Absorption, transport and metabolism of Vitamin B2

Humans cannot synthesize vitamin B2 and thus must
obtain the vitamin from exogenous sources via intestinal
absorption. The intestine is exposed to fl avins
from two sources: (1) the diet and (2) the bacterially
synthesized fl avins in the large intestine. Whether the
latter source of vitamin B2 is available to the host tissues
(apart from the colonic epithelial cells) in nutritionally
signifi cant amounts is unknown.
12.4.1 Digestion and absorption of dietary
vitamin B2
The FMN and FAD present in the ingested food are
released from noncovalent binding to fl avoproteins as
a consequence of acidifi cation in the stomach and gastric
and intestinal proteolysis. Ribofl avin is similarly
released from its association with binding proteins
(Merrill et al., 1981).
The fl avin coenzymes are hydrolysed in the upper
small intestine to free ribofl avin, which is then absorbed.
Hydrolysis of both FMN and FAD is effected
by alkaline phosphatase (EC 3.1.3.1), which has a
broad specifi city and is located on the brush-border
membrane of the enterocyte (Daniel et al., 1983a).
Two additional brush-border enzymes, FMN phosphatase
and FAD pyrophosphatase, participate in
the degradation of the fl avin coenzymes (Akiyama
et al., 1982). The considerably smaller amounts of
covalently bound fl avins are released as 8α-(peptidyl)
ribofl avins, which are absorbed along with the free
ribofl avin (Chia et al., 1978).
In vitro studies using rat everted jejunal sacs have
shown that absorption of ribofl avin takes place by a
saturable, energy-dependent process at physiologically
relevant concentrations and by simple diffusion
at higher concentrations (Daniel et al., 1983b; Said et
al., 1985; Middleton, 1990). This dual process of absorption
has been confi rmed under in vivo conditions
(Feder et al., 1991). Casirola et al. (1994) showed that
the ribitol side chain and the NH group at position 3
of the isoalloxazine moiety are essential for ribofl avin
binding to specifi c sites on the brush-border membrane
of rat small intestine.
Transport of ribofl avin across brush-border and
basolateral membrane vesicles prepared from rabbit
small intestine was found to be independent of sodium
and electroneutral in nature (Said et al., 1993a,b).
Using human-derived Caco-2 intestinal epithelial
cells, Said & Ma (1994) confi rmed the involvement of
a carrier-mediated process in the initial phase of ribofl
avin uptake (a 3-min incubation time). Ribofl avin
uptake was Na+- and pH-independent and the initial
phase occurred without metabolic alteration of the
transported ribofl avin. Inhibitors of anion transport
did not produce inhibition of ribofl avin uptake by
Caco-2 cells, thus ribofl avin does not appear to act as
an anion with regard to its intestinal transport.
Some of the absorbed ribofl avin is phosphorylated
to FMN within the cytosol of the enterocyte by fl avokinase
(ATP:ribofl avin 5´-phosphotransferase, EC
2.7.1.26) and most of the FMN is further converted
to FAD by FAD synthetase (ATP:FMN adenylyltransferase,
EC 2.7.7.2). Both of these metabolic steps require
ATP, i.e. they are energy-dependent. Gastaldi et
al. (1999) investigated the energy dependency of the
ribofl avin uptake process in isolated rat enterocytes
by comparing de-energized cells (cells treated with
rotenone) with normal cells. Short (3 min) and long
(20 min) incubation times were selected as these
times represent membrane events and intracellular
metabolic events, respectively. The results showed
that in the initial (3 min) phase, the saturable uptake
of [3H]ribofl avin is mainly an energy-independent
process with high affi nity and low capacity, whereas in
the later (20 min) phase the saturable uptake is strictly
energy-dependent and has an increased capacity. The
presence of a saturable mechanism even when intracellular
metabolism is blocked, as in de-energized
cells, suggests that the transport across the membrane
is due solely to ribofl avin binding to carrier proteins
on the brush-border membrane. Saturable uptake in
the later phase is due to high-affi nity binding of ribofl
avin to the cytosolic enzymes fl avokinase and FADsynthetase.
The conversion of ribofl avin to FMN
and FAD by these enzymes accounts for the energy
dependency of the transport process.
Additional evidence that intracellular phosphorylation
is important for the absorption of physiologically
relevant concentrations of ribofl avin is the observation
that ribofl avin analogues that are absorbed at low
concentrations by the saturable transport process are
good substrates for fl avokinase, whereas analogues
that are absorbed solely through simple diffusion at
all concentrations are poor substrates for this enzyme
(Kasai et al., 1990). Moreover, both membrane and
intracellular events in ribofl avin absorption are inhibited
by ribofl avin analogues that are readily phosphorylated
(Gastaldi et al., 1999).
To summarize, the small intestine is well adapted to
completely extracting the small amounts of ribofl avin
that are largely bound within the ingested fl avin coenzymes.
The coenzymes are dephosphorylated in the
lumen and the liberated ribofl avin is extracted very effi
ciently by a high-affi nity, carrier-mediated transport
system, which is distributed along the entire length of
the small intestine. The uptake mechanism is Na+-independent
and electroneutral in nature. After uptake,
some of the ribofl avin is metabolically trapped within
the enterocyte as FMN. The energy used in ribofl avin
absorption is not required for membrane uptake, but
rather for ribofl avin metabolism within the enterocyte.
Thus, intracellular metabolism is probably the
driving force behind the internalization of ribofl avin.
The vitamin is dephosphorylated to permit exit of
ribofl avin across the basolateral membrane; this also
takes place by a carrier-mediated, Na+-independent
and electroneutral mechanism.