A nnals o f C l in ic a l L aboratory Sc ie n ce , Vol. 1, No.

8
Copyright © 1971, Institute for C lin ic a l S c ie n c e

The Biochemistry o f Folic Acid and Vitamin B12

M . M IC H A E L L U B R A N , M .D ., P h .D .

Harbor General Hospital

Torrance, CA 90509

Folic acid and vitamin B 12 are the p re­ tissues and hum an red cells as a polygluta­
cursors of coenzymes involved in many mate, typically containing three to seven
im portant biochemical reactions. Most of glutamic acid residues linked by gamma-
our knowledge of the biochemistry of these peptide bonds. H um an small intestinal
vitamins has been obtained from the study juice contains the enzyme folic conjugase
of bacterial metabolism: there have been which is necessary for the hydrolysis and
fewer studies of animal or hum an tissues. absorption of the poly glutamates. Absorp­
The role of these vitamins in m an has been tion is an active process th at occurs mainly
determ ined mainly by the investigation of in the duodenum and jejunum.
patients with folate or B J2 deficiency. There Folic acid itself has no biological activity.
is still m uch th at is not known about their It is the precursor of a group of coenzymes
behavior in man; nor is it certain th at all derived from tetrahydrofolic acid in which
the biochemical reactions in which they the N atoms at 5 and 8 and the C atoms
participate have been discovered. Reactions a t 6 and 7 of the pyrazine ring have been
involving folic acid and B 12 in micro-or­ reduced. The tetrahydrofolate derivatives
ganisms and even animals do not neces­ contain groups attached to N-5, N-10 or
sarily occur in man. In particular, megalo­ both atoms by a bridge (table 1). The
blastic anemia as seen in the hum an does coenzyme is attached to its specific enzyme
not occur in animals deficient in folate or through the pyrimidine ring.
Bi2. The major biochemical reactions of Folic acid is reduced to dihydrofolic acid
folic acid and vitamin B i2 will be described and then to tetrahydrofolic acid by the
here w ith emphasis on those known or b e­ enzyme dihydrofolic acid reductase, which
lieved to be im portant in hum an m etabo­ requires pyridine nucleotides (N A D H 2 or
lism. The literature on the subject is vast. NADPH2). Dihydrofolic acid, which is
Recent reviews are cited in the references. the natural substrate, is reduced more
readily than folic acid by this enzyme.
F olic Acid Folic acid reductases, which convert folic
acid to dihydrofolic acid only, have been
Folic acid is pteroylglutamic acid (figure described in some bacteria. Their role in
1). It consists of a pteridine portion linked man is uncertain. Dihydrofolic acid reduc­
through p-aminobenzoic acid to L-glutamic tase is widely distributed in bacterial and
acid. It is found in plants (its name is de­ animal cells. It is strongly inhibited by
rived from the Latin folium, a leaf), animal folic acid antagonists such as am inopterin
236
 BIOCHEM ISTRY O F FOLIC ACID AND V ITA M IN B 12 237

Table I. The Tetrahydrofolate

H C00H
Y ' ¿;|r C H 2 - N - < f 'y C 0 - N - C H - C H 2-C H 2-C 00 H Coenzymes
Substituent Formula Attached to N Oxidation level

pteridine ^-aminobenzoic L-glutamic Methyl ch3 5 Methanol

acid acid Methylene c h 2- 5 and 10 Formaldehyde

pteroic acid Methenyl CH = 5 and 10 Formate

V---------------------------------------------- y-----------------------------------------------/ Formyl CHO 5 or 10 Formate
pteroylglutamic acid (PteGlu) Formimino CH=NH 5 Form ate

F ig u r e 1. Structural formula of folic acid. Folate coenzymes include tetrahydrofolate and substituted
tetrahydrofolates

(4-aminopteroylglutamic acid) and m etho­

trexate (4-amino- 10-methylpteroylglutamic
ac id ).
Functions of Folic Acid Coenzymes
The major function of the folate co­
enzymes is the transfer of one-carbon
groups in a variety of synthetic reactions,
which depend upon the state of oxidation
of the group transferred. The lowest level
of oxidation is th at of methanol (m ethyl
group); next is formaldehyde (m ethylene
group); highest is formate (methenyl,
formyl, formimino groups). Coenzymes
containing these groups are readily inter­
convertible by specific enzymes, except for
5-methyltetrahydrofolate. The m ethylene
level is reduced to methyl by 5, 10-
m ethylenetetrahydrofolate reductase,
flavo-protein requiring FADH2 (reduced
flavin adenine dinucleotide); the reverse
reaction does not occur in man. Regenera­
tion of tetrahydrofolate from methyltetra-
hydrofolate is B j 2 dependent and occurs
during the synthesis of methionine. This
process is described in the section on B i 2.
the folate coenzymes are derived from the
following compounds: serine (/3-C) mainly,
formate and histidine to a small extent only.
Single carbon atoms derived from these
groups are found in: serine (/3-C), formate,
formaldehyde, purines (C-2 and C-8),
thymine (5-methyl C ) and methionine (5-
m ethyl C ). The reactions involved are
described below. All are believed to take
place in hum an metabolism.
(1) L - serine-glycine interconversion
Serine hydroxymethyltransferase, tetra­
hydrofolate and pyridoxal-5-phosphate re-
versibly convert L-serine to glycine. The
enzyme is present in hum an red cells.
C H , O H -C H -C O O H + H , PteC lu ¡ a C H , C O O H + 5 .1 0 -C H .-H , P teC lu
I I
NHa NHS
serine tetrahydrofolate glycine m ethylenetetra-
hydrofolatc
a
2) Formimino glutamic acid (FIGlu) con­
version
FIG lu is an interm ediate in the enzy­
matic degradation of histidine in animals.
It is converted to glutam ic acid by FIG lu
formiminotransferase and tetrahydrofolate.
COOH - C H - C H *-C H 8 COOH + H 4 PteClu -> C O O H -C H -C H a-C H a COOH

The NAD P-dependent enzyme, 5,10-methy- NH NH.

I
lenetetrahydrofolate dehydrogenase, brings NH = CH
formim inoglutamic acid glutam ic acid
about the interconversion of coenzymes 5 -C H N H -iri PtcGlu
formim inotetrahydrofolate
containing groups at the form aldehyde and
formate levels of oxidation. M ethenyl and The formiminotetrahydrofolate produced in
formyl groups are interconverted by a cyclo- this reaction is converted by cyclodeaminase
hydrolase; methenyl and formimino deriva­ into 5,10-methenyltetrahydrofolate; this in
tives by a cyclodeaminase. turn is converted by cyclohydrase into 10-
The single carbon groups transferred by formyltetrahydrofolate. In patients with
238
folic acid deficiency, the catabolism of
FIG lu is decreased and its excretion in the
urine is increased. Excretion is more m arked
in the presence of a histidine load. Vitamin
B 12 deficiency may also give rise to in­
creased urinary excretion of FIGlu, b u t the
increase is not as m arked as in folic acid
deficiency.
3) Utilization of formate
Tetrahydrofolate formylase (formate-ac-
tivating enzym e), ATP and tetrahydrofolate
give rise, reversibly, to 10-formyltetrahy-
drofolate; this coenzyme transfers the
formyl group to appropriate substrates (for
example, in the synthesis of purines).
H* P teG lu + H C O O H + ATP *=» l()-C H O -H 4 PteGlu + ADP + P i
formyltetrahydrofolate
Only 10-formyltetrahydrofolate participates
in formate transfer in purine synthesis. The
energy of hydrolysis of 5-formyltetrahydro-
folate is too low for this transfer to occur.
This enzyme can be converted into the 10-
formyl form by ATP and magnesium ions.
4) Formylation of glutamate
This takes place through the action of
glutam ate transformylase and 5-formyltetra-
hydrofolate; 10-formyltetrahydrofolate is in­
active in this reaction.
G l u t a i n a t e 5 - C H O - H i PteGlu formylglutamide -j- I I , PteGlu
The reverse reaction is im portant in for­
m ate metabolism, since formylglutamate is
an interm ediate in the conversion of the
a-C atom of glycine into active formate.
Urinary formate excretion rises in folic acid
deficiency. Excretion is increased by oral
tryptophan b u t not histidine.
c h2 - nh2
I 5,10-CH ■>
\ H4 PteGlu
NH
I I
R ib o s e - P
GAR
LUBBAN
Syntheses Involving Folic Acid
Coenzymes
Folic acid coenzymes are involved in the
synthesis of purines, pyrimidines ( and thus,
indirectly, in the synthesis of DNA) and
methionine. The last synthesis also involves
vitamin B]2 and will be described later.
1) Purine synthesis
Folic acid is concerned in the introduc­
tion of carbon atoms into positions 8 and 2
in the purine ring; different coenzymes are
involved. C-8 is introduced by the forma­
tion of formylglycinamide ribonucleotide
(FG A R) from glycinamide ribonucleotide
(GAR) by 5,10-methenyltetrahydrofolate
and GAR transformylase (figure 2). Tetra­
hydrofolate is formed. The formyl group of
FGAR later condenses with the am ide N
to form an imidazole ring.
C-2 is introduced by formylation of 5-
amino-4-imidazole-carboxamide ribonucleo­
tide (AICAR) w ith 10-formyltetrahydro­
folate and AICAR transformylase to give
4-formamido-5-imidazoecarboxamide ribo­
nucleotide (FAICAR) and tetrahydrofolate
(figure 3). AICAR undergoes ring closure
to form inosinic acid, which is subsequently
converted to adenylic, xanthylic and guany-
lic acids by appropriate enzyme systems.
Folate inhibitors ( aminopterin, metho­
trexate) do not inhibit the folate dependent
steps of purine synthesis.
2) Pyrimidine synthesis
Folic acid is not concerned in the syn­
thesis of the pyrim idine ring, but in the
introduction of the m ethyl group of thy-
c h 2 - nh
' \ * HQ
\ + H ,P le G lu FICUII 2, ,„ tll, j „ c.
NH tion of carbon atom 8
into purine ring.
R ib o s e - P
FG A R
 BIOCHEM ISTRY O F FOLIC ACID AND V IT A M IN B ]2
0 0
II
r
HoN C— N
2 II
H2N
Ribose-P
F ig u r e 3. Introduc­
tion o f carbon atom 2
AICAR
into purine ring.
mine (figure 4) and, in phage infected E.
coli, the hydroxymethyl group of 5-hy-
droxymethylcytosine. The folate coenzyme
is 5,10-methylenetetrahydrofolate. In the
synthesis of thymidylate, C H 2 is transferred
from the folate coenzyme by thym idylate
synthetase and simultaneously reduced to
CH 3; concurrently the tetrahydrofolate
initially formed is oxidized to dihydrofolate
which is then reduced to tetrahydrofolate
by dihydrofolate reductase and NADPH2.
Recycling of the folic acid and the con­
tinuance of pyrimidine synthesis thus de­
pends upon the activity of dihydrofolate
reductase. Folic acid antagonists which
inhibit the action of this enzyme inhibit
thym ine synthesis; further, there is an ac­
cumulation of dihydrofolate and a decrease
in the am ount of tetrahydrofolate available
for conversion into other folate coenzymes.
Thus, other metabolic functions of folic
fl| : |l
F ig u r e4. Introduc­
tion of m ethyl group into
a pyrimidine.
IS N
d R ib o se -P
deoxyuridylic acid
239
II
* h2nTCvc
^ I O - C H O - H 4 P te G lu
8CH ------------ 2------ > 8CH + H4 PteGlu
/
0 =C H ^ C - N
i
j Ribose-P
FAICAR
0
CH
Ribose-P
INOSINIC ACID
acid are im paired by the antagonists. The
synthesis of thym idylate is believed to be
the rate limiting step in DNA synthesis.
Vitamin B i 2
Vitamin B i 2, although found in most
animal tissues, is almost exclusively syn­
thesized by micro-organisms, either com­
mensals in the animal’s digestive tract or
ingested w ith animal food. The hum an is
wholly dependent on ingested B i 2, being
unable to utilize any of the vitam in which
m ight be synthesized in the intestine.
Vitamin B i 2 probably occurs in nature in
its coenzyme form linked to a specific
protein. Cyanocobalamin, the form of the
vitam in containing a cyanide group, does
not occur in the body, b u t is form ed dur­
ing the extraction and purification pro­
cedures for obtaining the vitam in (active
OH
+ 5 ,IO - C H o - H 4 P te G lu > ^ ¡P 0^ H2 PieGlu
°SV
I
d R ib o se -P
thymidylic acid
240 LUBRAN
charcoal is used, which contains cyanide).
However, most assay procedures, w hether
microbiological or radioisotopic, use cy-
anocobalamin as the reference material; the
various forms of the vitamin are first con­
verted to it during the prelim inary extrac­
tion procedures.
Chemistry of Vitam in Bn
Vitamin B12 has the structure shown in
figure 5. It consists of two major portions:
a planar group closely, bu t not completely,
resembling the porphyrins, and a nucleotide
lying in a plane almost at right angles to
it. The porphyrin- like structure is term ed
the corrin nucleus; unlike the porphyrins,
there is a direct linkage betw een the two
a-carbon atoms of rings A and D. The cen­
tral cobalt atom, which is trivalent and
positively charged, is linked to the four
nitrogen atoms of the reduced pyrrol rings,
C0A/Hz
to the nitrogen atom of the nucleotide and
to the cyanide group. Thirteen of the nine­
teen carbon atoms of the corrin nucleus are
fully substituted w ith methyl groups or
acetam ide or propionam ide residues. The
groups attached to the bridge carbon atoms
are in the plane of the corrin nucleus; the
other groups lie above or below the plane
of the nucleus.
The nucleotide in vitam in B12 differs
from the nucleic acid nucleotides in two
respects: the base is neither a purine nor
pyrimidine, b u t 5,6-dimethylbenziminazole;
the ribose linkage is «-glycosidic, not ¡3-gly-
cosidic as in the nucleic acids. The ribose
is phosphorylated at C-3. The phosphate is
esterified w ith l-amino-2-propanol, which is
combined through an amide link with the
propionic acid residue of ring D at C-17.
The third hydroxyl group of the phosphate
is ionized; the negative charge on the O
atom and the positive charge on the Co
atom make vitam in B12 an inner salt.
The B 12 structure w ithout the cyanide
group is term ed a cobalamin. Other
groups may take the place of cyanide. Im ­
portant cobalamins are: hydroxocobalamin,
which contains an O H linked to the Co
atom; aquocobalamin, which has H 20
linked to the cobalt (it is the form in which
hydroxocobalamin occurs in neutral or acid
solution); sulphito, chloro, nitrito, bromo
and thiocyanato groups m ay be attached
to the Co by appropriate means.
Coenzyme B i2, finked to a specific pro­
tein, is the principal form in which B12
occurs in hum an and animal tissues. Two
coenzymes are known: 5'-deoxyadenosylco-
balamin in which adenosine (m inus its OH
at C 5 ') is linked at this atom to Co, and
methylcobalamin, in which a methyl group
is directly attached to the cobalt atom.
Both coenzymes are light sensitive in the
presence of oxygen, yielding hydroxoco­
balamin. Cyano-, hydroxo- and other forms
of Bi2 are rapidly converted in vivo into the
coenzymes.
 BIOCHEM ISTRY O F FOLIC ACID AND V IT A M IN B 12 241

Biochemical Reactions of B 12 not in humans. The next two reactions are

Coenzymes
These m ay be divided into two major
groups: those requiring deoxyadenosylco-
balam in and those requiring methylco-
balamin. As w ith folic acid, most of our
knowledge of the biochemistry of B 12 has
been obtained through the study of micro­
believed to occur in man, as well as in
micro-organisms.
5) L-methylm alonyl-CoA mutase reaction*
This enzyme catalyzes the interconver­
sion of L-methylmalonyl-CoA ( an inter-
0 Co in this enzym e refers to coenzym e A, not

cobalt
organisms. Those reactions known or be­
lieved to occur in the hum an will be dis­
I. Glutamate mutose
cussed in some detail; the remaining
CH-*
reactions will be described briefly. I H
H C — C-COOH
Reactions requiring 5'-deoxyadenosylcobala- | nh2
m in COOH

In these reactions, the coenzyme-enzyme

complex combines w ith H from the sub­ 2. Dioldehydrase
strate and transfers it to the adjacent C
H -
1 H
atom. A group, originally attached to this H C -O fH HCH
I + H 20
C atom, migrates to the C atom from which ( h/ c- Io H HC^O
H ------
the H has been detached. These changes
3. Ethanolamine deaminase
result in an intram olecular rearrangem ent
of the substrate. In some cases, w ater or H ____ ,
HC-[NH^
H
HCH
ammonia may be eliminated. In the case ( h/ c - o 1h
+ NH,
H C =0
of the ribonucleotide reductase reaction, w H L-

which is more complex, the substrate is 4. j6-Lysine isomerase

reduced in addition (figure 6).

H H H H H II , H H H H H II
1) Glutamate mutase reaction H C -C ^ C -C -C -C -O H - - H C - C - C - C — C -C -O H
L W iH | » H I H | H
Glutamic acid is rearranged to form L- nh2 NH2 nh2
ihreo- ^-aspartic acid. 3 ,5 diaminohexanoic acid
5. L -M e)h ylm a lon yl-C o A mutase
2) Dioldehydrase reaction
Ethylene or propylene glycol is con­
verted to acetaldehyde or propionaldehyde
respectively.
3) Ethanolamine deaminase reaction COOH COOH

Ethanolam ine is converted to acetalde­ 6. Ribonucleotide reductase

hyde.
rI °H nH I H
4) ¡3-Lysine isomerase 8 a s e -C -C -C -C -C -0 -P -P -(P )-
H I I H H
Lysine is converted to butyrate, acetate OH OH

and ammonia by some clostridia. An inter­

mediate, /3-lysine, is converted to 3,5-di- I H H I H
Base - C - C - C - C - C - O - P - P - ( P )
aminohexanoic acid. The further stages in H I H H
OH
the degradation are not clear.
F ig u r e 6. Reactions requiring
These four reactions occur in bacteria, deoxyadenosylcobalamin.
242 LUBRAN
m ediate in the enzymatic conversion of
propionate to succinate) and succinyl-CoA.
The reaction involves the transfer of the
H from C-3 of the substrate to C-5' of the
deoxyadenosyl moiety of the coenzyme. The
CoA-thioester carboxyl group migrates from
C-2 to C-3 of the substrate and the H is
transferred from the coenzyme to C-2.
There is decreased activity of this enzyme
system in vitam in Bi 2 deficiency; urinary
excretion of methylmalonic acid is in­
creased from the normal value of less than
4 mg in 24 hours to over 6 mg m ethyl­
malonic acid excretion in normals is un­
affected by an oral dose of 10 gm of valine;
this dose causes an increased excretion in
patients w ith pernicious anemia. Increased
excretion of methylmalonic acid is not con­
sistently found in patients with other forms
of B 12 deficiency. It is normal in patients
with folic acid deficiency who do not have
concurrent B 12 deficiency (these two de­
ficiencies often occur together). Normal
methylmalonic acid excretion is restored in
patients w ith B ]2 deficiency who are treated
with parenteral Bi2. Folic acid administra­
tion in these patients is w ithout effect.
These clinical observations point strongly to
the existence of methylmalonyl-CoA mutase
in the human.
6 ) Ribonucleotide reductase reaction
This enzyme catalyzes the reduction of
ribonucleotides to deoxyribonucleotides,
which are required for DNA synthesis. The
substrate is a ribonucleotide diphosphate
or triphosphate. The reaction involves the
substitution of an OH group by H in the
2' position of the ribosyl moiety by a com­
plicated mechanism involving transfer of
H from a thiol group of a low molecular
weight protein to the C-5' of the coenzyme-
adenosyl moiety. H ere it is exchanged for
an OH. This is not an intramolecular re­
arrangement, as occurs in the other reac­
tions described, b u t transfer of H, via the
coenzyme, from a hydrogen donor.
Study of this reaction in bacteria has
shown th at two different reductases exist;
one is B 12 dependent, the other is indepen­
dent of Bi2. Mammalian ribonucleotide
reductase requires Mg++ and acts on
ribonucleotide disphosphate. I t is not
stim ulated by Bi 2 coenzyme and is prob­
ably not B 12 dependent. B i2-depleted bone
marrow, obtained from patients with
pernicious anemia, showed no stimulation
by B i 2 or the deoxyadenosyl coenzyme .5
Thus, there is no evidence to show th at
ribonucleotide reductase activity in m an is
dependent on deoxyadenosylcobalamin.
Reactions Requiring Methylcobalamin
These reactions involve transfer of a
m ethyl group from 5-methyltetrahydro-
folate to an appropriate substrate. M ethyl­
cobalamin, bound to a reducing enzyme,
acts as interm ediate transfer agent in the
reaction. Only the first reaction described
below is known to occur in man.
1) M ethionine synthesis
In this reaction, m ethionine is formed
from homocysteine by transfer of a methyl
group from 5-methyltetrahydrofolate;
FA D H 2, S-adenosylmethionine and methyl­
cobalamin are required. The de novo syn­
thesis of the m ethyl group takes place as
described earlier through the formation of
5-methyltetrahydrofolate. Methionine syn­
thesis serves as the means of renewing the
tetrahydrofolate.
SH.OH2.CH2.C H N H .C O O H + 5 - C H ,- H 4 PteGlu -*
homocysteine
C H ,S.C H 2.C H ,.C H N H o.COOH + H 4 PteGlu
methionine
T he reaction mechanism is complex. Ini­
tially, the reducing enzyme is combined
w ith a cobalamin. In the presence of the
other factors, the Co is reduced to the
m onovalent state, in which it readily ac­
cepts the C H 3 group from the S-adenosyl­
methionine, forming enzyme-bound methyl-
 BIOCHEM ISTRY O F FO LIC ACID AND V ITA M IN B 12
cobalamin. The m ethyl group is transferred
from this as a carbonium ion to homo­
cysteine and the cobalamin is rem ethylated
by a methyl group donated by the 5-methyl-
tetrahydrofolate. In the reaction, methyl
groups are donated by 5-methyltetrahydro-
folate; S-adenosylmethionine acts as a
prim er (i.e., it initiates the reaction) and
m ethylcobalamin is the transfer agent. A
Bi2-independent pathw ay for methionine
synthesis exists in some bacteria. It is of
fundam ental importance to determine
w hether m ethionine synthesis can occur
independently of B]2 in the human. This
question is discussed in the next section.
2) M ethane formation
M ethanol is converted to m ethane by
bacteria in sewage sludge. M ethylcobalamin
is involved. The mechanism is similar to
th at described for m ethionine synthesis.
3) Actetate synthesis
Both 5-methyltetrahydrofolate and BJ2
are involved in the total synthesis of
acetate from C 0 2. As in the two reactions
described above, a protein-bound methyl­
cobalamin complex is formed as an inter­
mediate.
Interrelationships of Vitamin Bi2 and
Folic Acid
Folic acid deficiency and pernicious
anemia produce the same cellular changes
in the hum an bone marrow. Tissue cul­
ture studies of normal hum an m arrow and
megaloblastic marrow, involving incorpora­
tion of tritium -labelled precursors into
DNA and RNA of megaloblasts and
erythroblasts, suggest that, in these defici­
ency states, there is a failure to complete
DNA synthesis prior to mitosis, i.e. there is
prolongation of the S and G-2 phases of
the cell cycle. These observations suggest
a close association betw een B12 and folate
during hematopoiesis.
243
Of the many biochemical reactions in­
volving B 12, only two are believed to occur
in man: the isomerisation of L-methyl-
malonyl-CoA to succinyl-CoA (not affected
by folate; disturbance of this enzyme action
is postulated as the cause of the degenera­
tive disease of the nervous system due to
B 12 deficiency), and the methylation of
homocysteine to methionine, which is folate
dependent. It is this latter reaction which
forms the common ground betw een folate
and vitamin Bi2. The folate coenzymes are
interconvertible, with the exception of 5-
methyltetrahydrofolate. The probable way
in which tetrahydrofolate is regenerated is
through the synthesis of methionine.
The “m ethyltetrahydrofolate trap” hy­
pothesis postulates that, in fact, 5-methylte-
trahydrofolate can be converted to other
folate derivatives only by the cobalamin-
dependent methyltransferase reaction; this
reaction is seriously diminished in Bi2 de­
ficiency. The result is th at the total body
pool of folate is largely trapped in the
form of m ethyltetrahydrofolate and reac­
tions depending on the other folate co­
enzymes are diminished. In particular,
purine and pyrim idine biosynthesis are
diminished and therefore DNA production
is impaired. Megaloblastosis results. The
hypothesis depends upon the dem onstra­
tion that the cobalamin-independent p ath­
way, known to exist in some bacteria, does
not exist in man, or is of little significance.
This has not, as yet, been satisfactorily
determ ined by direct methods.
Some indirect evidence supports the hy­
pothesis. M ethyltetrahydrofolate is the
principal monoglutamate coenzyme of
plasma and liver. In Bi2 deficiency asso­
ciated w ith a normal intake of folate and
methionine, there is often an increase in
plasma methyltetrahydrofolate; this is con­
sistent with the hypothesis. The crucial
test of the m ethyltetrahydrofolate trap
hypothesis would be the demonstration of
tissue deficiency of folate derivatives, other
244 LUBRAN

than methyltetrahydrofolate, in patients the clinically docum ented relationship be­

w ith pernicious anemia in relapse, on ade­
quate dietary folate and methionine.3 Di­
rect measurements on m arrow and liver
have not been performed. C urrent methods
have inadequate sensitivity.
However, indirect evidence of folate
enzyme levels in tissues can be obtained
by studying in these patients enzyme reac­
tions which are folate dependent. The in­
creased excretion of FIG lu and amino-
imidazolecarboxamide in the urine of Bi2
deficient patients, diminished by treatm ent
with folate, is in keeping w ith the hypoth­
esis. In vitro experiments on the thymid-
ylate synthetic activity of cells from bone
marrow aspirates suggest th at there is a
deficiency of m ethylenetetrahydrofolate in
the marrow cells of cobalamin-deficient as
well as folate deficient patients. Although
there are some contradictory experiments,
the m ethyltetrahydrofolate trap hypothesis
provides at present the best explanation for
tween vitamin B i 2 and folic acid in cell
metabolism.
References
1. C h a n a r i n , I.: The M egaloblastic Anaemias:
F. A. D avis Co., Philadelphia, 1969.
2. H u e n n e k e n s , F. M.: F olic acid coenzym es in
the biosynthesis of purines and pyrimidines.
Vitamins Hormones 26:375-394, 1968.
3. N i x o n , P. J. a n d B e r t i n o , J. R.: Interrelation­
ships of vitamin Bi2 and folate in man. Amer.
J. Med. 48:555-561, 1970.
4. S i l b e r , R. a n d M o l d o w , C. F.: The bio­
chemistry of Bi2 -m ediated reactions in man.
Amer. J. M ed. 48:549-554, 1970.
5. S i l b e r , R., F u j i o k a , S ., M o l d o w , C. F . , a n d
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