Absorption, transport and metabolism of folate

Absorption, transport and
metabolism of folate
Domina Petric, MD

Absorption
I.
April 19, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
2

Hydrolysis of folyl polyglutamates
Under fasting conditions, folic acid,
5-methyl-FH4 and 5-formyl-FH4 are
virtually completely absorbed.
Most polyglutamyl folates are
absorbed at efficiencies in the
general range of 60-80%.
April 19, 2018
3

Hydrolysis of folyl polyglutamates
• The majority of food folates occur as
reduced polyglutamates.
• They must be cleaved to the mono-
or diglutamate forms for absorption.
• This is accomplished by the action of
an exocarboxypeptidase folyl
γ-glutamyl carboxypeptidase, more
commonly called folyl conjugase.
April 19, 2018
4

Folyl conjugase
• Conjugase activity is widely distributed in the
mucosa of the proximal small intestine, both
intracellularly and in association with the
brush border.
These appear to be different enzymes:
• 75 kDa intracellular enzyme is localized in the
lysosomes and has an optimum of pH 4,5-5,0.
• 700 kDa brush border enzyme has an
optimum of pH 6,5-7,0.
April 19, 2018
5

Folyl conjugase
• Folyl conjugase activities have
also been found in bile,
pancreatic juice, kidney, liver,
placenta, bone marrow,
leukocytes and plasma.
• In the uterus, conjugase activity
is induced by estrogen.
April 19, 2018
6

Folyl conjugase
Loss of conjugase activity
results in impaired folate
absorption.
Conjugase activity is reduced
by nutritional zinc deficiency
or by exposure to naturally
occurring inhibitors in foods.
April 19, 2018
7

Folyl conjugase
• Natural conjugase inhibitors are
contained in certain foods: cabbage,
oranges, yeast, beans (red kidney,
pinto, lima, navy, soy), lentils and
black-eyed peas.
• The presence of conjugase inhibitors reduces
folate bioavailability.
• This effect appears to be the basis for
the low availability of the vitamin in
orange juice.
April 19, 2018
8

Folyl conjugase
Folate absorption can also be reduced by
certain drugs including:
• cholestyramine (which binds folates)
• ethanol (which inhibits deconjugation)
• salicylazosulfapyridine
• diphenylhydantoin
• aspirin and other salicylates
• several nonsteroidal anti-inflammatory
drugs
April 19, 2018
9

Uptake
Dietary folates are absorbed as folic acid,
5-methyl-FH4 and 5-formyl-FH4.
The overall efficiency of folate absorption
appears to be ∼50% (10-90%).
Malabsorption of the vitamin occurs in
diseases affecting the intestinal mucosa.
April 19, 2018
10

Microclimate hypothesis
Folate absorption is dependent on the pH
of the proximal jejunum, with an optimum
at pH 6,0-6,3.
Elevated absorption of folate in individuals with
pancreatic exocrine insufficiency may be due to
the low pancreatic excretion of bicarbonate in
that condition.
Loss of buffering capacity renders the intestinal
lumenal milieu slightly more acidic, minimizing the
charge on the folate molecule, thus facilitating its
diffusion across the brush border membrane.
April 19, 2018
11

Uptake
• Under more basic conditions
(pH>6.0), folate absorption falls off
rapidly.
• Two jejunal brush border folate-
binding proteins (FBPs) have been
isolated.
• Three mechanisms are involved in
folate absorption.
April 19, 2018
12

Folate transporter
• Folic acid is actively transported across
the jejunum, and perhaps the duodenum,
by an Na+-coupled, carrier-mediated
process that is stimulated by glucose and
shows a pH maximum at about pH 6.
• The transporter is a transmembrane
protein with much greater affinities for
folic acid than for reduced folates.
April 19, 2018
13

Folate transporter
The active transport process is maximal at
lumenal folate concentrations of 10-20 µM.
Its expression is suppressed by
exposure to alcohol.
Hereditary folate malabsorption,
apparently involving failure of transporter
expression, has been reported.
April 19, 2018
14

Folate receptor
• A high-affinity, folate-binding protein, also
called the folate receptor (FR), has been
identified in the intestine and other tissues.
• The intestine contains three isoforms, each
of which is associated with the brush border.
• Two isoforms (α- and ß-) are
glycosylphosphatidylinositol (GPI)-anchored
to the membrane.
• β-type is a soluble protein.
April 19, 2018
15

Folate receptor
• The GPI-anchored receptor FRα has
been shown to cycle between
intracellular and extracellular
compartments while remaining
bound to the membrane, thus
effecting the high-affinity uptake of
folate and nonmolar concentrations.
April 19, 2018
16

Diffusion
• Folic acid can also be absorbed
passively, apparently by diffusion.
• This nonsaturable process is
linearly related to lumenal folate
concentration and can account for
20-30% of folate absorption at
high folate intakes.
April 19, 2018
17

Methylation
• Folic acid taken up by the intestinal
mucosal cell is reduced to FH4, which
can either be transferred without
further metabolism to the portal
circulation or alkylated (by
methylation to 5-methyl-FH4) before
being transferred.
April 19, 2018
18

Transport
II.
April 19, 2018
19

Free in plasma
Folate is transported to the tissues mostly
as monoglutamate derivatives in free
solution in the plasma.
Plasma folate concentrations in humans
are typically in the range of 10-30 nM.
Most circulates in free solution, but some is
bound to low-affinity protein binders such as
albumin, and others are bound to a soluble
form of the high-affinity FBP.
April 19, 2018
20

Free in plasma
Erythrocytes contain greater
concentrations of folate: 50-100 nM.
These stores are accumulated during
erythropoiesis.
The mature erythrocyte does not take
up folate.
April 19, 2018
21

Free in plasma
The folate levels of both
plasma and erythrocytes are
reduced by cigarette
smoking.
Habitual smokers show
plasma folate levels that are
more than 40% less than
those of nonsmokers.
April 19, 2018
22

Free in plasma
• More than 80% of impoverished
chronic alcoholics show abnormally
low serum levels and some 40%
show low erythrocyte levels.
• This corresponds to a similar
incidence (34-42%) of
megaloblastosis of the bone
marrow in alcoholic patients.
April 19, 2018
23

Cellular uptake
• The cellular uptake of folates occurs
exclusively with monoglutamate
derivatives found in the plasma, as
the polyglutamates can not cross
biological membranes.
• The cellular uptake of folate involves
the three processes outlined above
for enteric absorption.
April 19, 2018
24

Folate transporter
The folate transporter is expressed
in most tissues.
It has affinities for various folates varying
both between tissues and between the
apical and basolateral membranes.
Its affinities for folic acid are two orders
of magnitude greater than those for
reduced folates.
April 19, 2018
25

Folate receptor
A high-affinity FR is
expressed in many tissues.
It mediates the uptake of
folate in the kidney.
April 19, 2018
26

Cellular uptake
Within cells, FH4 is methylated
to yield 5-methyl-FH4, which is
bound to intracellular
macromolecules.
As a result of methylation in
the mammary gland, 5-methyl-
FH4 comprises three-quarters of
the folates in breast milk.
April 19, 2018
27

Cellular uptake
• Folate is held in cells by conversion
to folyl polyglutamates.
• Polyglutamation traps folates inside
cells at concentrations greater (by
one to two orders of magnitude)
than those of extracellular fluids.
April 19, 2018
28

Folate-Binding Proteins
• Folate-binding proteins (FBPs) have
been identified in plasma, milk,
erythrocytes, leukocytes, intestinal
mucosa, kidney, liver, placenta, choroid
plexus and urine.
• Each binds folates noncovalently with
high affinity, such that the complex
does not dissociate under physiological
conditions.
April 19, 2018
29

Tissue distribution
The total body content of folate is 5-
10 mg, about half of which resides in
the liver in the form of tetra-, penta-,
hexa- and heptaglutamates of
5-methyl-FH4 and 10-formyl-FH4.
April 19, 2018
30

Tissue distribution
• In tissues with rapid cell division
(intestinal mucosa, regenerating liver,
carcinoma), relatively low concentrations
of 5-methyl-FH4 are found, usually with
concomitant elevations in 10-formyl-FH4.
• In contrast, in tissues with low rates of
cell division (normal liver), 5-methyl-FH4
predominates.
April 19, 2018
31

Tissue distribution
• Brain folate (mostly 5-methyl-FH4)
levels tend to be very low, with a
subcellular distribution (penta- and
hexaglutamates mostly in the
cytosol and polyglutamates mostly
in the mitochondria) the opposite of
that found in liver.
April 19, 2018
32

Metabolism
III.
April 19, 2018
33

Reduction of the pteridine ring system
• Reduction of the pterin ring from the
two nonreduced states, folic acid
and dihydrofolic acid (FH2), to the
fully reduced form tetrahydrofolic
acid (FH4) that is capable of
accepting a single-carbon unit is
accomplished by the cytosolic
enzyme 7,8-dihydrofolate reductase.
April 19, 2018
34

Reduction of the pteridine ring system
• This activity is found in high amounts
in liver and kidney and in rapidly
dividing cells (tumor).
• The reductase is inhibited by several
important drugs including the cancer
chemotherapeutic drug methotrexate,
which appears to exert its antitumor
action by inhibiting the reductase
activity of tumor cells.
April 19, 2018
35

Reactions of the polyglutamyl side chain
• The folyl monoglutamates, that are taken
up by cells, are trapped therein as
polyglutamate derivatives that can not
cross cell membranes.
• Polyglutamate forms are also mobilized
by side-chain hydrolysis to the
monoglutamate.
• These conversions are catalyzed by two
enzymes: polyglutamation and cellular
conjugases.
April 19, 2018
36

Acquisition of single-carbon moieties at certain
positions (N-5 or N-10) on the pterin ring
• Folate is metabolically active as a
variety of derivatives with single-
carbon units at the oxidation levels
of formate, formaldehyde or
methanol substituted at the N-5
and/or N-10 positions of the
pteridine ring system.
April 19, 2018
37

Acquisition of single-carbon moieties at certain
positions (N-5 or N-10) on the pterin ring
• The main source of single-carbon
fragments is serine
hydroxymethyltransferase, which
uses the dispensable amino acid
serine as the single-carbon donor.
• Each folyl derivative is a donor of its
single-carbon unit in metabolism.
April 19, 2018
38

April 19, 2018
39
Catabolism
and excretion

Catabolism
Tissue folates appear to turn over by
the cleavage of the polyglutamates at
the C-9 and N-10 bonds to liberate
the pteridine and
p-aminobenzoylpolyglutamate moieties.
This cleavage probably results from
chemical oxidation of the cofactor
in both the intestinal lumen
(dietary and enterohepatically
recycled folates) and the tissues.
April 19, 2018
40

Catabolism
• Once formed, p-aminobenzoylpolyglutamate
is degraded, by the action of folyl conjugase,
and is acetylated to yield
p-acetaminobenzoylglutamate and
p-acetoaminobenzoate.
• Urinary levels of p-acetoaminobenzoate are
correlated with the total body folate pool.
• Folate breakdown is greatest under
hyperplastic conditions: rapid growth,
pregnancy.
April 19, 2018
41

Excretion
• Intact folates and the water-soluble
side-chain metabolites
p-acetaminobenzoylglutamate and
p-acetaminobenzoate are excreted
in the urine and bile.
• The total urinary excretion of folates
and metabolites is small: ≤1% of
total body stores per day.
April 19, 2018
42

Excretion
• Fecal concentrations of
folates are usually high.
• These concentrations
represent mainly folates of
intestinal microfloral origin.
April 19, 2018
43

Literature
• Combs GF. The Vitamins. Fundamental Aspects in
April 19, 2018 44

Absorption, transport and metabolism of folate

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