Date : July, 01 „ 2010 Publication : The Specialist Forum Page Number: 49-53
V - Vitamins - B12 And Folate
Part I - Physiology And Pathology
Ravnit Grewal and Johan Van Wyk - Senior Registrars
Peler Jacobs - Professor and Director
Lucille Wood - Senior Lecturer and Haematology Coordinator
Divisions of Haematopathology and Clinical Haematology
Stellenbosch University - Tygerberg Academic Hospital
- and The Department of Haematology and Bone Marrow Transplant Unit
incorporating
The Si-.nil Research Laboratory for Cellular and Molecular Biology,
Constantlaberg Medi-Cllnlc
This is the twenty-third in our monthly series of clinical vignettes (hat are centred on everyday presentations. Each emphasises practical
aspects ol team-based care having relevance for general practitioners, specialists and paramedical professionals alike.
D
isorders of [he blood and bone marrow result in
some of the commonest symptoms and signs for
which patients seek medical advice. A carefully taken
history and meticulous physical examination, complemented by thoughtful and judicious use of laboratory
tests, provides the clinical basis for a working diagnosis. Specialised haematology may be needed lo cxlend
evaluation to the bone marrow and plasma while supplementary imaging or radionuclide technology further link primary care practitioner or specialist to the
experienced haematologist. Such a, multidisciplinary
and fully interactive approach offer* the preferred way
to problem solving and optimum management.
A GENERAL COMMENTARY
Maintenance of good health is critically dependent on optimal
provision of trace elements such as iron as well as a variety of
vitamins. Nutritional inadequacy, in certain cases, such as
with folic acid, can be linked with increased incidence of
malignancy. In stark contrast is worldwide preoccupation with
dietary supplements, more often than noi in an irrational
endeavour lo maintain or promote a sense of well-being.
Despite such popularity it is not always realised that regulation of these products is limited and there may be unappreciated risks to offset any benefit, indeed adverse events occur
and these have been extensively studied by the American
Food and Drug Administration. It is therefore prudent for
individuals, and medical professionals alike, to balance the
perceived benefils of ihesc practices against enthusiastic, as
well as widespread, ingestion of supplements.
INTRODUCTION
Vitamin B12 and folate deficiencies are the main cause of
megaloblastic haematopoicsis. Both disorders are characterised by specific morphologic features in all cell lines readily evident in the bone marrow. Comparable disturbances in
cytomorphology. reflecting defective DNA synthesis but having normal levels of these two essential nutrients, arc the
prcleukaemic or myelodysplasia syndromes and retroviral
infections.
Maintenance of good health l i critically
dependant on optimal provision of
trace elements such as iron as well
as a variety of vitamins
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Date : July, 01 , 2010 Publication : The Specialist Forum Page N u m b e r : 49­53
PHYSIOLOGY
VITAMIN B12 (COBALAMINI
Biologic importance
A unique character!si\C is (he presence of cobalt in the mol­
ecule, giving rise to the alternative name of cobalamin.
There arc two main naturally occurring forms. 5'
dcoxyadenosylcobalaniin, which is located in the mito­
chondria, functions is a co­factor for the enzyme methyl­
malonyl CoA mutasc in the conversion of methylmalonyl
CoA to succinyl CoA. which then enters the citric acid
cycle. Methylcobalamin is found in plasma and the cyto­
plasm, accepts a methyl group from N5­methyl­tcirahydro­
folate (5­MTH). The litter is an essential step in the folate
pathway, and acts as the cofactor for the enzyme methion­
ine synthase in the conversion of homocysteine to methio­
nine. This pathway is the first step by which folate enters
the bone marrow and other cells from the plasma (Figure
I). Methionine donates methyl group to various reactions
in the body. Recall thai other forms, cyanocobalamin and
hydroxycobalamin exist.
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Intake, absorption and internal exchange
Recovery from external environment occurs in two ways.
Small amounts arc passively transported through the mucous
membranes including the na.sal mucosa, duodenum, ileum
and sublingualis The other main mode is active. Here peptic
digestion occurs in the stomach releasing (he vitamin from
food for uptake by a low affinity or R­binder, which is relal­
cd to iranscobalamin I and III. On reaching the alkaline envi­
ronment of the duodenum, the protein is degraded by the pan­
erratic trypsin and the liberated vitamin binds to an intrinsic
factor, which is generated by the parietal cells in the fundus
and body of the stomach. Intcrnalisation takes place in the
ileum via enlerocytcs having special receptors on the
microvillus membranes (Figure 2).
In plasma, there are 3 specific binding proteins, transcohal­
amin I. I I . and III. Iranscobalamin II is the essential protein
for transferring vitamin B12 into the cells through associa­
tion with specific cell membrane receptors. The amount
bound to iranscobalamin II is normally very low (about
25%). because most serum BI2 is bound to iranscobalamin I
which does not transfer readily across cell membranes.
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Distribution and requirements
j Loss to the environment
Cobalamin is exclusively produced by micro­organisms
There is a coniinuous secretion into the biliary system, most
and therefore is found only in meat, fish and dairy prod­
j of which is recovered and reused with excess found in faeces.
ucts. Unlike folate, this molecule is heat stable and is not
destroyed during cooking. H epatic stores are relatively
large at 2­3 mg. considering that only a small daily amount
Cobalamin is exclusively produced by
of 1­3 ug is required in a healthy diet. An average balanced
microorganisms
and t h e r e f o r e is found only
diet provides 5 to 30 ug. primarily derived from animal ori­
in
me
at,
fish
and diary products
gin, with small additions via bacterial synthesis in the gas­
trointestinal tract.
Figure 1: The effect of cobalamin and folate on nucleic acid synthesis
N ­5­ METHYL THF
^ _ r"METH uMNi"
^
j
SYH
NT ASE
POLYGLUTAMATEDTHF
COBALAMIN
/ " " 4—N
V­_^ _ J
METHIONINE
\"
METHYl­CBL
H
C 3­" " I ^ ­ f
TRANSFERASE ; Q _
•••'
ONS methylation
)P
HOMOCYSTEINE
DNA synthesis
and repair
▼ m tD c.
• • • • N­5­N10METH YLENETH F
The primary role of the intracellular metabolic routes ot cobalamin and folate is Ihe sequential transfer of single carbon units for the syn­
thesis and repair of DNA as well as DNA methylation through the metabolism of methionine.
TS = thymtdytaie synthase
THF = Tetrahydrofolate
CBL = Cobalamin
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Date : July, 01 „ 2010 Publication : The Specialist Forum Page Number: 49­53
Intake, absorption a n d internal exchange
Figure 2: Physiologic steps in cobalamin
absorption
Lumen
Q
Peptic Digestion
STOMACH
■
Free Cobalamin Bltndstor­Binders Of
Transcooalamin l
Q:
Pancreatic Enzyme Digestion and
Transfer to Intrinsic Facte*
DUODENUM
Intrinsic Factor: Cobalamin Uptake and
Transport by Transcobalamm I I
DISTAL ILEUM
MUCOSA
Internal Redistribution by
Transcooalamin m
BLOOD
Storage in Hepatocytes and Granulocyte
TranscoWijimin in
STORES
9Q:
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Q
Recovery from the diet is mostly in the polyglutamate form,
which is hydrolyse d to a monoglutamate by the e nzyme
hydrolase for the purposes of recovery occurring in the brush
border, mostly in the uppe r small inte stine , with de clining
capacity in the ile um and jejunum. Actual move me nt is via
simple diffusion or by binding to folate transport prote ins.
Further enzymatic breakdown of monoglutamate takes place in
the enterocyte before it is released in the methylated form (N5­
mcthyl te trahydrofolate /5­MTHF) into the portal circulation.
Most of this vitamin then circulates loosely bound to albumin
or to a lesser degree to a high­affinity binding protein. Cellular
uptake is facilitated by folatc­rcccptors (Figure 3).
Loss to the environment
Unbound plasma folate isfilteredand then reabsorbed by the
proximal re nal tubule s with urinary loss re fle cting only a
small percentage of the dietary intake.
Biliary e xcre tion is estimated to be 100 ug/day. most of
which is reabsorbed in an emcrohepalic circulation. Presence
in faeces appears to be similar in type and quantity to urinary
lasses.
C0BALAMIN-F0LATE INTERDEPENDENCE
Cobalamin Is released from lood source through peptic digestion
of animal proteins. Absorption is dependent on interaction with
intrinsic tactor and further transportation. Storage occurs through
binding to the transcobalamins.
Excretion in ihe urine w i l l occur only i f the concentration
exceeds the binding capacity o f the transport proteins.
T H E FOLATES
Adequate and simultane ous bioavailability of the se hae ma­
tinics have intrace llular inte rde pe nde nce . Folic acid is cru­
cially linke d to B12 me tabolism by transfe rring a me thyl
group from 5­MTHF to cobalamin ­ a central ste p in repair
and synthe sis of de oxyribonucle ic acid (DNA). Inside the
cells, folic acid is polygluiamate d to the biological active
form, and cobalamin simultane ously be come s me thylate d.
Figure 3: The pathway for folate absorption
DIETARY POLYGIUTAMATES
Biologic importance
Folic acid is the trivial name for pieroy(glutamic acid, which is
the pare nt compound of a large group of compounds, the
folates, that occurs in nature conjugate d to polyglutamate
chains. Humans are unable to synthesisc these structures, and
therefore re quire pre forme d naturally occurring vitamin for
many functions in the body, including purine and pyrimidine
synthesis and inte grity of the homocyste ine pathway.
Deficiency re sults in defective DNA synthesis that impairs the
ability of all prolife rating ce lls to synthesise enough nucle ic
acid during the ce ll cycle , with subse que nt asynchronous
nuclear and cytoplasmic maturation. This manifests with inef­
fective haematopoiesis and impaired cell proliferation.
Distribution and requirements
The main sources of folate arc liver, spinach and other dark
green, leafy ve ge table s. In most countries cereal fortification
often be come s the major dietary source. Adults require 100 ­
200 ug/day. with increased requirements of up to 500 ug/day
during pre gnancy. Childre n and infants have lowe r re com­
mended daily requirements of 25 ­ 100 ug.
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FOUCACIO
MONOGLUTAMATE
BRUSH BORDER
COMMON POOL
ENTEROCYTE
PORTAL BLOOD TO LIVER
As dietary folates are ingesled as potyglulamates, they are decon­
jugated by a hydrolase present in the brush border of jejunal and
ileal epithelial cells to the monoglutamated form for absorption. In
the enterocyte monoglutamate folate is reduced to tetrahydrofo­
late (THF) and then methylated to N5­methyl­THF where it forms
the common pool of enterocyte folate, before release into the por­
tal circulation.
Date : July, 01 , 2010 Publication : The Specialist Forum Page Number: 49­53
Methylcobalamin acts as a coenzyme in the remethylation
cycle, where single carbon units arc transported to other pro­
teins, with thereconversionof homocysteine hack to methio­
nine.
Due to the large size, the polyglulamated folate form can­
not diffuse across membranes, thereby retaining it for physi­
ological intracellular function. Specifically, a methylene
group is added lo fonn N­5­NlO­mclhylene­TH F. which is
involved in the nucleic synthesis. This compound provides
the methyl group needed in the conversion of deoxy uridine
monophosphate (dUMP> to deoxythymidine monophosphate
(dTMP). one of the building blocks of DNA. Folate is con­
served intracellular!)' by two mechanisms. It can be reduced
back to polyglulamated TH F after demcihylation. or N­5­
NIO­mclhylcne­THF can cnzymaiically be reduced to N5­
mcihyl­THF (FJgurt 11.
During cobalamin deficiency, intracellular folate concentra­
tion falls due to the lack of polyglutamatc formation, as 5­
MTHF demethylation cannot OCCUT The plasma folate rises
as the monoglutamatc form diffuses out of the cell.
Homocysteine accumulates, as it is dependent on cobalamin
for its conversion to methionine. Similarly a folate deficien­
cy would cause failure of the methyl group donation to cobal­
amin and therefore accumulation of homocysteine in the
cells. Folate and cobalamin arc biochemically intertwined
and impaired DN \ synthesis will occur when either vitamin
is deficient. Distinctions do exist and can be assessed bio­
chemically.
I THE FOLATES
: Congenital
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May be due lo defective transmembrane transpon or sub­
optimal utilisation, resulting in early appearance of severe
megaloblastic anaemia, which responds to treatment.
Unfortunately, the associated mental retardation improves
less satisfactorily because of inability to maintain adequate
folate levels in the spinal fluid. Malfunction of the enzyme
mcthylene­tetrahydrofolalc reductase is characterised by
mental retardation, seizures, and schizophrenic syndromes.
often with vasculopathy. but not necessarily with any
haematologic abnormality.
j Acquired
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Daily attract more attention as a result of the high inci­
dence of developmental abnormalities of the neural tube in
neonates and children. In addition, a variety of neurologic.
metabolic, cardiovascular, neoplastic, and epithelial
lesions arc becoming more prominent in the elderly, with
increasing concern about general poor nutrition, drug and
alcohol abuse. Malabsorptive States, as seen with inflam­
matory bowel disease, sprue or short bowel syndrome and
certain drugs, like methotrexate, trimethoprim and pheny­
tion also hinder absorption. Furthermore failure to meet
increased demands, as in pregnancy, exfoliative dermatitis
and chronic haemolysis, also leads to folate deficiency.
| CLINICAL FEATURES
PATHOLOGY
VITAMIN B12 (COBALAMIN)
Congenital
Defects can he due to single gene mutations and are trans­
mitted as recessive traits affecting absorption, transport, or
intracellular metabolism of cobalamin. Imerslund­Ciracsbcck
syndrome is one such example where whole families present
with selective intestinal malabsorption uninfluenced by
intrinsic factor function. It is associated with proteinuria and
structural genitourinary abnormalities. Found typically in
infancy, biochemical disturbances, especially methylmalonic
aciduria, result in neurologic defects.
Acquired
Gastric disease accounts for most deficiencies including gastri­
tis, pancreatitis and small bowel diseases exemplified by
Crohn's disease. Infrequently occur in entire populations, and
may be found in groups that practice veganism or where poor
diet is found, as among the institutionalised elderly. More usu­
ally, autoimmune destruction of foregut mucosa leads to
decreased production of intrinsic factor in Addisonian
Pernicious Anaemia: extensive resection of stomach or terminal
ileum may have a similar result. Competition for vitamin Bl2
by fish tapeworm, or intestinal stasis syndrome and sprue are
alternatives that should not be overlooked. Numerous medica­
tions can limit absorption, and include neomycin, biguanides
and proton pump inhibitors.
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j tures of both deficiencies arc similar, causing megaloblas­
| tic anaemia. Vitamin B12 deficiency, however, develops
! less rapidly than folate due lo the larger reserve stores
I often taking years to manifest. In contrast, folate depletion
j can occur within months when intake is disturbed.
! Presentation is with non­specific symptoms and signs due
j to tissue hypoxia and compensatory mechanisms. Mild
j jaundice due to increased haemoglobin reflects breakdown
j secondary to the ineffective haematopoicsis. Rapidly grow­
■ ing cells, like epithelium, exhibit abnormal growth and
; lead lo glossitis, angular stomatitis and malabsorption with
| weight loss. Melanin pigmentation and purpura, due to sec­
i ondary thrombocytopenia, are less frequent skin manifes­
; tations.
j Cobalamin deficiency
| Neurological problems are usually seen in severe deficicn­
i cy. including subacute combined degeneration of the cord
: and a progressive, n mmetrical neuropathy.
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Folate and cobalamin are biochemically
intertwined and impaired DNA synthesis w i l l
occur when either vitamin is deficient
Date : July, 01 „ 2010 Publication : The Specialist Forum Page Number: 49-53
The bruni falls on the lower limbs initially as paresthesia
and ataxia, but can progress to spasticity and paraplegia.
Rarely, optic atrophy or psychiatric disorders arise, among
which arc depression, mania or even psychosis, although
may include memory loss, irritability and dementia. It is
important to note that normal peripheral full blood count
docs not exclude clinically important deficiency and. particularly in the elderly, levels should be interpreted in this
context.
The skeleton is frequently overlooked when osteoblast
function is suppressed in the face of cobalamin deficiency,
increasing risk for osteoporosis and hip and spine fractures.
Pernicious anaemia is more commonly associated with
elderly females of northern European descent, but can also
occur in younger, and especially. African women. This
condition carries increased risk for other auto-immunc diseases and stomach carcinoma. The coincidence of iron
deficiency can mask the macrocytosis. and affected individuals may not necessarily be anaemic. Hyptrsegmcntcd
neutrophils documented on peripheral smear should alert
clinicians (o (his condition.
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Folate deficiency
Although clinically similar to cobalamin deficiency, there
is usually a stronger association with substance abuse and
poor diet upon presentation. There are strong implications
for occurrence of neural lube and other congenital defects
during pregnancy. It also carries increased risk of epithelial
and other cancers including colon as well as neuroblastoma
in the young age group.
In the older population cardiovascular disease is correlated with hypcrhomocystcinaemia, with increased incidence
of myocardial infarcts, peripheral and cerebral vascular
disease such as venous thrombosis and atherosclerosis. The
benefits of folate supplementation in reducing the rate of
myocardial infarction or stroke have not yet been established. Less clearly defined are disturbances in platelet
function that may accelerate thrombosis in parallel with
hyperhomocysteinacmia.
References are available on request.