Supplementary Information Submitted to the SCN Working Group

on Micronutrients Report 2006

Vitamin B12 and folate deficiency: megaloblastic anemia and

beyond
Dr Jagdish Chandra, MD, FIAP
Prof. of Pediatrics,
Lady hardinge Medical College, and
Kalawati Saran Children’s Hospital
New Delhi.
Deficiency of vitamin B12 and folate most commonly results in megaloblastic anemia
(MA). Other less common manifestations resulting from deficiency of these
hematopoeitic micronutrients in children include neuro-developmental effects and
abnormal movements. Neural tube defects result from deficiency in mothers during
pregnancy. Bone loss resulting in osteopenia, osteoporosis and pathological fractures
and cardiovascular effects predisposing to coronary artery disease are observed in
adults and elderly patients (1,2).
Megaloblastic anemia (MA) is a distinct type of anemia characterized by macrocytic
RBCs and typical morphological changes in RBC precursors. The precursors are
larger than the cells of same stage and maturation and exhibit disparity in nuclear-
cytoplasmic maturation. Basic underlying pathogenetic mechanism in MA is
deficiency of folic acid (FA) and/or vitamin B12 at the cellular level with resultant
impairment of DNA synthesis.
This article reviews the certain important aspects of MA and other effects of
deficiency of vitamin B12 and folate.
Prevalence of MA is on the rise ?
Answer to this question may not be forthcoming easily as varying criteria have been
used in literature to point towards anemia resulting from deficiency of FA or vit B12.
Some studies have considered macrocytosis of red cells as indicative of this type of
anemia while others have defined MA by presence of megaloblastic changes in the
bone marrow. Still others have used subnormal micronutrient levels to define this
type of anemia. Table I shows the proportion of cases contributed by macrocytic/
anemia/MA/ FA- vit. B12 deficiency as observed in various series on nutritional
anemia in children or during survey of micronutrient deficiency.While proportion of
these cases seems to be increasing over the years, of particular interest is the
observation by Gera et al which shows an almost four fold rise in proportion of
macrocytic anemia cases over less than a decade at one center-2 % in 1991 and 7.8%
in 1999 (Table I). A recent report shows 46.9% of non-anemic adult subjects having
subnormal levels of B12 or folate- B12 deficiency being five times more common than
folate.(10)
Further, the timing of the reports reveals a renewed interest in the subject over last
10-15 years. Observations on adults and children from other developing countries
including Pakistan, Zimbabve, Mexico and Guatemala during this period shows that
the trend in India may be reflecting a more generalized phenomenon (11-14). The
increase in prevalence of MA is also supported by observation that MA accounts for
maximum/ a large number of cases of pancytopenia in many Indian series (see details
below(15-18) .

Table I : Prevalence of MA, macrocytic anemia or B12 /folate deficiency

No and Series Year Proportion of cases

1. Ghai et al(3) 1956 19.1%
2. Dalal et al (4) 1969 18.0%
3. Ghai et al (5) 1977 02.0%
4. Sharma et al(6) 1985 24.0%
5. Gomber et al (7) 1998 42.1%
6. Chaudhry et al (8) 2001 27.1%
7.Gera et al (9) 1991 02.0%
1999 07.8%

Table II: Relative prevalence of vitamin B12 and folate deficiency

Indian Series: Year / Country Population/ Fola B12 Combined

Study group te def. deficiency
Def. (%) (%)
(%)
Bhende et al (19) 1965 Nutr. MA 54.9 7.0 5.3
Mittal et al (20) 1969 MA, child 57.1 22.4 10.2
Sarode et al (15) 1989 MA, all ages 6.8 76.4 8.8
Gomber et al(21) 1998 MA, children 10.0 50.0 20
Gomber et al (7) 1998 Preschool 2.2 36.6 2.2
Chaudhry MW(8) 2001 Anemic child 12.0 19.0 14.0
Chandra et al (22) 2002 MA, children 20.0 32.0 20.0
Khanduri et al(10) 2005 Gen. Population 6.8 33.0 8.3
Other countries
Mukibi et al (12) 1992, MA, all ages 17.0 51.0 32.0
Zimbabwe
Maddood-ul- 1995, Pakistan MA, all ages 8.0 56.0 20.0
Mannan et al (11)
Allen et al (14) 1995, Mexico Gen. population - 19-41 -
Casterline etal (13)
1997, Lactating 9.0* 46.7* -
Guatemala
Garcia-Casal et al 2005, Gen. Population 30.0 11.4 -
 (23) Venezuala Pregnancy 36.3 61.3 -
*Includes deficienct and low levels of folate and cobalamin

Relative prevalence of deficiency -B12 / Folate:

Deficiency of B12 or folate can not be differentiated easily from one another as the
clinical and hematological features resulting from the deficiency of both
micrnutrients are similar. Estimation of serum levels are required to be certain of
the deficient micronutrient. Serum B12 and folate assay used to be cumbersome
dependent upon biological methods , hence they were not performed widely.
Currently most widely used methods is radioimmunoassay which because of its
high cost is not routinely available in developing countries. HPLC based assay
have also become available. The problem is further compounded by the wide
range over which the levels are obtained in normal individuals and normal values
obtained in patients with frank megaloblastic morphology and severe anemia.
Combined deficiency is also seen in many cases (24).
TableII shows the proportion of cases according to micronutrient deficiency (the
studies have not been differentiated whether they are on cases with megaloblastic
anemia or population based studies as the proportions are under comparison). The
Indian series from 1965 shows that isolated B12 or combined deficiency was
present in nearly 5% and 60 % instances while folate deficiency accounted for
nearly 80% (19)However, Sarode et al from PGIMER Chandigarh, reported B 12
deficiency in nearly 85 % cases with megaloblastic anemia ( adults included, 15)
The later studies from Delhi/ New Delhi have also highlighted that B12 deficiency
is far more common than folate deficiency. A study from our hospital on cases
with nutritional anemia shows B12 deficiency in 19 % cases and folate deficiency
in 12 %. In addition nearly 35 % cases had levels of B12 which could be classified
as low (8).

Higher prevalence of B12 deficiency among population groups and cases with MA from
India also reflects a more widespread trend. A study from Mexico showed B12
deficiency in 19% -41% of various population subgroups while no cases with folate
deficiency were observed(14). Another study on Guatemalan lactating mothers
revealed B12 deficiency in 46% compared to 9% prevalence of folate deficiency (13)
Series on MA patients from Pakistan and Zimbabve revealed B12 deficiency in over
50% cases while folate deficiency was seen in only 8% and 17 % cases respectively
(11,12). Considering the fact that most common cause of B12 deficiency in young
children is the maternal deficiency leading to decreased stores at birth and its
consequences, these studies including adults may have a direct relevance to pediatric
population.
Etiology of MA/B12-folate deficiency:
In India and other developing countries, most cases of MA are caused by nutritional
deficiency of folate, B12 or both. Pernicious anemia due to intrinsic factor deficiency,
malabsorption resulting in folate deficiency (celiac disease) and certain inborn errors
of metabolism eg. mthylmalonic aciduria (B12 deficiency) and methylene
tetrahydrofolate reductase deficiency (folate) account for minority of cases.
Nutritional deficiency is far more common in vegetarian than in non-vegetarian
families (24,25). B12 deficiency seen in infants and young children has been
particularly related to maternal deficiency which results in poor body stores at the
time of birth. These underprivileged infants who are exclusively/ predominantly
breastfed for prolonged period (in some instances as much as 3-5 years) tend to
develop B12 deficiency as the breast milk content of B12 in these mothers is far below
normal ( poverty- vegetarianism-exclusive prolonged breast feeding axis). Cobalamin
content of breast milk is lower in vegetarian mothers and is positively correlated with
their serum cobalamine levels (13,26). We have documented a good co-relation
between serum levels of the mothers and their suckling infants and young children
(22). From the developed countries, cases of MA are being reported among infants
born to vegetarian mothers (25,27).
Increasing B12 and folate deficiency is not entirely related to poverty. Biochemical
evidence of recent B12 malabsorption has been documented in some populations.
Giardia infection, particularly acquired in Asian countries has been demonstrated to
cause folate malabsorption. H.pylori infection has been incriminated to cause B12
malabsorption among adults (14).

Clinico-hematological profile: Anemia and Beyond:

Most cases of MA are seen in poor vegetarian families. Infants and young children
who are not adequately weaned are particularly susceptible. Hence most cases tend
to be moderately or severely malnourished. Anemia, anorexia, irritability, easy
fatigability are clinical features common to other causes of anemia.
Clinical features peculiar to MA include hyperpigmentation of knuckles and
terminal phalanges (observed in Asian communities), enlargement of liver and
spleen (seen in upto30-40% cases). Petechial and other hemorrhagic manifestations
have been reported in upto 25% cases. Presence of bleeding with severe anemia
makes them clinically indistinguishable from aplastic anemia. Cases with MA may
mimic acute leukemia due to presence of hepatosplenomegaly (19,21,22,28).
Tremors have been described to occur as a distinct entity in children of north and
central India – The Infantile Tremors Syndrome (29,30) . These cases mostly have
macrocytic anemia and developmental regression in addition to tremors. Cases of
MA not presenting with tremors also exhibit developmental retardation/ regression
in association with severe anemia. Recently a group of infants has been described
having microcephaly and developmental retardation even before anemia became
clinically evident. Occurrence of abnormal movements in association with
hypotonia, psychomotor retardation, apathy and failure to thrive is being reported in
western literature as well (31,32). These cases have been shown to have diffuse
frontotemporoparietal cortical atrophy on MRI of brain (25,32). Some studies report
persistence of neurological consequences even after treatment (33). Impairment of
cognitive function is also described in children and adolescents with B12 deficiency
even in absence of anemia (34).
Laboratory Findings :
Laboratory investigations reveal macrocytic normochromic RBCs. On the
electronic cell counter, MCV is increased and RBC count is decreased
proportionate to degree of anemia. Nucleated red cell precursors may be seen and
they show megaloblastic changes. In the WBC series, hypersegmentation of
neutrophils has been described as an early feature of folate-B12 deficiency. In
addition, neutropenia is seen in 17- 49 % cases in various series. Similarly, platelet
counts are decreased – observed in as many as 44-80 % cases. It is thus not
surprising that the cases with MA manifest with pancytopenia and mimic aplastic
anemia (15,21,22,28). From India, in various series of patients presenting with
pancytopenia, MA has been found to be more common cause compared to aplastic
anemia and acute leukemia. Sarode et al were among the first to report this
observation in patients of all age group from a referral hospital (15). In another
series from a referral hospital, MA accounted for 22.28% cases of pancytopenia
seen over 6 years. This was second only to aplastic anemia (18). Khunger et al have
observed MA accounting for over 72% cases (all age groups) with pancytopenia
(17). In our series of 109 patients of pediatric age group presenting with
pancytopenia, MA was the single most common cause (28.4%). Aplastic anemia
and acute leukemia accounted for 20% and 21% cases respectively (16).
Increased levels of homocysteine have been documented in folate- B12 deficiency
and are being linked to coronary artery disease.

Diagnosis and Differential Diagnosis:

Other causes of macrocytosis should be considered in the differential diagnosis of
MA. These causes include aplastic anemia and other marrow failure syndromes
(pure red cell aplasia, transient erythroblastopenia of childhood etc particularly
during recovery phase), congenital dyserythropoietic anemia, cold agglutinin
disease, chronic liver disease and hypothyroidism. These conditions are obviously
less frequent and have only mild to moderate macrocytosis ( higher the MCV, more
are the chances that the case will be of MA). Presence of anisocytosis, macro-
ovalocytes and tear drp cells are most prominent in MA and help differentiating it
from other causes of macrocytic anemias (35). Red cell distribution width (RDW)
which is an indicator of anisocytosis has been found to be significantly higher in
cases with MA as compared to aplastic anemia (36). However Saxena et al have
reported normal RDW in 31% cases with pernicious anemia. In cases with
increased RDW, a progressive fall was noted during recovery with treatment. Some
cases showed an initial increase before a fall in RDW was observed (37).
Diagnosis rests on demonstration of megaloblastic changes in the circulating red
cells precursors or bone marrow aspiration.
Levels of folate and B12 are required to identify the deficient micronutrient. Cut off
levels of 100-150 pg/ml of B12 and 3-5 ng/ml of folate have been used to define
respective deficiency states (21,22). Serum levels of homocystiene are increased in
deficiency of folate and B12 but levels of methylmalonic acid (MMA) are increased
in B12 deficiency only ( even increased urinary levels of MMA are considered
diagnostic of B12 deficiency). Very high specificity of methyl-malonicaciduria in
B12 deficiency has recently been confirmed. The authors also observed high
specificity of FIGLU test in folate deficiency. The authors advocated for using these
relatively inexpensive tests for diagnosis of B12 and folate deficiency (13,38). This
assumes significance in light of the fact that levels of B 12 and folate are quite
variable, difficult to measure and influenced by even subclinical hepatic
dysfunction.
Levels of lactate dehydrogenase (LDH) have been reported to be elevated in MA. In
a recent Indian study, levels above 3000 IU/l were found to be diagnostic of MA.
Reverse isoenzyme pattern (LDH1 > LDH2) was found to be a good adjuvant in the
differential diagnosis of MA from hemolytic anemia. (39)
Schilling test and its modifications are required in cases where the diagnosis of
pernicious anemia is in question.
Treatment:
Like any other deficiency state replacement therapy is required. Anemia and other
cytopenias respond to administration of very small doses of drugs. However, since
folate is available as a 5 mg tablet and its over dose is not associated with adverse
effects, 5 mg daily dose has been used.
For B12 deficiency, parenteral administration of B12 is usually recommended.
Intramuscular 1mg dose has been traditionally used but with this high dose cases
have developed tremors and other extrapyramidal symptoms (31). We observed
development of tremors in 6 out of 51 cases within few hours of administration of 1
mg dose. However, these tremors and other neurological signs have been self-
limiting but their appearance may influence the compliance. Sudden increase in the
levels of neurotransmitters has been offered as the plausible explanation for this
neurological phenomenon (24,31) Currently we are using a dose of 250 µ g in
infants and 500 µ g in older children.
The duration of treatment is not as standardized as in case of iron deficiency
anemia. Six to eight weeks treatment is usually described as sufficient. Decrease in
MCV, reticulocytosis, and improvement in platelet and neutrophil counts are
observed within few days. In our follow up, some cases have developed
thrombocytosis which resulted in stroke in one patient. Hence careful monitoring of
blood counts is required during treatment.
B12 and folate deficiency in adults/ elderly:
Levels of both -B12 and folate have been shown to decrease with age. In elderly, this
fall may lead to precarious levels. The low levels may in turn lead to vasculotoxic
hyperhomocysteinemia(1,2). As homocysteine levels increase with deficiency of
either nutrient, tackling of deficiency of both micronutrients is being advocated.
Some recent work suggests that vasculotoxic effect of folate may be independent of
homocysteine levels (2).
Brittleness of bones in elderly due to decreased bone mineral density (BMD) is
increasingly being reported in adults with folate and/or B12 deficiency. Initial
reports suggested that this effect also could be due to increased levels of
homocysteine, but recent data suggests a more direct action independent of
homocysteine levels (40).
Conclusions:
It is very obvious that there is “resurgence” of articles on folate-B12 deficiency/MA
over the last 10-15 years. This clearly reflects renewed interest in the subject as
more and more cases are being seen in clinical practice. The phenomenon appears
to be widespread. Many developing countries eg. India have anemia control/
prophylaxis programs and in addition there is overall improvement in general
standard of living. In light of these facts, the causes for increased incidence of
folate-B12 deficiency and /or MA needs to be elucidated. As pointed out earlier,
over the last few years, B12 deficiency has taken over as more common
micronutrient deficiency as compared to folate. Even this shift needs to be
explained. Role of malabsorption particularly giardiasis and H. pylori needs to be
clearly understood.

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