Tuesday, July 3, 2007

Brain homeostasis for Vitamin B6

Vitamin B6 levels in brain are homeostatically regulated.
Although it is relatively easy to produce symptomatic
vitamin B6 deficiency in animals, levels of the
vitamin in brain (and heart) are somewhat better
maintained in defi ciency states than they are in liver
and kidney. Conversely, massive parenteral doses
(daily intravenous injections of 200 mg kg–1 of PN for
3 days to rabbits) elevated the brain levels of PLP by an
average of only 39% (Spector & Shikuma, 1978).
In brain, vitamin B6 exists predominantly in the enzymatically
active forms PLP and PMP at concentrations
much higher than in plasma and cerebrospinal
fl uid (CSF). In rabbits, the concentrations of vitamin
B6 in plasma, CSF, brain and choroid plexus were, respectively,
0.30, 0.39, 8.90 and 15.10 μmol L–1 or kg–1
(Spector, 1978a).
Spector studied the in vitro uptake and release of
tritium-labelled vitamin B6 in rabbit brain slices and
isolated choroid plexuses (Spector, 1978b; Spector
& Greenwald, 1978). Uptake of [3H]PN by both tissues
was inhibited by (1) low temperature (2°C) and
dinitrophenol, demonstrating energy dependence;
(2) pyridoxal azine, demonstrating dependence on
the activity of intracellular pyridoxal kinase; and (3)
unlabelled non-phosphorylated B6 vitamers and, to
lesser extent, phosphorylated B6 vitamers, demonstrating
saturability of the uptake system. There was
no detectable metabolism of [3H]PN to [3H]pyridoxic
acid in brain slices or choroid plexus. From 70 to 80%
of the labelled vitamin B6 in both tissues was phosphorylated
after a 30-minute incubation in [3H]PN.
Phosphorylated B6 vitamers were taken up much less
readily than non-phosphorylated vitamers. These
studies are not conclusive in separating active transport
from metabolic trapping because both pyridoxal
kinase and active transport require ATP, and therefore
depletion of ATP could affect either process. Furthermore,
dinitrophenol is known to inhibit mammalian
pyridoxal kinase as well as preventing ATP synthesis
by uncoupling oxidative phosphorylation from electron
fl ow through the electron-transport chain.
The activity of pyridoxal kinase in brain is unimpaired
by moderate and severe vitamin B6 defi ciency
(McCormick et al., 1961). Spector & Shikuma (1978)
showed that pyridoxal kinase activity and vitamin B6
accumulation by brain slices and choroid plexus are
not affected by various drugs that alter the concentrations
of PLP or biogenic amines in brain.
Spector (1978a) showed that, during one pass
through the cerebral circulation, [3H]PN was cleared
from the circulation no more rapidly than mannitol.
Mannitol, a molecule of similar size and shape to
PN, is known to be transported by diffusion. Spector
(1978a) also confi rmed in vivo, by injection directly
into the ventricular CSF of rabbits, that non-phosphorylated
B6 vitamers enter brain cells by a saturable
accumulation process. Kinetic studies conducted by
Spector & Greenwald (1978) revealed marked differences
in the uptake of vitamin B6 by choroid plexus
and brain. The half-saturation concentrations and
rate maxima for accumulation were ~0.2 μM and
1.0–2.0 μmol kg–1 per 30 min for brain slices and
7.0 μM and 40 μmol kg–1 per 30 min for isolated
choroid plexus. Assuming the absence of a membrane
carrier for vitamin B6 uptake, the kinetic constants
refer to the binding of substrate to pyridoxal kinase
and are therefore values of Km and Vmax. The differences
in the constants for vitamin B6 uptake by brain
cells and choroid plexus are presumably due to factors
(e.g. intracellular pH) that cause variations in enzyme
This makes the choroid plexus, rather than brain cells,
the likely source of the phosphorylated B6 vitamers
in CSF.
Vitamin B6 is transported in the reverse direction
(i.e. from brain and/or CSF into blood) more rapidly
than mannitol (Spector, 1978a). This suggests that the
transport mechanism for vitamin B6 in this direction
involves a mechanism other than simple diffusion.
In conclusion, Spector’s data show that circulating
vitamin B6 can enter the brain via the blood–CSF
barrier (choroid plexus). The fi nding that PN was
extracted no more rapidly than mannitol during one
pass through the cerebral circulation argues against
signifi cant entry of PN via the blood–brain barrier.
There is a saturable transport system (Km = 0.7 μM)
within the choroid plexus that regulates the entry
of free (unbound) non-phosphorylated B6 vitamers
from plasma into the CSF. The vitamers fi nds their
way into the extracellular space of brain and enter
brain cells by a high-affi nity (Km = ~0.2 μM) saturable
accumulation system. The transport system in both
choroid plexus and brain cells appears to be simple
(or possibly facilitated) diffusion accelerated by the
concentration gradient created by phosphorylation of
the transported B6 vitamers (metabolic trapping). PN,
PL and PM have comparable affi nity for the vitamin
B6 transport systems, as they also do for pyridoxal
kinase. Excessive concentrations of phosphorylated
B6 vitamers within brain cells are dephosphorylated
intracellularly and transported out of the cells.

No comments: