirements for folate are increased in patients with increased erythropoiesis, particularly when there is ineffective erythropoiesis with a high turnover of primitive cells. Occasional patients, presumably those with a poor folate intake, develop megaloblastic anaemia, particularly in sickle cell anaemia, thalassaemia major, hereditary spherocytosis, and warm-type autoimmune haemolytic anaemia; prophylactic folic acid is usually given in these disorders.

Primary myelofibrosis

Megaloblastic haemopoiesis was reported in as many as one-third of patients. Circulating megaloblasts, increased transfusion requirements, severe thrombocytopenia, or pancytopenia may be the first indication that folate deficiency has developed. Polycythaemia vera is not a cause of folate deficiency.

Sideroblastic anaemia

Folate deficiency, usually mild, may occur in about half of acquired cases. Megaloblastosis, refractory to folate or vitamin B12, also occurs in the acquired form as in other myelodysplastic diseases.

Inflammatory diseases

Folate deficiency has been described in patients with tuberculosis, malaria, Crohn’s disease, psoriasis, widespread eczema, and rheumatoid arthritis. The degree of deficiency is related to the extent and severity of the underlying disorder. Increased demand for folate probably is a factor but reduced appetite is also important in those who develop megaloblastic anaemia.

Metabolic

Homocystinuria

(see Chapter 12.2)

Patients with the most common form of this disorder, due to cystathionase deficiency, may show folate deficiency, possibly due to excess conversion of homocysteine to methionine and thus excess utilization of the folate coenzyme concerned.

Excess urinary loss of folate

Urine folate excretion of 100 µg a day or more occurs in some patients with congestive cardiac failure or active liver disease causing necrosis of liver cells. It is presumed that losses are due to release of folate from damaged liver cells. Haemodialysis and peritoneal dialysis remove folate from plasma. Folic acid (e.g. 5 mg/week) is now usually given prophylactically to patients with renal failure who require long-term dialysis.

Drugs

Dihydrofolate reductase (DHFR) inhibitors

Methotrexate, aminopterin, pyrimethamine, and trimethoprim all inhibit DHFR but have different relative activities against the human, malarial, and bacterial enzymes. Methotrexate is converted to polyglutamate forms, which increases its activity against DHFR and also increases its retention in cells. These methotrexate derivatives invariably impair human folate metabolism. Trimethoprim, used as an antibacterial agent, may aggravate pre-existing folate or vitamin B12 deficiency but does not in itself cause megaloblastic anaemia.

Alcohol

Folate deficiency may occur in spirit-drinking alcoholics. The main factor is poor nutrition and it is likely that alcohol interrupts the enterohepatic circulation for folate. It also has a direct effect on haemopoiesis, causing vacuolation of normoblasts, impaired iron utilization, sideroblastic changes, macrocytosis, megaloblastosis, and thrombocytopenia, even in the absence of folate deficiency. Beer drinkers usually appear to avoid folate deficiency because of the high folate content of beer. The usual macrocytosis in nonanaemic alcoholics is not related to folate deficiency.

Anticonvulsants, barbiturates

Diphenylhydantoin, primidone, and barbiturate therapy may be associated with folate deficiency. The more severe deficiency is associated with poor dietary intake of folate and prolonged drug therapy at high doses. The mechanism of the deficiency is unknown and double-blind trials have shown no effect of folic acid supplementation on the frequency of seizures.

Other drugs

Nitrofurantoin, triamterene, proguanil, and pentamidine have been reported to cause folate deficiency.

Liver disease

Folate deficiency occurs most commonly in alcoholic cirrhosis where alcohol, poor nutrition, release of stored folate with excess urine losses may all be important. The deficiency is less frequent in other types of liver disease.

Laboratory investigation of megaloblastic anaemia

This consists of three stages: (1) recognition that megaloblastic anaemia is present; (2) distinction between vitamin B12 or folate deficiency (or rarely some other factor) as the cause of the anaemia; (3) diagnosis of the underlying disease causing the deficiency (Table 22.5.6.7).

Table 22.5.6.7 Laboratory diagnosis of megaloblastic anaemia

1. General tests

Peripheral blood film and count

Bone marrow

Serum bilirubin, iron, LDH

2. Tests for B12 or folate deficiency

Serum B12 and folate; red-cell folate

Serum homocysteine and methylmalonic acid levels

3. Tests for cause of B12 or folate deficiency

B12 deficiency:

Serum antibodies to parietal cell, intrinsic factor

Serum immunoglobulins

Gastric secretion; intrinsic factor, acid

Serum gastrin

Endoscopy, gastric biopsy

Barium meal + follow-through

Proteinuria, fish tapeworm ova, intestinal flora, etc.

Folate deficiency:

Transglutaminase, endomysial antibodies

Small-intestinal function

Duodenal or jejunal biopsy

Barium follow-through

Tests for many underlying conditions

Recognition of megaloblastic anaemia

Peripheral blood

The mean corpuscle volume (MCV) is raised to between 100 and 140 fl. Oval macrocytes are seen in the blood film. In mild cases, macrocytosis is present before anaemia has developed. Cabot rings (composed of arginine-rich histone and nonhaemoglobin iron) and occasional Howell–Jolly bodies (DNA fragments) may occur due to extramedullary haemopoiesis in the liver and spleen. The MCV may be normal if there is associated iron deficiency, when the blood film appears dimorphic, or if the anaemia (usually due to folate deficiency or antimetabolite drug therapy) develops acutely over the course of a few weeks. The MCV is also normal in some severely anaemic cases involving excess red-cell fragmentation. The reticulocyte count is low for the degree of anaemia, usually of the order of 1 to 3%.

The peripheral blood also shows hypersegmented neutrophils (which have nuclei with more than five lobes; Fig. 22.5.6.6) and the leucocyte count is often moderately reduced in both neutrophils and lymphocytes, although the total leucocyte count rarely falls to less than 1.5 × 109/litre. The lymphocyte CD4/CD8 ratio is reduced. The platelet count may be moderately reduced but rarely falls below 40 × 109/litre.

Fig. 22.5.6.6 Megaloblastic anaemia. Hb 4.0 g/dl, MCV 120 fl. Hypersegmented neutrophil, oval macrocytes, and a small lymphocyte to show size of macrocytes. The fragmentation of advanced megaloblastosis is present. Thrombocytopenia is marked.

Fig. 22.5.6.6
Megaloblastic anaemia. Hb 4.0 g/dl, MCV 120 fl. Hypersegmented neutrophil, oval macrocytes, and a small lymphocyte to show size of macrocytes. The fragmentation of advanced megaloblastosis is present. Thrombocytopenia is marked.

Biochemical changes

These are confined to the anaemic patient and include a mild rise in serum bilirubin (up to 50 µmol/litre), mainly unconjugated, a rise in serum lactic dehydrogenase of up to 10 000 IU/litre. The serum iron and ferritin are also raised and fall with effective treatment. The serum cholesterol is low and alkaline phosphatase mildly reduced. Absence of haptoglobins is usual. In severe cases, free haemoglobin may be present in plasma, Schumm’s test for methaemalbumin in serum is positive, and haemosiderin and fibrin degradation products are present in urine. The direct antiglobulin test is weakly positive in some patients, due to complement.

Bone marrow

The bone marrow is hypercellular in moderate or severely anaemic cases. The myeloid–erythroid ratio is often reduced or reversed. The erythroblasts are larger than normal and show asynchronous maturation of nucleus and cytoplasm, nuclear chromatin remaining primitive with an open, lacy, fine granular pattern despite normal maturation and haemoglobinization of the cytoplasm. Excessive numbers of dying cells, and nuclear remnants including Howell–Jolly bodies, mitoses, and multinucleate cells may be present. Because of death (by apoptosis) of later cells, there is a disproportionate accumulation of early cells. Giant and abnormally shaped metamyelocytes and megakaryocytes with hypersegmented nuclear lobes are also usually present (Fig. 22.5.6.7).

Fig. 22.5.6.7 Megaloblastic anaemia. Bone marrow aspirate showing megaloblasts at different stages and giant metamyelocytes.

Fig. 22.5.6.7
Megaloblastic anaemia. Bone marrow aspirate showing megaloblasts at different stages and giant metamyelocytes.

The severity of these changes parallels the degree of anaemia. In milder cases, changes, described as ‘intermediate’, ‘transitional’, or ‘moderate’, are principally in the size and nuclear chromatin pattern of the erythroblasts, with giant metamyelocytes present; hypercellularity and gross dyserythropoiesis may be absent. In very mild cases, megaloblastic changes are difficult to recognize. In patients with severe anaemia but only mild megaloblastic changes, some additional cause for the anaemia should be sought.

Chromosomes

Changes found in marrow and other proliferating cells include: (1) random chromatin breaks; (2) exaggeration of centromere constriction; and (3) thin, elongated, uncoiled chromosomes.

Ineffective haemopoiesis

The increased cellularity of the marrow with degenerate forms, and the low reticulocyte count suggest that many developing cells are dying in the marrow. This occurs by apoptosis, especially of late erythroblasts. The raised uncongugated serum bilirubin, lactic dehydrogenase, and lysosyme are all due to ineffective haemopoiesis.

Differential diagnosis

Other causes of macrocytosis include a high reticulocytosis (e.g. haemolytic anaemia or regeneration of blood after haemorrhage), aplastic anaemia, red-cell aplasia, liver disease, alcoholism and myxoedema, the myelodyplastic syndromes, myeloid leukaemias, cytotoxic drug therapy, chronic respiratory failure, myelomatosis, and other paraproteinemia. Once a bone marrow biopsy has been done, the principal differentiation is from other causes of megaloblastosis, particularly myelodysplasia. Other causes of megaloblastic anaemia not due to vitamin B12 or folate deficiency are listed in Table 22.5.6.6.

Some patients with rapidly developing megaloblastic anaemia, particularly due to folate deficiency, may develop almost complete aplasia of the red-cell series, and the peripheral blood and bone marrow may resemble that of acute myeloid leukaemia.

Diagnosis of vitamin B12 or folate deficiency

The peripheral blood and bone marrow appearances are identical in folate or vitamin B12 deficiency. Special tests are, therefore, needed to distinguish between the two deficiencies.

Vitamin B12 deficiency

The assay of serum vitamin B12 content of serum is now by competitive binding assays. The reference range, depending on the assay, is from 160–200 to 960–1200 ng/litre. The concentrations are low in vitamin B12 deficiency, being extremely low in patients with neurological disease. Unfortunately, using competitive-binding luminescence assays, false normal results have been reported in some patients with untreated pernicious anaemia and intrinsic factor antibodies in serum, which interfere with the test. Subnormal serum vitamin B12 concentrations in the absence of tissue vitamin B12 deficiency have been reported in pregnancy, in inherited mutations of TCI (haptocorrin), in severe nutritional folate deficiency, in subjects taking large doses of vitamin C, and occasionally in iron deficiency. In the elderly, low serum vitamin B12 concentrations usually in the range 100–160 ng/litre may occur in the absence of anaemia or macrocytosis. In some research studies serum holo TCII levels have been measured to diagnose vitamin B12 deficiency.

Raised serum vitamin B12 concentrations, if not due to therapy, are most commonly caused by a rise in TCI as in a leucocytosis due to a myeloproliferative disease. Raised haptocorrin also occur in association with some tumours, especially hepatoma and fibrolamellar tumour of the liver. In benign leucocytosis, the rise is mainly of TCIII and this is often not accompanied by a high serum vitamin B12. Raised serum TCII concentrations occur in conditions where macrophages are stimulated, e.g. autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, in Gaucher’s disease and in some monocytic or monoblastic leukaemias, in histiocytic lymphomas, and inflammatory bowel disease. In active liver diseases, serum vitamin B12 leaks from the liver with saturation of the serum vitamin B12 binders.

A second and less widely used test for vitamin B12 deficiency is serum methylmalonic acid (MMA). Serum MMA levels are raised in vitamin B12 deficiency but not in folate deficiency but raised levels may occur in renal failure. Rare cases of congenital methylmalonicaciduria have been described, owing to a variety of enzyme defects.

A sensitive method of measuring MMA in serum was introduced and combined with serum homocysteine assay for the diagnosis of vitamin B12 or folate deficiency. The significance of minor rises in serum MMA concentration found particularly in older people in the absence of macrocytes or anaemia with or without borderline vitamin B12 concentrations suggests ‘biochemical’ vitamin B12 deficiency which does not progress to megaloblastic anaemia. Randomized trials are needed to assess the value of preventing or treating disease due to putative vitamin B12 deficiency in these subjects.

Folate deficiency

Direct tests include the serum and red-cell folate assay. The serum folate is always low in folate deficiency (and is normal or raised in vitamin B12 deficiency unless folate deficiency is also present). Raised levels occur after folate therapy and also in vitamin B12 deficiency and in the stagnant-loop syndrome. Red-cell folate is a better guide than the serum folate to tissue folate stores but is low in a proportion of patients with megaloblastic anaemia solely due to vitamin B12 deficiency. Serum homocysteine levels are usually raised in folate and vitamin B12 deficiency and many other situations (see Chapter 12.2).

Diagnosis of the cause of vitamin B12 deficiency

Although the clinical and family history and the clinical findings may point to PA or some other cause of vitamin B12 deficiency, it is important to establish this for certain. A brief dietary history will rapidly establish whether or not the patient is a vegan or takes a very inadequate diet.

Endoscopy and gastric biopsy will show features of gastric atrophy and help to exclude gastric carcinoma. Follow-through radiographic examination of the small intestine will help to exclude a small-intestinal lesion, e.g. duodenal or jejunal diverticulosis.

The serum gastrin concentration is raised in patients with PA and the serum is tested for antibodies to IF, parietal cells, and thyroid; serum immunoglobulins are measured in view of the association with hypogammaglobulinaemia.

Diagnosis of the cause of folate deficiency

An inadequate diet is usually at least partly implicated, but an exact estimate of dietary intake from the clinical history is impossible because of variation in folate content of foods, losses in cooking, and size of portions. Often it is the general social circumstances that suggest a poor intake. Drug intake, particularly of barbiturates, is important. Many underlying inflammatory or malignant diseases may exaggerate the tendency to folate deficiency in patients with inadequate diets. The main cause of malabsorption of folate is gluten-induced enteropathy; in patients with severe folate deficiency, tests for transglutaminase and endomysial antibodies and a duodenal biopsy are usually necessary. In certain tropical countries, sprue may cause a generalized malabsorption syndrome in which folate deficiency commonly occurs.

Treatment of megaloblastic anaemia

Therapy is aimed at correcting the anaemia, completely replenishing the body of whichever vitamin is deficient, treatment of the underlying disorder, and prevention of relapse. In most cases, it is possible to diagnose which deficiency is present before starting therapy.

Vitamin B12 deficiency

Hydroxocobalamin 1000 µg intramuscularly given six times at several days’ interval over the first few weeks will restore normal vitamin B12 stores. There is no evidence that patients with vitamin B12 neuropathy derive greater benefit from more frequent doses, although many physicians use these for 6 months or so.

Response to therapy

The patient feels better within 24 to 48 h, and the mild fever, if not due to infection, abates. A painful tongue and uncooperative, disorientated state may also be improved in 48 h. The white-cell count becomes normal by 3 to 7 days and the platelet count rises and may reach levels of 500 to 1000 × 109/litre before falling to normal at about 10 to 14 days. The bone marrow reverts to normoblastic by 36 to 48h, although giant metamyelocytes persist for 10 to 12 days.

The neuropathy always improves with therapy but residual deficits remain in some patients; this applies usually to those with the longest histories or the most severe manifestations, particularly where there is subacute combined degeneration of the spinal cord and spastic paraparesis.

Maintenance

Hydroxocobalamin, 1000 µg intramuscularly, is given once every 3 months for life in PA and most other causes of vitamin B12 deficiency to prevent relapse. The life expectancy in PA once treated, is as good as that in the general population in women, and slightly lower in men, probably due to the increased incidence of carcinoma of the stomach. In a few patients with vitamin B12 deficiency, the underlying cause can be reversed; e.g. expulsion of the fish tapeworm, improvement of vegan diet, surgical correction of an intestinal stagnant loop. A few micrograms of vitamin B12 can be absorbed each day in PA from oral doses of 1000 µg or more by passive diffusion, but this maintenance therapy is usually reserved for those who cannot have injections—e.g. those with a bleeding disorder, or who refuse them—and for the extremely rare individual who is allergic to all injectable forms of vitamin B12. Vegans may be maintained on much smaller oral doses of vitamin B12 each day, such as 50 µg as a tablet or syrup.

Prophylaxis

Vitamin B12 therapy should be given from the time of operation after total gastrectomy or ileal resection. Patients with PA tend to develop iron-deficiency anaemia and they may also develop thyroid disorders or carcinoma of the stomach. It is advisable that a regular blood count be made once a year. Routine endoscopy is not warranted but these diseases must be particularly borne in mind if relevant symptoms or signs develop.

It is unclear whether vitamin B12 should be given orally or parenterally to those with biochemical vitamin B12 deficiency (see Chapter 11.2) without anaemia or macrocytosis or clinical symptoms. Trials are needed.

Folate deficiency

This is corrected by giving 5 mg folic acid by mouth daily. It is essential to first exclude vitamin B12 deficiency so that precipitation of a neuropathy is avoided. It is usual to continue for at least 4 months until there is a completely new set of red cells, although body stores will theoretically be normal within a few days of therapy. In patients with severe malabsorption of folate, larger oral doses of folic acid (e.g. 5 mg three times a day) may be used but it is not necessary to give parenteral folate except for those unable to swallow tablets. The response to therapy is as described for vitamin B12. The decision whether or not to continue folic acid beyond 4 months depends on whether or not the cause can be corrected. In practice, long-term folic acid is usually needed only in patients with severe haemolytic anaemias (e.g. sickle cell anaemia and thalassaemia major), myelofibrosis, and in gluten-induced enteropathy when a gluten-free diet is either unsuccessful or not feasible.

Prophylactic folic acid

This should be given to all pregnant women to prevent megaloblastic anaemia and reduce the incidence of neural tube defects (doses of 300 to 400 µg/day are used). Only 19% of women in a Northern Ireland study had taken folic acid pre-conception, resulting in a lower red cell folate and thus increased risk of NTD, during the first trimester, than in women who had taken preconception folic acid. Studies in the USA show that black and Hispanic women have a lower dietary intake of folate than white non-Hispanic women and, therefore, particularly need folic acid supplements peri-conception. Doses of 5 mg/day would have a greater effect but currently need a medical prescription in the United Kingdom. They are given if there has been a previous infant with an NTD. Folic acid is given to patients undergoing regular haemodialysis or peritoneal dialysis, to premature infants weighing less than 1.5 kg at birth, and to selected patients in intensive care units or receiving parenteral nutrition. In young children exposed to a high risk of malaria combined iron and folic acid supplements may be harmful and should be avoided.

Folate therapy has been shown to improve chromosomal stability in the fragile X syndrome, even though these patients do not have folate deficiency or a demonstrable defect of folate metabolism.

Food fortification

Mandatory fortification of cereals and grains with folic acid (140 μ‎g/100 g cereal grain) aimed at reducing the incidence of NTDs began in the United States of America in 1997 and is now also practised in Canada, Chile, and other countries amounting to about 20% of the world’s population. Median serum folate in clinical specimens in United States rose from 12.6 to 18.7 μ‎g/litre between 1997 and 1998. There was also a fall in serum homocysteine levels. The theoretical side effects of fortification are largely in patients with unsuspected vitamin B12 deficiency who, it has been suggested, might present with neuropathy if the extra folate consumed prevents the development of anaemia due to vitamin B12 deficiency. There is, however, no evidence for an increased incidence of nonanaemic subjects with low serum vitamin B12 levels in the United States since fortification. In the United Kingdom fortification of flour with folic acid (240 μ‎g/100 g flour) has been recommended but not implemented. The possible effects of fortification on cardiovascular disease and cancer have been discussed. Fortification of grain with vitamin B12 has also been suggested to reduce the incidence of NTD, but this has not been implemented in any country.

Folinic acid (5-formyl-THF)

This reduced folate is used to prevent or treat toxicity due to methotrexate or other dihydrofolate reductase inhibitors.

Severely ill patients

Some patients, usually elderly, are admitted to hospital severely ill with megaloblastic anaemia, perhaps in congestive heart failure or with pneumonia. In this case, it is necessary to commence therapy immediately after obtaining blood for vitamin B12 and folate assay, before it is known which deficiency is present. Both vitamins should be given simultaneously in large doses. Heart failure and infection should be treated in conventional fashion but blood transfusion should be avoided, except in cases of extreme anaemia, when 1 to 2 units of packed cells may be given slowly, accompanied by removal of a similar volume of blood from the other arm, and diuretic therapy.

Other therapy

Hypokalaemia has been reported to occur during initial therapy but is, rarely, if ever, clinically important. An attack of gout has been reported on the days 6 to 7 of therapy. Most patients develop hyperuricaemia at this stage but the clinical disease probably only occurs in those with a strong gouty tendency. Iron deficiency commonly develops in the first few weeks of therapy and this should be treated initially with oral ferrous sulphate in the usual way.

Megaloblastic anaemia due to inborn errors of folate or vitamin B12 metabolism

Folate

A number of babies have been described with congenital deficiency of one or other enzyme concerned in folate metab-olism: 5-methyltetrahydro-folate, methylene THF-reductase, FIGLU-transferase, methenyl-THF cyclohydrolase. Some of the babies had multiple congenital defects including the heart and cerebral ventricles and nearly all showed impaired mental development. In the methylfolate transferase deficiency, megaloblastic anaemia was present.

Vitamin B12

Congenital deficiency of TCII was first reported as an autosomally recessive disease in 1971 in two siblings who developed megaloblastic anaemia requiring therapy with large daily doses of vitamin B12 at 3 and 5 weeks of age. Similarly affected families have been described. A spectrum of mutations in the gene for TCII have been detected. In some cases, TCII is undetected; in others, often presenting later in life functionally inactive TCII has been detected. The serum vitamin B12 concentration is normal, vitamin B12 being bound to TCI. Absorption of vitamin B12 is impaired. Treatment is with massive doses of vitamin B12 (e.g. 1000 µg intramuscularly three times each week). Delay in treatment may allow a neuropathy to occur. In contrast, in subjects with rare, inherited, mutations of TCI, low serum vitamin B12 concentrations occur, but haemopoiesis is normal.

Children with one form of congenital methylmalonicaciduria, which responds to vitamin B12 therapy in large doses, have been shown to have a defect in conversion of hydroxocobalamin to adocobalamin. They do not show megaloblastic anaemia. In a few, this defect has been associated with a defect of formation of methylcobalamin and with homocystinuria, but some of the children have also surprisingly not shown megaloblastic anaemia. Neurological abnormalities are usual. Homocystinuria and megaloblastic anaemia without methymalonicaciduria have also been reported. In some cases, the defect appears to be in maintaining vitamin B12 bound to methionine synthase in the reduced state.

Megaloblastic anaemia due to acquired disturbances of folate or vitamin B12 metabolism

Folate

Therapy with dihydrofolate reductase inhibitors may cause megaloblastic anaemia. This is usual with methotrexate and less likely with pyrimethamine unless high doses are used or the patient is already folate deficient. Trimethoprim and triamterene are very weak folate antagonists in man, but may precipitate megaloblastic anaemia in patients already vitamin B12 or folate deficient (see earlier).

Vitamin B12

Nitrous oxide (N2O)

This anaesthetic gas oxidizes vitamin B12 from the active fully reduce cob(I)alamin form to the inactive cob(II)alamin and cob(III)alamin forms, inactivating methylcobalamin and hence methionine synthase. Megaloblastosis develops within several hours in humans. This recovers over several days when exposure to N2O is discontinued. After many weeks exposure to N2O, monkeys develop a neuropathy resembling vitamin B12 neuropathy in humans; peripheral neuropathies and more severe neurological disease have also been described in humans (e.g. dentists and anaesthetists) repeatedly exposed to the gas. When N2O is used as anaesthetic for patients with low vitamin B12 stores, megaloblastic anaemia or neuropathy may be precipitated months later, due to failure to replenish vitamin B12 stores by absorption. Recovery from N2O exposure needs new cobalamin and also synthesis of new apoenzyme (methionine synthase) because this protein is also damaged by active oxygen derived from the N2O–cobalamin reaction. Methylmalonicaciduria has not been found in animals or humans exposed for short periods to N2O, as methylmalonic CoA mutase does not need reduced vitamin B12.

Megaloblastic anaemia not due to folate or vitamin B12 deficiency or metabolic defect

Congenital

Orotic aciduria

This is a very rare, recessive disorder involving two consecutive enzymes (orotidsylic pyrophosphatase and orotidylic decarboxylase) in pyrimidine synthesis and presents with megaloblastic anaemia in the first few months of life. The diagnosis is made if needle-shaped, colourless crystals of orotic acid are found in the urine, daily excretion ranging from 0.5 to 1.5 g. Heterozygotes excrete slightly raised amounts of orotic acid but show no haematological disorder. Treatment with uridine (1–1.5 g/day) leads toa haematological response, restoration of normal haemopoiesis and growth, and reduction in orotic acid excretion.

Lesch–Nyhan syndrome

A few patients with this rare disorder of purine synthesis have shown megaloblastic change but whether this was due to associated folate deficiency or a direct result of reduced purine synthesis is not certain (see Chapter 12.2).

Vitamin E deficiency

This has been reported to cause megaloblastosis in a group of children with kwashiorkor. However, many were also folate deficient.

Vitamin C deficiency

Megaloblastic appears to be due to associated folate deficiency.

Thiamine responsive

About 12 cases have been well documented. They have also shown sideroblastic change and a defect in phosphorylation of thiamine has been implicated. Diabetes mellitus and sensineural deafness are additional features. There is a fault in thiamine phosphorylation due to a genetic defect of the phosphorylase enzyme.

Responding to large doses of vitamin B12 and folate

A single patient has been reported who needed both vitamins in large doses, but the site of the defect was not elucidated.

Congenital dyserythropoietic anaemia

Some cases of congenital dyserythropoietic anaemia show megaloblastic changes not due to vitamin B12 or folate deficiency.

Acquired

Megaloblastic changes are often marked in acute myeloid leukaemia and less commonly in other forms of acute myeloid leukaemia. They also occur in the myelodysplastic syndromes. The exact site of block in DNA synthesis in these syndromes is unknown.

Drugs that directly inhibit purine or pyrimidine synthesis (e.g. cytosine arabinoside, 5-fluorouracil, hydroxyurea, 6-mercaptopurine, or azathioprine and azidothymidine (AZT)) may cause megaloblastic anaemia. Alcohol has also been found to have a direct effect on the bone marrow, causing megaloblastosis in some cases even in the absence of vitamin B12 or folate deficiency. On the other hand, drugs that inhibit mitosis (e.g. colchicine or daunorubicin) or alkylate preformed DNA (e.g. cyclophosphamide, chlorambucil, or busulfan) do not cause megaloblastosis.

Other deficiency anaemias

Vitamin C

Anaemia is usual in scurvy but the pathogenesis is complicated. It is likely that vitamin C has a direct effect on erythropoiesis but folate and iron deficiencies, haemorrhage, or haemolysis often complicate the picture.

Biochemical and nutritional aspects

Vitamin C is needed for collagen synthesis by its involvement in the hydroxylation of protein and for maintenance of intercellular substance of skin, cartilage, periosteum, and bone. It may also have a general role in oxidation–reduction systems, e.g. glutathione, cytochromes, pyridine, and flavin nucleotides. Although vitamin C is also thought to be needed for maintaining body folates in the reduced active state, the exact reactions involved are unclear. Vitamin C has a particular role in iron metabolism, iron excess causing increased utilization of vitamin C and in extreme cases clinical scurvy, whereas iron deficiency is associated with a raised leucocyte ascorbate concentration. Vitamin C is needed for incorporation of iron from transferrin into ferritin and for iron mobilization from ferritin. Vitamin C therapy increases iron excretion in patients receiving subcutaneous desferrioxamine infusions and also, at least in experimental animals, affects iron distribution by increasing parenchymal relative to reticuloendothelial iron. Minimum adult daily requirements for vitamin C are about 10 mg but 30 to 70 mg is recommended; utilization, and therefore requirement, are relatively higher in infants, children, and pregnant and lactating women. Vitamin C may be excreted as such but is also broken down to oxalate.

Vitamin C is present in food as its reduced (ascorbic acid) and oxidized (dehydroascorbic acid) forms, the highest concentrations occurring in green vegetables, fruits, tomatoes, liver, and kidney. Potatoes are not a rich source but provide a substantial proportion of normal dietary intake. Cooking, particularly in alkaline conditions with large volumes of water, destroys the vitamin, which is also lost on storage with exposure to the air. Absorption occurs through the length of the small intestine and deficiency is never solely due to malabsorption.

The anaemia of scurvy is typically normochromic, normocytic with a slightly raised reticulocyte count (to 5 to 10%) and a normoblastic marrow with erythroid hyperplasia. This suggests a direct role for vitamin C in erythropoiesis but not all patients with clinical scurvy are anaemic. Extravascular haemolysis with mild jaundice and increased urobilinogen excretion occurs in many of the patients. Moreover, in many the anaemia is complicated by folate deficiency (due to inadequate folate intake) with a megaloblastic marrow, or in a few by iron deficiency due to external haemorrhage, reduced diet intake, and possibly reduced iron absorption. In a few patients placed on a low-folate diet, response of megaloblastic haemopoiesis to vitamin C alone has been described. In others, response of the megaloblastic anaemia to folic acid alone on a diet low in vitamin C has occurred but in most such cases, both vitamin C and folic acid have been found necessary.

Vitamin B6

This, as its coenzyme form pyridoxal-5-phosphate, is involved in many reactions of the body, especially transaminases and decarboxylases. It is also a cofactor in the important rate-limiting reaction in haem synthesis, δ‎-aminolaevulinic acid (ALA)-synthase (see Section 12). It occurs in natural tissues in three major forms: pyridoxine, pyridoxamine, and pyridoxal phosphate. Red cells are capable of interconverting them. Anaemia due purely to vitamin B6 deficiency has been produced in animals. It is hypochromic and microcytic with a raised serum iron and increased iron in erythroblasts, with some partial or complete ring sideroblasts. A similar anaemia has occurred in humans with malabsorption, pregnancy, or haemolysis but has not been fully documented to respond to physiological doses of vitamin B6 alone. Vitamin B6-responsive anaemia is, however, well documented among patients with sideroblastic anaemia of all types. Pyridoxine responses occur particularly in the inherited form (when it is assumed that a fault in one or other enzyme of haem synthesis, e.g. ALA-synthase, increases the need for pyridoxal phosphate as cofactor) and when sideroblastic anaemia occurs in patients receiving pyridoxine antagonists, such as antituberculous drugs. The value of pyridoxine dietary supplements in lowering serum homocysteine and reducing the incidence of cardiovascular disease has yet to be proven.

Riboflavin

On the basis of studies in experimental animals and humans fed a deficient diet together with a riboflavin antagonist, deficiency of this vitamin is known to cause a normochromic, normocytic anaemia associated with a low reticulocyte count and red-cell aplasia in the marrow, sometimes with vacuolated normoblasts. The exact biochemical basis is undecided. Clinically, a similar anaemia may occur in pure form but is usually associated with the anaemia due to protein deficiency, as in kwashiorkor or marasmus. Other clinical features of riboflavin deficiency—dermatitis, angular cheilosis, and glossitis for example—may be present.

Thiamine

For discussion, see under megaloblastic anaemia not due to folate or vitamin B12 deficiency or metabolic defect.

Nicotinic acid, pantothenic acid, and niacin

Deficiencies of these vitamins cause anaemia in experimental animals, but anaemia purely due to one or other of these deficiencies has not been established to occur in humans.

Vitamin E

This vitamin is needed for preventing peroxidation of cell membranes. A haemolytic anaemia responding to vitamin E has been reported in premature infants. Less well documented is a macrocytic anaemia due to vitamin E deficiency in protein–calorie-deficient infants and aggravation of anaemia in patients with thalassaemia major because of vitamin E deficiency.

Protein deficiency

(see Chapter 11.3)

Anaemia is usual in both ‘pure’ protein deficiency (kwashiorkor) and in protein–calorie malnutrition (marasmus). It has been reported in many parts of the world where malnutrition, especially in children and pregnant women, is common. The anaemia also occurs in patients with gastrointestinal disease and severe malabsorption. The anaemia is typically normochromic, normocytic, and of the order of 8.0 to 9.0 g/dl. The reticulocyte count is usually reduced and the marrow may show a selective reduction in erythropoiesis. Experimental studies in animals suggest that the anaemia is largely due to reduced serum erythropoietin levels consequent on a lack of stimulus for erythropoietin secretion. Lack of amino acids for synthesis of erythropoietin or globin is not the cause. In many patients, the anaemia is complicated by infection, folate or iron deficiency, and possibly other vitamin deficiencies (e.g. riboflavin, vitamin E) and then it may be more severe and show additional morphological abnormalities in the blood and marrow.

Further reading

General

Clarke R (2012). Folic acid and vitamin B12 for prevention of cognitive decline in older people with depression. Aging Health, 8, 115–7.Find this resource:

    Lewerin C (2008). Serum biomarkers for atrophic gastritis and antibodies against Helicobacter pyloric in the elderly: Implications for vitamin B12, folic acid and iron status and response to oral vitamin therapy. Scand J Gast, 143, 1502–08.Find this resource:

      Moore EM, et al. (2014). Among vitamin B12 deficient older people, high folate levels are associated with worse cognitive function: combined data from three cohorts. J Alzheim Dis, 39, 661–8.Find this resource:

        Morris MS, Selhub J, Jacques PF (2012). Vitamin B-12 and folate status in relation to decline in scores on the mini-mental state examination in the framingham heart study. J Am Geriatr Soc, 60, 1457–64.Find this resource:

          Wald DS, et al (2011). Serum homocysteine and dementia: meta-analysis of eight cohort studies involving 8669 participants. Alzh Dement, 7, 412–7.Find this resource:

            Whitehead VM (2006). Acquired and inherited disorders of cobalamin and folate in children. J Haematol, 134, 125–36.Find this resource:

              Vitamin B12

              Bunn HF (2014). Vitamin B12 and pernicious anemia—the dawn of molecular medicine. N Engl J Med, 370, 773–6.Find this resource:

                Carmel R (2011). Biomarkers of cobalamin (vitamin B12-12) status in the epidemiologic setting: A critical overview of context, applications, and performance characteristics of cobalamin, methylmalonic acidand holotranscobalamin 11. Am J Clin Nutr, 97, 541–56.Find this resource:

                  Carmel R (2012). Subclinical cobalamin deficiency. Curr Opin Gastroenterol, 28, 151–8.Find this resource:

                    Carmel R, Agrawal YP (2012). Failures of cobalamin assays in pernicious anemia [letter]. N Engl J Med, 367, 385–6.Find this resource:

                      Carmel R, Parker J, Kelman Z (2009). Genomic mutations associated with mild and severe deficiencies of transcobalamin I (haptocorrin) that cause mildly and severely low serum cobalamin levels. Br J Haematol, 147, 386–91.Find this resource:

                        Dali-Youcef N, Andres E (2009). An update on cobalamin deficiency in adults. QJM, 102, 17–28.Find this resource:

                          Hershko C, et al. (2006). Variable hematological presentation of autoimmune gastritis: age related progression from iron deficiency to cobalamin depletion. Blood, 107, 1673–9.Find this resource:

                            Mills JL (2011). Do high blood folate concentrations exacerbate metabolic abnormalities in people with low vitamin B12 status. Am J Clin Nutr, 94, 495–500.Find this resource:

                              Quadros EV (2009). Advances in the understanding of cobalamin assimilation and metabolism. Brit J Haematol, 148, 195–204.Find this resource:

                                Toh B-H, et al. (2012). Cutting edge issues in autoimmune gastritis. Clin Rev Allerg Immunol, 42, 269–78.Find this resource:

                                  Weir DG, Scott JM (1997). Brain function in the elderly: role of B12 and folate. Br Med Bull, 55, 669–82. [A large review of this important topic.]Find this resource:

                                    Yin Y, et al. (2007). NALPI in vitiligo-associated multiple auto immune disease. N Engl J Med, 356, 1216–25.Find this resource:

                                      Folate

                                      Bergen NE, et al. (2012). Homocysteine and folate concentrations in early pregnancy and the risk of adverse pregnancy outcomes: the Generation R Study. Br J Obstet Gynaecol, 119, 739–51.Find this resource:

                                        English M, Snow RW (2006). Iron and folic acid supplements and malaria risk. Lancet, 367, 90–1.Find this resource:

                                          Ford AH, Almeida OP (2012). Effect of homocysteine lowering treatment on cognitive function: a systematic review and meta-analysis of randomized controlled trials. J Alzheimers Dis, 29, 133–49.Find this resource:

                                            Hoffbrand AV (2014). Professor John Scott, folate and neural tube defects. J Alzheimers Dis, 29, 133–49.Find this resource:

                                              Kibar Z, et al. (2007). Mutations in VANGLI associated with neural tube defects. N Engl J Med, 356, 1432–7.Find this resource:

                                                Matherly LH, Goldman ID (2003). Membrane transport of folates. Vitam Horm, 66, 403–56.Find this resource:

                                                  Mills JH, et al. (2003). Low B12 concentrations in patients without anemia: the effect of folic acid fortification of grain. Am J Clin Nutr,77, 1474–7.Find this resource:

                                                    Qui A, et al. (2006). Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell, 127, 917–28.Find this resource:

                                                      Salojin KV, et al. (2011). A mouse model of hereditary folate malabsorption: deletion of the PCFT gene leads to systemic folate deficiency. Blood, 117, 4895–904.Find this resource:

                                                        Wright AJA, Dainty JR, Finglas PM (2007). Folic acid metabolism in human subjects revisited: potential implications for proposed mandatory folic acid fortification in the U.K. Br J Nutr, 98, 667–75.Find this resource:

                                                          Zhao R, et al. (2007). The spectrum of mutations in the PCFT gene, coding for an intestinal folate transporter that are the basis for hereditary folate malabsorption. Blood, 110, 1147–52.Find this resource:

                                                            Neural tube defects

                                                            Botto L, et al. (1999). Neural-tube defects. N Engl J Med, 341, 1509–18. [A major review of all aspects of NTD.]Find this resource:

                                                              Correa A, et al. (2012). Lack of periconceptional vitamins or supplements that contain folic acid and diabetes mellitus-associated birth defects. Am J Obstet Gynecol, 206, 218.e1–18.13.Find this resource:

                                                                De Wals P, et al. (2007). Reduction in neural tube defects after folic acid fortification in Canada. N Engl J Med, 357, 135–42.Find this resource:

                                                                  McNulty B, et al. (2011). Women’s compliance with current folic acid recommendations and achievement of optimal vitamin status for preventing neural tube defects. Hum Reprod, 26, 1530–6.Find this resource:

                                                                    Molloy AM, et al. (2009). Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification. Pediatrics, 123, 917–23.Find this resource:

                                                                      Pangilinan F, et al. (2010). Transcobalamin II receptor polymorphisms are associated with increased risk for neural tube defects. J Med Genet, 47, 677–85.Find this resource:

                                                                        Cardiovascular disease

                                                                        Ji Y, et al. (2013). Vitamin B supplementation, homocysteine levels and the risk of cerebrovascular disease. A meta-analysis. Neurology, 81, 1298–307.Find this resource:

                                                                          Wald DS, et al. (2011). Reconciling the evidence on serum homocysteine and ischaemic heart disease: a meta-analysis. PLoS ONE, 6, e1643.Find this resource:

                                                                            Yang H-T, et al. (2012). Efficacy of folic acid supplementation in cardiovascular disease prevention. An updated meta-analysis of randomized controlled trials. Europ J Int Med, 23, 745–54.Find this resource:

                                                                              Cancer

                                                                              Logan RFA, et al. (2007). Aspirin and folic acid for the prevention of recurrent colorectal adenomas. Gastroenterology, 134, 29–38.Find this resource:

                                                                                Vollset SE, et al. (2013). Effects of folic acid supplementation on overall and site - specific cancer incidence during randomized trials: meta-analysis of data on 50,000 individuals. Lancet, 381, 1029–36.Find this resource:

                                                                                  Miscellaneous

                                                                                  Adams EB (1970). Anemia associated with protein deficiency. Semin Haematol, 7, 55–66. [An excellent review of the role of protein deficiency in causing anaemia.]Find this resource:

                                                                                    Cox EV (1968). The anaemia of scurvy. Vitam Horm, 26, 635–52. [An excellent review of the role of vitamin C in haemopoiesis.]Find this resource:

                                                                                      Rindi G, et al. (1994). Further studies of erythrocyte thiamin transport and phosphorylation in seven patients with thiamin-responsive megaloblastic anaemia. J Inher Metabol Dis, 17, 667–77. [Shows the mechanism of thiamine responsive anaemia.]Find this resource: