# 22.6.6 Megaloblastic anaemia and miscellaneous def

# 22.6.6 Megaloblastic anaemia and miscellaneous deficiency anaemias 5407 A.V. Hoffbrand

22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5407
these conditions may mobilize iron from macrophage stores, easing 
functional iron deficiency and facilitating erythropoiesis. ESAs are 
widely used to treat patients with chronic kidney disease in whom 
endogenous erythropoietin production is inadequate to maintain 
erythropoiesis in the context of declining haemoglobin concentra-
tions. However, use of ESAs for routine treatment of anaemia of in-
flammation is no longer recommended, although these drugs may be 
valuable in selected cases where serum erythropoietin levels have been 
measured and are inappropriately low. The goal should be to gradually 
increase haemoglobin concentrations to a target range between 100 
and 120 g/​l, as rapid increases or higher levels have been associated 
with cardiovascular risk among patients with renal failure and cancer.
Prognosis and outcome
Anaemia of inflammation will persist for as long as erythroblasts are 
deprived of iron by the effects of hepcidin, or via direct cytokine-​
mediated suppression of erythropoiesis. Resolution or control of 
the underlying condition should relieve these processes and facili-
tate erythropoiesis. Some emerging data suggest that following re-
covery from inflammation, rebound erythropoiesis may transiently 
suppress hepcidin, facilitating enhanced iron absorption and release 
from macrophages, and optimizing recovery from anaemia.
Special circumstances
Anaemia of the elderly
Elderly individuals have a higher prevalence of anaemia compared 
with the younger population. For example, in Australia 16% of indi-
viduals older than 75 years are anaemic compared with fewer than 
5% of younger adults. The prevalence of anaemia increases with age 
and among individuals in hospital or in care. Anaemia in the eld-
erly is consistently correlated with impaired physical and cognitive 
performance, and increased frailty. Epidemiological studies indicate 
that about one-​third of cases of anaemia of the elderly are due to 
deficiencies of iron and other haematinics. A further third are due 
to anaemia of inflammation due to identifiable medical comorbid-
ities and renal impairment. The causes of the final third are more 
difficult to ascertain, and it is often termed ‘unexplained anaemia 
of the elderly’. Some cases may also represent underlying malig-
nant haematological diseases such as myelodysplasia. The mech-
anisms of unexplained anaemia require further investigation, but 
may be multifactorial, including reduced erythroid potential of the 
haematopoietic stem cell compartment, hepcidin-​dependent and -​
independent mechanisms of anaemia of inflammation, reduced 
erythropoietin production in response to reduced haemoglobin 
concentrations, and in men, hypoandrogenism. Elderly patients 
with anaemia should be appropriately investigated to identify po-
tential therapies and exclude serious underlying conditions.
Areas of uncertainty and controversy
Treatment of anaemia of inflammation remains challenging, as few 
specific therapies are presently available. Further work is needed to 
assess the role, timing, and optimal regimens of intravenous iron and 
ESAs. The safety of and optimal targets for treatment with ESAs re-
quire investigation in nonrenal-​ or cancer-​related anaemia. The role 
of measurement of hepcidin in diagnosis of anaemia of inflammation 
and its role in guiding treatment need development. The epidemi-
ology and mechanisms of anaemia of the elderly requires clarification.
Future developments
The discovery that hepcidin is a key mediator of anaemia of inflam-
mation has stimulated interest in development of novel therapeutics 
which either directly inhibit its action or potentially, prevent its expres-
sion. For example, hepcidin expression can be prevented by heparin, 
and heparin-​derived compounds (e.g. glycol-​split or oversulphated 
heparins) have been shown to reduce hepcidin levels and alleviate an-
aemia of inflammation in experimental animals. Likewise, antihepcidin 
monoclonal antibodies and the antihepcidin L-​ribonucleic acid 
aptamer (NOX-​H94) both neutralize circulating hepcidin, reducing 
the anaemia of inflammation in animal models. Finally, recombinant 
erythroferrone (the erythroid-​derived suppressor of hepcidin) may 
prove to be a useful strategy for reducing hepcidin expression and 
treating anaemia of inflammation in the future.
FURTHER READING
Camaschella C, Pagani A (2018). Advances in understanding iron 
metabolism and its crosstalk with erythropoiesis. Br J Haematol, 
182(4), 481–94.
Cullis JO (2011). Diagnosis and management of anaemia of chronic 
disease: current status. Br J Haematol, 154, 289–​300.
Ganz T (2019). Anemia of inflammation. N Engl J Med, 381, 1148–57.
National Blood Authority (2012). Patient blood management guide-
lines: module 3 medical. National Blood Authority, Canberra.
Thomas DW, et al. (2013). Guideline for the laboratory diagnosis of 
functional iron deficiency. Br J Haematol, 161, 639–​48.
Weiss G (2015). Anemia of chronic disorders: new diagnostic tools and 
new treatment strategies. Semin Hematol, 52, 313–​20.
Weiss G, Ganz T, Goodnough LT (2019). Anemia of inflammation. 
Blood, 133, 40–50.
22.6.6  Megaloblastic anaemia and 
miscellaneous deficiency anaemias
A.V. Hoffbrand
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 vitamin 
B12 or folate metabolism, or affects DNA synthesis independent of 
vitamin B12 or folate.


section 22  Haematological disorders
5408
Biochemical and nutritional aspects of vitamin  
B12 and folate
Vitamin B12—​synthesized by bacteria; in humans the daily re-
quirement of 1 to 2 μg is acquired from secondary animal sources 
including fish, eggs, milk, and meat. Processing within the body oc-
curs 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 vitamin 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 lyso-
somal 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 des-
troyed by cooking. The daily requirement is about 100 μg, with ab-
sorption occurring through a proton-​coupled folate transporter in 
the proximal small intestine and duodenum. Attached glutamate res-
idues are cleaved, releasing methyl tetrahydrofolate into the portal 
plasma.
Biochemical basis of megaloblastic anaemia—​(1) folate defi-
ciency reduces the availability of the coenzyme 5,10-​methylene 
tetrahydrofolate (THF) polyglutamate, thus inhibiting synthesis 
of thymidylate, which is the rate-​limiting step in DNA synthesis. 
(2) Vitamin B12 deficiency impairs DNA synthesis indirectly because it 
is needed for conversion of methyl-​THF entering cells from plasma to 
THF, the substrate of active folate coenzymes (folate polyglutamates) 
including 5,10 methylene-​THF.
Causes of megaloblastic anaemia
Vitamin B12 deficiency—​(1) malabsorption—​including (a)  gastric 
causes (e.g. acquired pernicious anaemia, gastrectomy); (b) intestinal 
causes (e.g. bacterial overgrowth, ileal resection); and (2) nutritional 
(e.g. vegans).
Folate deficiency—​(1) poor diet—​e.g. poverty, alcoholism; (2) mal-
absorption (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. anti-
convulsants); and (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); and 
(c) drugs (e.g. antimetabolites, hydroxycarbamide).
Laboratory investigation
This consists of three stages: (1) recognition that megaloblastic an-
aemia is present—​the mean corpuscle volume is raised to 100 to 
140 fl, and the peripheral blood shows hypersegmented neutrophils. 
The bone marrow (if examined) 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, 
transglutaminase antibody, and pursuing clinical clues to other pos-
sible causes. Subclinical deficiency of both vitamins is more frequent 
than overt megaloblastic anaemia.
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 autoimmune gastritis and failure of absorption 
of vitamin B12.
Clinical features—​anaemia usually develops gradually, and symp-
toms 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, and 
(5) features of an associated disorder (e.g. vitiligo, thyroid disease). 
Complications include (1) peripheral sensorimotor neuropathy and 
(2) subacute combined degeneration of the spinal cord—​manifest as 
loss of proprioception and pyramidal weakness.
Treatment and prevention of megaloblastic anaemia
Vitamin B12 deficiency—​may be treated with intramuscular 
hydroxocobalamin (1-​mg doses, six given in the first 2–​3 weeks, then 
every 3 months). Oral therapy is practised by a minority and is un-
likely to be useful in pernicious anaemia. Neurological complications 
are irreversible unless treated early.
Folate deficiency—​high-​dose oral folic acid (5 mg daily) over-
comes 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 by 
methotrexate, oral or parenteral folinic acid is given to restore DNA 
synthesis.
Prevention—​dietary folate fortification is an accepted and highly 
effective public health measure in many countries (none in Europe) 
for reducing the incidence of neural tube birth defects.
Introduction
The megaloblastic anaemias are a group of disorders characterized 
by a macrocytic anaemia and distinctive morphological abnormal-
ities of the developing haematopoietic cells in the bone marrow. In 
severe cases, the anaemia may be associated with leucopenia and 
thrombocytopenia. Megaloblastic anaemia arises because of in-
hibition of DNA synthesis in the bone marrow, usually due to de-
ficiency of one or other of two water-​soluble B vitamins: vitamin 
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 disturb-
ance 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 men-
tioned later in this chapter.
Folic acid supplements in pregnancy and food fortification with 
folic acid are aimed at preventing neural tube defects. Possible 
relations between folate and vitamin B12, and cardiovascular or 


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5409
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. 22.6.6.1 and Table 22.6.6.1). 
5′-​Deoxyadenosyclobalamin (adocobalamin) accounts for about 
80% of vitamin B12 inside mammalian cells and is located mainly 
in mitochondria; methylcobalamin is a minor cellular compo-
nent but the main form in plasma. Both are extremely light sensi-
tive 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 re-
duced 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.
Vitamin B12 is known to be involved in only three reac-
tions 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. 22.6.6.2). In some bacteria, but not in humans, vitamin B12 
has a direct role in DNA synthesis by virtue of its involvement in 
ribonucleotide reductase.
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 to 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 glyco-
protein 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. Gastric pepsin and pancre-
atic trypsin degrade this protein and so releases vitamin B12 for at-
tachment to intrinsic factor (IF) and subsequent absorption. IF is 
a glycoprotein produced mainly by the gastric parietal cells (Table 
22.6.6.2). The normal stomach produces a vast excess of IF, meas-
ured in units (1 unit binds 1 ng vitamin B12). Vitamin B12 in bile 
is also attached to IF and reabsorbed through the ileum. At neu-
tral pH, in the presence of calcium ions, the vitamin B12–​IF com-
plex 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 pre-
sent 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–​B12 complex. 
Mutations of cubilin or AMN underlie hereditary malabsorption of 
vitamin B12 (discussed later in this chapter).
After cubilin–​AMN-​mediated endocytosis, IF undergoes lyso-
somal degradation. After a delay of 3 to 5 h, vitamin B12 appears in 
portal blood, with a peak concentration 8 h after ingestion, com-
plexed 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 
 N
N
CH2
CH2
CH2
CH3
CO
NH
CH
O
O –
P
N
A
C
B
CO+
OH
C
D
N
N
N
N
C
C
O
O
O
C
CH2OH
CH3
CH3
Fig. 22.6.6.1  The structure of cyanocobalamin.


section 22  Haematological disorders
5410
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 duo-
denal mucosae without the involvement of the IF pathway and this 
forms the basis for treating malabsorption of vitamin B12 with large 
oral doses of cyanocobalamin.
Transport
Vitamin B12 in plasma is 70 to 90% attached to a glycoprotein, 
transcobalamin I (TCI), and 0 to 10% to transcobalamin III (TCIII), 
which do not enhance cell uptake of vitamin B12 (Table 22.6.6.2). 
TCI and III belong to a group of glycoproteins, the R binders or 
haptocorrins (previously mentioned), that are present in many tis-
sues and fluids; these molecules have the same amino acid compos-
ition 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 excre-
tion in the bile. Genetic mutations of the gene TCN1, which codes 
for TCI, cause subnormal serum vitamin B12 levels.
The most important plasma vitamin B12-​binding protein, TCII, 
is synthesized in macrophages, the liver, the ileum, and possibly the 
endothelium. TCII is loaded with vitamin B12 from the ileum and 
by release of vitamin B12 from the liver and other organs. It is nor-
mally 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 internal-
ized by endocytosis; vitamin B12 is released by proteolytic cleavage 
in lysosomes but TCII is not reutilized (Table 22.6.6.1). TCII has a 
20% amino acid homology and greater than 50% nucleotide hom-
ology 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 de-
rivatives of folic acid (pteroylglutamic acid), which has the structure 
Table 22.6.6.1  Vitamin B12 and folate
Vitamin B12
Folate
Parent form
Cyanocobalamin (cyano-​B12), molecular weight 1355
Folic acid (pteroylglutamic acid), molecular weight 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
Deconjugation, 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
(2) S-adenosylhomocysteine
Methionine
Homocysteine
THF
Methyl THF
(3) α-leucin
B-leucine
adocobalamin
(1) Propionyl CoA
adocobalamin
Succinyl CoA
Methylmalonic Acid
Isoleucine
Odd-chain fatty acids
Thymidine
Valine
methylmalonyl
CoA mutase
Methylmalonyl CoA
S-adenosylmethionine
Fig. 22.6.6.2  Biochemical reactions of vitamin B12 (cobalamin) in 
human tissues. THF, tetrahydrofolate.


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5411
of a pteridine, a para-​aminobenzoic acid moiety and L-​glutamic 
acid (Fig. 22.6.6.3). 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
In human and other mammalian cells, the number of glutam-
ates is mainly four, five, or six. Polyglutamate forms of folate are 
the active coenzymes; they show increased affinity or lowered Km 
values compared to the monoglutamate equivalent compounds, 
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 22.6.6.3. 
In each there is transfer of a single carbon group, methyl (–​CH3), 
formyl (–​CHOH), methenyl (≡CH), methylene (=CH2), or formi­
mino (=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. 22.6.6.4). During its reactions, folate is not 
completely reutilized, some degradation at the C9–​N10 bond oc-
curs to nonfolate compounds. Thus, folate utilization is increased 
and folate deficiency likely when cell turnover and DNA synthesis 
are increased.
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 250 µg, 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 tis-
sues 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 je-
junum. 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 22.6.6.1). 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 200 µg, as it is a poor sub-
strate for reduction by dihydrofolate reductase. A proton-​coupled 
folate transporter (PCFT) with high affinity and a low pH op-
timum is essential for absorption of reduced folates and folic acid. 
It is expressed particularly in the apical brush border of the en-
terocytes 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) 
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.
Table 22.6.6.2  Vitamin B12-​binding proteins
Intrinsic factor
Transcobalamin I and IIIa
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)
Glycoprotein
Polypeptide
Normal total binding capacity
30–​110 μg/​litre
700–​800 ng/​litre
900–​1000 ng/​litre
Vitamin B12 content
No vitamin B12
300–​400 ng/​litre vitamin B12
30–​60 ng/​litre vitamin B12
Function
Vitamin B12 absorption  
(not itself absorbed)
? Storage of vitamin B12
Vitamin B12 delivery to marrow, placenta, brain, 
and other tissues,
Vitamin B12 absorption
? Protection of vitamin B12
Binding of vitamin B12 analogues
a Related ‘R’ binders (haptocorrins) occur in other tissues and secretions, e.g. milk, gastric juice, saliva, and tears.
N
H
O
C
COOH (α)
CH
COOH (γ)
1
2
3
6
7
8
9
10
N
N
N
N
N
H
N
5
4
Fig. 22.6.6.3  The structure of pteroylglutamic (folic) acid.


section 22  Haematological disorders
5412
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 ex-
changer. The gene is located at chromosome 21q 22.2. The protein 
has 12 transmembrane domains and both N-​ and C-​termini are dir-
ected to the cytoplasm. It is ubiquitously expressed on normal tis-
sues 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, des-
ignated α, β, and γ, localized to chromosome 11q13.3 to 11q13.5. 
FRα and FRβ are both glycosylphosphatidylinositol (GPI)-​anchored 
proteins. The physiological role of the FRs is not clear. They are ex-
pressed 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 physio-
logical significance of this is unknown.
Biochemical basis of megaloblastic anaemia
All known causes of megaloblastic anaemia, whether drugs, de-
ficiencies, or inborn errors of metabolism, inhibit DNA synthesis 
by reducing the activity of one of the many enzymes concerned in 
Table 22.6.6.3  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 synthase
2.
Oxidation–​reduction
Oxidized or dihydrofolates converted to tetrahydrofolates
Dihydrofolate reductase
3.
Amino acid interconversions
(a) Homocysteine → methioninea
5-​Methyl-​THF methyltransferase (methionine synthase)
Methyl THF →THF
(b) 5-​Formiminoglutamic acid (Figlu) → glutamic acid
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 synthase
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
THF, tetrahydrofolate; DHF, dihydrofolate; GAR, glycinamide ribotide; AICAR, 5-​amino-​4-​imidazolecarboxamide ribotide.
a See Figs. 22.6.6.2 and 22.6.6.4.
Reaction (6) has been demonstrated only in vitro and may not take place in vivo.


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5413
purine or pyrimidine synthesis or by inhibiting DNA polymeriza-
tion 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 mamma-
lian 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 intra-
cellular supply of THF, from which the natural folate coenzymes, 
folate polyglutamates, are made. Methyl-​THF cannot act as a sub-
strate for synthesis of folate polyglutamates in human cells. When 
vitamin B12 is deficient there is reduced activity of all reactions re-
quiring folate coenzymes, including those involved in DNA syn-
thesis (Fig. 22.6.6.4). Misincorporation of the base uracil, because 
of the accumulation of dUMP (Fig. 22.6.6.4) 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 22.6.6.4–​22.6.6.6), 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.
Pernicious anaemia (Addisonian pernicious anaemia, 
Biermer’s anaemia)
Definition
An autoimmune disease in which there is atrophy of the stomach 
with severely reduced or absent IF and acid secretion with conse-
quent 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 con-
trols 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 DRB104 has 
been reported in Finnish and Italian populations. 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 improve-
ment in mucosal appearance and function with corticosteroid 
therapy, the presence of antibodies in serum and gastric juice dir-
ected against parietal cells and IF, and of cell-​mediated immunity 
to IF. Parietal cell antibody is present in the serum of 85 to 90% 
of patients. The autoantigens are the α-​ and β-​subunit of the gas-
trin 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 in gastric juice 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 black patients and Hispanic patients may have a higher inci-
dence of IF antibodies. The antibodies to IF are virtually specific for 
PA but parietal cell antibodies occur 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 hypogammaglobulinaemia or with se-
lective 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.
DNA
dATP
dGTP
dTTP
dCTP
dTDP
thymidine
kinase
Thymidine
dTMP
thymidylate synthase
dUMP
DHF-polyglutamate
dihydrofolate
reductase
Methylation
of myelin, basic
proteins, lipids,
DNA, amines
S-adenosyl-
methionine
S-adenosyl
homocysteine
THF-polyglutamate
THF
Methionine
Homocysteine
Methyl THF
(synthesized by
small intestine
from dietary
folates)
5,10 methylene THF –
polyglutamate
Deoxyuridine
(dU)
Fig. 22.6.6.4  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.


section 22  Haematological disorders
5414
The relationship of PA, autoimmune gastritis, and Helicobacter 
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 ex-
cept 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 an-
aemia usually develops gradually, perhaps over several years, and 
symptoms may not occur until it is severe. The most common com-
plaints are due to the anaemia, but loss of mental and physical drive, 
numbness, or difficulty in walking suggest neurological complica-
tions. 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 inter-
current infection may precipitate severe anaemia and thus symp-
toms. 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 de-
velop a widespread brown pigmentation, affecting nail beds and 
skin creases particularly, but not mucous membranes. This is re-
versible 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. 
Table 22.6.6.4  Causes of vitamin B12 deficiency and malabsorption 
of vitamin B12
Causes of severe 
vitamin B12 
deficiency
(a) Nutritional:
Vegans
Long-​continued extremely poor diet (rarely)
(b) Malabsorption:
Gastric causes:
(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
Fish tapeworm
Transcobalamin II deficiency
Causes of 
malabsorption 
of vitamin B12 
usually without 
severe vitamin 
B12 deficiency
Malabsorption of food vitamin B12 (due to simple 
atrophic gastritis, gastric bypass, proton pump 
inhibitors, H. pylori infection); severe chronic 
pancreatitis,
Zollinger–​Ellison syndrome, adult gluten-​induced 
enteropathy, giardiasis, HIV disease, graft-​versus-​host 
disease
Drugs: p-​aminosalicylic acid, colchicine, neomycin, 
slow K, ethanol, metformina, phenformin, 
anticonvulsants
a Recent studies suggest metformin lowers serum vitamin B12 by reducing the level of TCI.
Table 22.6.6.5  Causes of folate deficiency
Poor diet
Especially poverty, psychiatric disturbance, alcoholism, 
dietary fads, scurvy, kwashiorkor, goats’ milk anaemia
Malabsorption
Gluten-​induced enteropathy (child or adult or 
associated with dermatitis herpetiformis)
Tropical sprue
Congenital specific malabsorption
Minor factors: jejunal resection, inflammatory bowel 
disease, systemic infections
Drugs: cholestyramine, sulphasalazine, methotrexate, ? 
others (see ‘Drugs’).
Excessive 
requirements
Physiological:
Pregnancy
Prematurity and infancy
Pathological:
(a) Malignancies—​leukaemia, carcinoma, lymphoma, 
myeloma, etc.
(b) Blood disorders—​haemolytic anaemia 
(especially sickle-​cell anaemia, thalassaemia major), 
myeloproliferative diseases
(c) Inflammatory—​tuberculosis, malaria, Crohn’s disease, 
psoriasis, exfoliative dermatitis, rheumatoid arthritis, etc.
(d) Metabolic—​homocystinuria (some cases)
Excess urinary 
excretion
Congestive heart failure, acute liver damage, chronic 
dialysis
Drugs
Mechanism uncertain
Anticonvulsants (diphenylhydantoin, primidone, 
barbiturates)
? Alcohol
Also drugs causing malabsorption of folate (see 
‘Malabsorption’ )
Liver disease
Mixed causes as above, and poor storage


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5415
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 myxoe-
dema. Other thyroid disorders, vitiligo, carcinoma of the stomach 
(incidence three times that of controls), Addison’s disease, and 
hypoparathyroidism, may precede, occur simultaneously with, or 
follow the onset of the anaemia. A few cases of PA with gastric at-
rophy, achlorhydria, and IF antibodies have occurred in children. 
They may show associated autoimmune conditions, for example, 
myxoedema, hypoparathyroidism, Addison’s disease, or chronic 
mucocutaneous candidiasis.
Neurological complications of vitamin B12 deficiency
Vitamin B12 deficiency may cause a symmetrical neuropathy, af-
fecting the lower limbs more than the upper, which usually pre-
sents 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 biochem-
ical 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 re-
sults from accumulation of S-​adenosyl homocysteine (caused by the 
block in conversion of homocysteine to methionine) with inhibition 
of transmethylation of biogenic amines, proteins, phospholipids, 
and neurotransmitters in the spinal cord and brain. Methionine has 
been shown to prevent the neurotoxicity caused by N2O in experi-
mental animals.
General tissue effects of vitamin B12 and folate deficiencies
Both deficiencies cause macrocytosis and related cytopathic effects 
in 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 regen-
eration 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 im-
paired intracellular killing has been described in vitamin B12 but not 
in folate deficiency. Vitamin B12 deficiency reduces serum concen-
trations of the osteoblast-​related proteins alkaline phosphatase and 
osteocalcin, but whether clinically important bone disease occurs is 
unknown.
Neural tube defects
Folic acid supplements at the time of conception and in early (first 
weeks) of pregnancy reduce the incidence of neural tube defects 
(NTDs) (anencephaly, encephalocoele, and spina bifida) in the 
first and subsequent pregnancies when such a malformation has 
occurred previously.
Folic acid fortification of the diet has led to a substantial reduc-
tion of incidence of NTDs (e.g. in North America). The explanation 
for the effect of folic acid on NTDs is not certain. Women carrying 
affected fetuses have on average 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 NTDs 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 NTDs. Folic acid prevention of NTDs (and in 
some studies cleft lip and palate), despite apparently normal serum 
and red cell folate concentrations, suggests that folic acid is over-
coming a metabolic abnormality in folate metabolism. Only one 
such defect, a mutated 5,10 methylene tetrahydofolate reductase 
(MTHFR) enzyme, has been identified. Periconceptional use of vita-
mins or supplements containing folic acid is also associated with a 
reduced incidence of birth defects associated with maternal diabetes 
mellitus.
Mutated MTHFR, a common thermolabile variant (677C→T) 
(Ala225Val) is associated with lower serum and red cell folate con-
centrations and with higher plasma homocysteine than in control 
subjects in the general population. The prevalence of the homozy-
gous state in the population is approximately 5% and in parents of 
fetuses with NTDs the prevalence is approximately 13%. The pres-
ence of this mutation can therefore account for only a small propor-
tion of NTDs. Mutations of other genes (e.g. VANGL1) not related to 
folate metabolism, have been found in NTD families. Serum vitamin 
B12 levels are also lower in sera of mothers with NTD infants than 
in controls. Also, TCII receptor polymorphisms are associated with 
Table 22.6.6.6  Megaloblastic anaemia not due to vitamin B12 or 
folate deficiency
Abnormalities 
of vitamin 
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
Independent 
of vitamin B12 
or folate
Congenital:
Orotic aciduria (responds to uridine)
Lesch–​Nyhan syndrome, ? responds to adenine
Thiamine-​responsive
Some cases of congenital dyserythropoietic anaemia
Acquired:
Erythroleukaemia, other myeloid leukaemias (some cases)
Myelodysplasia
Drugs:
Antimetabolites: 6-​mercaptopurine, cytosine arabinoside, 
hydroxycarbamide, 5-​fluorouracil, azathioprine, etc.


section 22  Haematological disorders
5416
increased risk for NTD. There are, however, no studies showing that 
vitamin B12 therapy or dietary fortification with vitamin B12 reduces 
the incidence of NTDs.
Mental deterioration
There is a more rapid decline in cognitive function in subjects with 
low serum vitamin B12 levels, serum holotranscobalamin, and raised 
serum MMA concentrations. Studies suggest that low serum folate 
levels are not associated with cognitive loss or depression in the eld-
erly. Meta-​analysis suggests administration of folic acid and vitamin 
B12 may have a mild effect on memory but does not improve or sta-
bilize cognitive function in older people with or without low serum 
vitamin B12 or folate concentrations. Although low serum vitamin 
B12 levels and raised serum homocysteine and methylmalonate 
levels have been reported to be more frequent in subjects with de-
mentia, including Alzheimer’s disease, than in controls, trials of 
vitamin B12 therapy have generally shown no benefit in treating the 
dementia or slowing its progression. Vitamin B12 has been used to 
treat chronic fatigue syndrome but there are no controlled trials 
which validate this.
Cardiovascular disease and stroke
McCully (1969) first implicated homocysteine as a cause of ath-
erosclerosis. 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 MTHFR 
(Fig. 22.6.6.5). In these children, plasma homocysteine concentra-
tions are raised to 10 to 100 times normal. It is now apparent that 
milder 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, oxi-
dize low-​density lipoprotein (LDL), and disturb collagen and extra-
cellular 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 
MTHFR, smoking, and alcohol consumption, as well as intake of 
various drugs.
Folate deficiency assessed by serum or red cell folate or by dietary 
folate intake is also associated with coronary vascular disease, myo-
cardial 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 homocyst-
eine level in serum than the wild type CC state, and ischaemic heart 
disease. Meta-​analysis of 75 studies showed an increased risk of is-
chaemic 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 car-
diovascular disease or all-​cause mortality, although it was linked to 
a decreasing trend in stroke risk. This was more marked in popu-
lations without mandatory fortification of the diet with folic acid 
(Yang et al. 2012). Huo et al. (2014) in the most recent meta-​analysis 
of 15 randomized trials (N = 55 764) found an overall reduction in 
stroke (relative risk 0.92; P = 0.04). This was more marked in those 
followed for more than 3 years, those without background fortifica-
tion of breakfast cereals with folic acid and those not taking statins. 
It may be relevant that a reduction in incidence of stroke occurred in 
the United States of America and Canada coinciding with the intro-
duction of dietary folic acid fortification, whereas no reduction in 
incidence occurred over the same period (1998–​2002) in England 
and Wales without fortification. A recent Chinese randomized con-
trolled study over 4.5 years has confirmed that folic acid 0.8 mg daily 
is effective in reducing primary occurrence of ischaemic stroke, par-
ticularly in those starting with lower folate levels, with the MTHFR 
TT mutation and for longer receiving folic acid (Huo et al. 2015). 
Those with the TT mutation had the highest stroke risk. Wald et al. 
(2011) have suggested that in trials of prevention of secondary cor-
onary disease, aspirin may be negating or reducing the effect of 
lowering homocysteine and folic acid prophylaxis. On this basis, 
folic acid would have a role in primary prevention of ischaemic heart 
disease in those not taking aspirin.
Malignancy
Positive and negative associations between the occurrence of various 
types of lymphoblastic or myeloblastic leukaemias in infancy and 
childhood and polymorphisms of folate-​metabolizing enzymes 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 for-
tification 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 nonrandomized 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 co-
lonic adenomas in women between controls and subjects receiving 
folic acid, vitamin B6, and vitamin B12 supplements.
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 disasso-
ciation between fortification and risk in colorectal cancer incidence 
Methionine
synthase
Methionine
Homocysteine
Cystathionine
synthase
Cystathionine
Tetrahydrofolate
5-Methyl
tetrahydrofolate
5,10-Methylene
tetrahydrofolate
reductase
5,10-Methylene
tetrahydrofolate
Fig. 22.6.6.5  The role of three enzymes (cystathione synthase, 
methionine synthase, and MTHFR) and three vitamins (vitamin B12, 
vitamin B6, and folate) in homocysteine metabolism.


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5417
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 for-
tification. 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, 10 for cardiovas-
cular disease prevention and 3 for colonic adenoma and cancer in-
cidence, 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 to 5 mg. 
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 supplemen-
tation (some included in the previous study).
Other effects
Complications of pregnancy that have been ascribed to folic acid, 
including miscarriage and multiple pregnancy, have no sound basis.
Malabsorption of vitamin B12
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.
Gastrectomy
All patients who have total gastrectomy 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. Iron deficiency usually 
accounts for the anaemia that occurs after partial gastrectomy. A mi-
nority 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. After gastric pli-
cation or Roux-​en Y surgery for obesity, vitamin B12 deficiency may 
occur and oral vitamin supplementation is often used. Monitoring 
for vitamin B12 deficiency is advisable.
Small-​intestinal lesions
Colonization of the upper small intestine with colonic bacteria, if 
sufficiently heavy as in the stagnant-​loop syndrome, leads to mal-
absorption of vitamin B12. The most common causes are listed in 
Table 22.6.6.4. It appears that the bacteria destroy IF. Infestation 
with the fish tapeworm (Diphyllobothrium latum) has a similar ef-
fect but is now almost completely eradicated; infestation is only suf-
ficiently marked in Finland and Russian lake regions to suggest a 
possible cause of megaloblastic anaemia.
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, neur-
opathy 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); and in Crohn’s disease, malabsorption of vitamin B12 is fre-
quent but severe vitamin B12 deficiency is unusual unless there is an 
ileal resection, fistula, or stagnant loop.
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 mu-
tation P1297L (FM1) in cubilin. A second less frequent mutation 
(FM2) activates a cryptic splice site with insertion of multiple stop 
codons in the CUB6 domain. Other mutations in cubilin have been 
described. In Norway at least six different mutations of the AMN 
gene have been reported in affected families. The proteinuria, pre-
sent in over 90% of cases, is benign, nonspecific, and persists after 
vitamin B12 therapy. The clinical presentation of the disease is iden-
tical to that of congenital IF deficiency.
Other causes of malabsorption of vitamin B12
The most frequent cause of subclinical vitamin B12 deficiency in the 
United Kingdom and United States of America, shown by a border-
line or low serum vitamin B12 level, and normal blood count with or 
without a raised serum homocysteine and methylmalonate levels, 
is malabsorption of dietary vitamin B12. This is thought to be due 
to failure of release of dietary vitamin B12 from its protein binding 
in food. It is usually due to an atrophic gastritis resulting in reduced 
pepsin activity. The gastritis may be associated with a positive par-
ietal cell antibody test or with H. pylori infection. The incidence is 
slightly more common in the elderly and the deficiency rarely pro-
gresses to megaloblastic anaemia or vitamin B12 neuropathy. Several 
other conditions and drugs may cause malabsorption of food 
vitamin B12, for example, proton pump inhibitors which very rarely 
cause deficiency of clinical severity.
Other causes of vitamin B12 malabsorption by unidentified 
mechanisms include p-​aminosalicylate, colchicine, neomycin, and 
‘slow’ potassium tablets. Recent studies suggest the fall in serum 
vitamin B12 level with metformin is due to a reduced serum level 
of TCI rather than malabsorption. In chronic pancreatitis and the 
Zollinger–​Ellison syndrome, there is failure to release vitamin B12 
from gastric haptocorrin due to absence or inactivation of pancre-
atic trypsin.
Serum vitamin B12 concentrations fall progressively in untreated 
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 the vitamin B12 malabsorption. 


section 22  Haematological disorders
5418
Malabsorption of vitamin B12 also occurs in inherited TCII defi-
ciency and temporarily after total-​body irradiation before stem 
cell transplantation. In chronic graft-​versus-​host disease affecting 
the gut, malabsorption of vitamin B12 is usual, due to the abnormal 
gut flora as well as to an 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 vegans. The incidence of overt meg-
aloblastic anaemia is much lower than the incidence of subclinical 
deficiency assessed by the serum vitamin B12 assay. These individ-
uals 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 vitamin B12 deficient if the mother’s stores are low. 
Dietary deficiency of vitamin B12 also occurs 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 seen when the anaemia is due to 
vitamin B12 deficiency, except that a neuropathy does not occur 
and the underlying aetiology tends to be different. Cognitive 
changes and depression may be caused by the deficiency, and 
neurological abnormalities do occur with inborn errors of folate 
metabolism and may be precipitated by antifolate drugs. Folate de-
ficiency 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 haematopoiesis’) over a short period, particularly if an 
infection supervenes.
Nutritional folate deficiency
Minor degrees of nutritional folate deficiency are frequent in 
most countries. Potentially millions of people in northern China, 
Bangladesh, Burma, Malaysia, Africa, or India have low levels of 
folate due to a poor dietary intake and nutritional folate deficiency 
is the main cause of megaloblastic anaemia, often presenting in 
pregnancy. In many countries—​for example, 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.
Severe folate deficiency has been estimated to account for about 
17% of all cases of megaloblastic anaemia in the United Kingdom, 
where it occurs mainly in the context of a poor diet and/​or alco-
holism. In some cases, barbiturates or consumption of spirits or 
cough mixtures or a physical abnormality such as rheumatoid arth-
ritis, 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. Goats’ milk anaemia is a nutritional folate 
deficiency due to the low (6 µg/​litre) folate content of goats’ milk.
Malabsorption
(Also see Diseases of the gastro-intestinal tract described in 
Section 15.)
Gluten-​induced enteropathy
Folate deficiency due to malabsorption of folates occurs in virtually 
all untreated patients, the serum folate being subnormal in virtu-
ally 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 chil-
dren 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 duo-
denal and jejunal abnormality; the severity of folate malabsorption 
and deficiency correlates with the severity of the intestinal lesion.
Tropical sprue
(Also see Chapter 15.10.8). Malabsorption of folate occurs in all 
severe, untreated patients in the acute phase and megaloblastic an-
aemia due to folate deficiency may develop within a few months. 
Not only does the anaemia respond to folate therapy but in many pa-
tients 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 re-
tardation, fits, athetotic movements) and present with megaloblastic 
anaemia responding to physiological doses of folic acid given paren-
terally 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 de-
grees 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 sulfasalazine have been suggested, on 
variable evidence, to cause malabsorption of folate in some subjects 
but none is definitely established except sulfasalazine.
Increased folate utilization
A general mechanism of increased folate utilization in conditions of 
increased cell turnover has emerged. This consists of partial degrad-
ation of folate at the C9–​N10 bond rather than complete recycling of 
the folate coenzymes required in DNA synthesis.
Pregnancy
This, associated with poor nutrition, is probably the most common 
cause of megaloblastic anaemia world-​wide, unless folic acid 


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5419
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 pregnan-
cies. 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 degrad-
ation 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 de-
ficiencies in pregnancy is probably due to a poor quality of the diet 
in certain women.
Prophylactic folic acid should now be given routinely in preg-
nancy; 400 µg/​day is recommended (mentioned previously) 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 concentra-
tions than adults. These fall to a nadir at about 6 weeks of age. In pre-
mature 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 22.6.6.5). In general, the severity correlates with the extent 
and degree of dissemination of the underlying disease. However, pa-
tients with megaloblastic anaemia due to folate deficiency are un-
usual and, supplementation tends to be avoided in the absence of a 
categorical indication for its use (e.g. anaemia, leucopenia, etc.) due 
to concerns regarding promoting tumour growth.
Blood disorders
Chronic haemolytic anaemia  Requirements for folate are in-
creased 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 in-
take, 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 haematopoiesis has been 
reported in as many as one-​third of patients. Circulating meg-
aloblasts, increased transfusion requirements, severe thrombo-
cytopenia, or pancytopenia may be the first indication that folate 
deficiency has developed. Polycythaemia vera is not a typical cause 
of folate deficiency.
Inflammatory diseases
Folate deficiency has been described in patients with tubercu-
losis, 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  Patients with the most common form of this dis-
order, 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 (see 
Chapter 12.2).
Excess urinary loss of folate
Urine folate excretion of 100 µg a day or more occurs in some pa-
tients 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 dia-
lysis remove folate from plasma. Folic acid (e.g. 5 mg/​week) is now 
usually given prophylactically to patients with renal failure who re-
quire long-​term dialysis.
Drugs
Dihydrofolate reductase inhibitors
Methotrexate, aminopterin, pyrimethamine, and trimethoprim 
all inhibit dihydrofolate reductase (DHFR) but have different rela-
tive 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 me-
tabolism. Trimethoprim, used as an antibacterial agent, may aggra-
vate pre-​existing folate or vitamin B12 deficiency but does not in 
itself cause megaloblastic anaemia.
Alcohol
Folate deficiency may occur in alcoholism. The main factor is poor nu-
trition and it is likely that alcohol interrupts the enterohepatic circu-
lation for folate. It also has a direct effect on haematopoiesis, causing 
vacuolation of normoblasts, impaired iron utilization, sideroblastic 
changes, macrocytosis, megaloblastosis, and thrombocytopenia, even 
in the absence of folate deficiency. Beer drinkers usually avoid folate de-
ficiency because of the high folate content of beer. The usual red cell 
macrocytosis in nonanaemic alcoholics is not related to folate deficiency.
Anticonvulsants and barbiturates
Diphenylhydantoin, primidone, and barbiturate therapy may be as-
sociated with folate deficiency. The more severe deficiency is associ-
ated 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 supplementa-
tion on the frequency of seizures.
Other drugs
Nitrofurantoin, triamterene, proguanil, and pentamidine have been 
reported to cause folate deficiency.


section 22  Haematological disorders
5420
Liver disease
Folate deficiency occurs most commonly in alcoholic cirrhosis 
where alcohol, poor nutrition, and release of stored folate with ex-
cess 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 an-
aemia is present; (2) distinction between vitamin B12 or folate de-
ficiency (or rarely some other factor) as the cause of the anaemia; 
and (3) diagnosis of the underlying disease causing the deficiency 
(Table 22.6.6.7).
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 haematopoiesis 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 de-
ficiency or antimetabolite drug therapy) develops acutely over the 
course of a few weeks. The MCV is also normal in some severely an-
aemic 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. 22.6.6.6) 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 platelet count may be moderately reduced 
but rarely falls below 40 × 109/​litre.
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 prod-
ucts are present in urine. The direct antiglobulin test is weakly posi-
tive 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 mat-
uration 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 dispropor-
tionate accumulation of early cells. Giant and abnormally shaped 
metamyelocytes and megakaryocytes with hypersegmented nuclear 
lobes are also usually present (Fig. 22.6.6.7).
Table 22.6.6.7  Laboratory diagnosis of megaloblastic anaemia
1. General tests
Peripheral blood film and count
Bone marrow
Serum bilirubin, iron, lactate dehydrogenase
2. Tests for 
vitamin B12 or 
folate deficiency
Serum vitamin B12 and folate; red cell folate
Serum homocysteine and methylmalonic acid levels
3. Tests for cause 
of vitamin B12 or 
folate deficiency
Vitamin B12 deficiency:
Serum antibodies to parietal cell, intrinsic factor
Serum gastrin
Gastric secretion; intrinsic factor, acid
Endoscopy, gastric biopsy
Upper gastrointestinal endoscopy
Proteinuria, fish tapeworm ova, intestinal flora, etc.
Folate deficiency:
Transglutaminase, endomysial antibodies
Small-​intestinal function
Duodenal biopsy
Barium follow-​through
Tests for many underlying conditions
Fig. 22.6.6.6  Megaloblastic anaemia. Hb 40 g/​litre 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. 22.6.6.7  Megaloblastic anaemia. Bone marrow aspirate showing 
megaloblasts at different stages and giant metamyelocytes.


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5421
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 pa-
tients 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 con-
striction; and (3) thin, elongated, uncoiled chromosomes.
Ineffective haematopoiesis
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 unconjugated serum bilirubin, lactic de-
hydrogenase, and lysozyme are all due to ineffective haematopoiesis.
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 myelodysplastic syndromes, myeloid leukaemias, 
cytotoxic drug therapy, chronic respiratory failure, plasma cell mye-
loma, and other paraproteinaemias. If a bone marrow biopsy has 
been performed, the principal differentiation is from other causes of 
megaloblastosis, particularly myelodysplasia. Other causes of meg-
aloblastic anaemia not due to vitamin B12 or folate deficiency are 
listed in Table 22.6.6.6.
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 by competi-
tive binding of intrinsic factor and immunochemiluminescence-​
based assays. The reference range, depending on the assay, is from 
160 to 200 mg/​litre to 960 to 1200 ng/​litre. Some report in pmol/​
litre (1 pmol = 1.355 ng/​litre). It is difficult to determine a normal 
range and each laboratory should establish this independently. The 
concentrations are low in vitamin B12 deficiency, being extremely 
low in patients with neurological disease. Unfortunately, using 
competitive-​binding 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 mu-
tations 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 usu-
ally in the range 100 to 200 ng/​litre may occur in the absence of an-
aemia or macrocytosis.
In some research studies, serum holo-​TCII levels have been meas-
ured 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 as-
sociation 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, for example, autoimmune diseases 
such as systemic lupus erythematosus, rheumatoid arthritis, in 
Gaucher disease, and in some monocytic or monoblastic leukae-
mias, and in inflammatory bowel disease. In active liver diseases, 
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 MMA. Serum MMA levels are raised in vitamin B12 defi-
ciency but not in folate deficiency. Raised levels may also occur in 
renal failure. Rare cases of congenital methylmalonic aciduria have 
been described, due 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 minor increases in serum MMA 
concentration found particularly in older people in the absence of 
macrocytosis or anaemia, with or without borderline vitamin B12 
concentrations, may suggest ‘biochemical’ vitamin B12 deficiency 
which does not progress to megaloblastic anaemia, but the normal 
ranges for MMA for different age groups are difficult to determine. 
Randomized trials are needed to assess the value of preventing or 
treating 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 (<3 µg/​litre, 7 nmol/​litre) in folate deficiency 
(and is normal or raised in vitamin B12 deficiency unless folate de-
ficiency 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 not now recommended for routine diagnostics but 
may be useful in some patients with probable folate deficiency in 
whom the serum folate is found to be normal. It is low in a propor-
tion 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.
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 diver-
ticulosis). The serum gastrin concentration is raised in patients with 
PA and the serum is tested for antibodies to IF, parietal cells, and 


section 22  Haematological disorders
5422
thyroid; serum immunoglobulins are measured in view of the asso-
ciation 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 be-
cause of variation in folate content of foods, losses in cooking, and 
size of portions. Often it is the general social circumstances that sug-
gest a poor intake. Drug intake, particularly of barbiturates, is im-
portant. Many underlying inflammatory or malignant diseases may 
exaggerate the tendency to folate deficiency in patients with inad-
equate 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 bi-
opsy are usually necessary. In certain tropical countries, sprue may 
cause a generalized malabsorption syndrome in which folate defi-
ciency 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 under-
lying disorder, and prevention of relapse. In most cases, it is possible 
to diagnose which deficiency is present before starting therapy.
Vitamin B12 deficiency
Hydroxocobalamin 1 mg 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 neur-
opathy 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 an uncooperative, dis-
orientated 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 48 h, 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, 1 mg intramuscularly, is given once every 
3 months for life in PA and most other causes of vitamin B12 de-
ficiency 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; for example, expulsion of the fish 
tapeworm, improvement of vegan diet, surgical correction of an in-
testinal stagnant loop. A few micrograms of vitamin B12 can be ab-
sorbed each day in PA from oral doses of 1 mg or more by passive 
diffusion, but this maintenance therapy is usually reserved for those 
who cannot have injections—​for example, those with a bleeding dis-
order, 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 war-
ranted but these diseases must be particularly borne in mind if rele-
vant symptoms or signs develop.
It is unclear whether vitamin B12 should be given orally or par-
enterally to those with biochemical (subclinical) vitamin B12 de-
ficiency without anaemia or macrocytosis or clinical symptoms. 
Trials are needed to clarify this.
Folate deficiency
This is corrected by giving 5 mg folic acid by mouth daily. It is essen-
tial 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 haemo-
lytic 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 NTDs (doses of 300–​400 µg/​
day are used). Only 19% of women in a Northern Ireland study had 
taken folic acid preconception, resulting in a lower red cell folate 
and thus increased risk of NTDs, during the first trimester, than 
in women who had taken preconception folic acid. Studies in the 
United States of America 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 periconception. 
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 pa-
tients 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.


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5423
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 1998 and is now also practised in 
Canada, Chile, and 70 other countries amounting to about 20% of 
the world’s population. Median serum folate in clinical specimens 
in United States of America 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. However, 
the small amounts of folic acid with each meal would be largely if 
not entirely converted to methyl-​THF by the small intestine and this 
folate is not able to affect haematopoiesis in vitamin B12 deficiency. 
In keeping with this, there is no evidence for an increased incidence 
of nonanaemic subjects with low serum vitamin B12 levels in the 
United States of America since fortification. In the United Kingdom, 
fortification of flour with folic acid (240 μg/​100 g flour) has been re-
commended but not implemented. As discussed previously, there 
is no evidence that fortification would affect the incidence in the 
population of any type of cancer. Fortification of grain with vitamin 
B12 has also been suggested to reduce the incidence of NTDs, 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 metho-
trexate 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.
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 com-
monly 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 de-
ficiency of one or other enzyme concerned in folate metab-
olism: 
5-​methyltetrahydro-​folate, 
methylene 
THF-​reductase, 
FIGLU-​transferase, methenyl-​THF cyclohydrolase. Some of the ba-
bies 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 megalo-
blastic anaemia requiring therapy with large daily doses of vitamin 
B12 at 3 and 5 weeks of age. Similarly affected families have been de-
scribed. A spectrum of mutations in the gene for TCII have been de-
tected. 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 mas-
sive doses of vitamin B12 (e.g. 1 mg intramuscularly, three times each 
week). Delay in treatment may allow a neuropathy to occur. In con-
trast, in subjects with rare, inherited, mutations of TCI, low serum 
vitamin B12 concentrations occur, but haematopoiesis is normal.
Children with one form of congenital methylmalonic aciduria, 
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 chil-
dren have also surprisingly not shown megaloblastic anaemia. 
Neurological abnormalities are usual. Homocystinuria and meg-
aloblastic anaemia without methylmalonic aciduria 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 megalo-
blastic 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 humans, but may precipitate megaloblastic anaemia 
in patients already B12 or folate deficient (mentioned 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 syn-
thase. Megaloblastosis develops within several hours in humans. This 
recovers over several days when exposure to N2O is discontinued. 
After many weeks of exposure to N2O, monkeys develop a neuropathy 
resembling vitamin B12 deficiency neuropathy in humans; peripheral 
neuropathies and more severe neurological disease have also been 
described in humans (e.g. dentists and anaesthetists) repeatedly ex-
posed to the gas. When N2O is used as anaesthetic for patients with 
low vitamin B12 stores, megaloblastic anaemia or neuropathy may 


section 22  Haematological disorders
5424
be precipitated months later, due to failure to replenish vitamin B12 
stores by absorption. Recovery from N2O exposure needs new co-
balamin and also synthesis of new apoenzyme (methionine synthase) 
because this protein is also damaged by active oxygen derived from 
the N2O–​cobalamin reaction. Methylmalonic aciduria has not been 
found in animals or humans exposed for short periods to N2O, as 
methylmalonic CoA mutase does not need reduced 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 en-
zymes (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 ex-
cretion 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 to a haematological re-
sponse, restoration of normal haematopoiesis and growth, and re-
duction 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 12.4).
Vitamin E deficiency
This has been reported to cause megaloblastosis in a group of chil-
dren 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 sensorineural 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 megalo-
blastic 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.
Drugs that directly inhibit purine or pyrimidine synthesis 
(e.g. cytosine arabinoside, 5-​fluorouracil, hydroxycarbamide, 
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, for example, gluta-
thione, cytochromes, pyridine, and flavin nucleotides. Although 
vitamin C is also thought to be needed for maintaining body fol-
ates 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 excre-
tion 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 require-
ments, 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, 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–​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 


22.6.6  Megaloblastic anaemia and miscellaneous deficiency anaemias
5425
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 
haematopoiesis 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. It occurs in natural tissues in three major forms: pyri-
doxine, 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 re-
sponses occur particularly in the inherited form (when it is as-
sumed 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 dis-
ease 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 an-
aemia 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 ani-
mals, 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 mem-
branes. 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
Anaemia is usual in both ‘pure’ protein deficiency (kwashiorkor) 
and in protein–​calorie malnutrition (marasmus). It has been re-
ported 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 malab-
sorption. The anaemia is typically normochromic, normocytic, 
and of the order of 80 to 90 g/​litre. The reticulocyte count is usu-
ally 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 con-
sequent 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
Devalia V, Hamilton MS, Molloy AM (2014). Guidelines for the diag-
nosis and treatment of cobalamin and folate disorders. Br J Haem, 
166, 496–​513.
Longo DL (2015). Drug-​induced megaloblastic anemia. N Engl J Med, 
373, 1649–​58.
McLean E, de Benoist B, Allen LH (2008). Review of the magnitude of 
folate and vitamin B12 deficiencies worldwide. Food Nutr Bull, 29 
Suppl 2, 538–​51.
Vitamin B12
Carmel R (2011). Biomarkers of cobalamin (vitamin B12-​12) status in 
the epidemiologic setting: A critical overview of context, applica-
tions, and performance characteristics of cobalamin, methylmalonic 
acid and holotranscobalamin 11. Am J Clin Nutr, 97, 541–​56.
Carmel R (2012). Subclinical cobalamin deficiency. Curr Opin 
Gastroenterol, 28, 151–​8.
Green R (2017). Vitamin B12 deficiency from the perspective of the 
practising hematologist. Blood, 129, 2603–11.
Green R, et al. (2017). Vitamin B12 deficiency. Nat Rev Dis Primers, 
3, 17040.
Hershko C, et al. (2006). Variable hematological presentation of auto-
immune gastritis: age related progression from iron deficiency to 
cobalamin depletion. Blood, 107, 1673–​9.
Lewerin C (2008). Serum biomarkers for atrophic gastritis and anti-
bodies 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–​8.
Mills JL (2011). Do high blood folate concentrations exacerbate meta-
bolic abnormalities in people with low vitamin B12 status. Am J Clin 
Nutr, 94, 495–​500.