3 16s Biochemical Society Transactions ( 1 99 1 ) 19
Ferric iron reduction and uptake by mouse
duodenal mucosa.
KISHOR B. RAJA, ROBERT J. SIMPSON and TIMOTHY J.
PETERS.
of iron by mouse duodenum has been reported previously 191.
The decrease in the rate of 59Fe uptake when ferrozirw is
present is quantitatively explained by the rate of appearmi(.(. (11
Fe(II1ferroineg conlplex (Table I ) . This suggests 1li;rt ;I
sequential reduction and uptake process operates for Fc(Ill1
uptake in mouse duodenum.
Department of Clinical Biochemistry. King’s College School ol‘
Medicine and Dentistry. Bessemer Road. London SE5 9PJ.
Body iron levels are maintained within narrow limits by
controlling duodenal absorption of dietary iron. The absorptive
process entails at least two distinct steps: 1) uptake of iron
across the brush-border membrane of intestinal enterocytes.
and (ii) following intracellular processing. transfer of sonie/all of
the iron to the portal circulation. Previous studies using ferriciron complexes 111 have demonstrated the uptake step to
respond adaptively to changes in body iron requirements i r i
mice. The niechanisni and regulation of this process however.
remains unclear. Recent reports 12.31 propose reduction of ferric
iron to be a prerequisite for cellular uptake. Support for
reduction of extracellular ferric-iron has been provided in rats
141. These workers claim the reduction to be dependent upon the
intestinal release of low-molecular weight reducing factors. In
the present study. we investigated the importance of a reductive
process, operating a t the intestinal level, for uptake of ferric iron
in mice.
An in vitro technique. described previously Ill. was used to
deterniine the initial rates of 59Fe uptake by niouse duodenurn.
Iron (250 pM1, was presented as a ferric chelate of
nitrilotriacetate (Fe:NTA. 1:2), 57Co-Cyanocobalan1in was used
as the extracellular fluid marker. Correction for non-niucosal
entry was made by perfomimg similar incubations with serosal
side-out tied-olT segments of duodenum [ 11.
Perturbation of the redox state of the intestinal mucosa by
inclusion of the cell impermeable oxidizing agent. ferricyanide
(ImMl. in the medium. resulted in a significant reduction
(p<O.O4)in the 59Fe uptake rate (3.820.4 pniol/mg/niin (n=5).
rnean+SEM). as cornpared to control values (5.4+0.5
pmol/rng/min (51). No such eKect was seen with ferrocyanitk
(5.0k0.6 pmol/mg/rnin (6)). Furthermore. a n almost complete
inhibition in duodenal uptake of 59Fe was seen when ferrozine.
(3-(2-pyridyl)-5,6-bis(4-phenylsulfo~c
acid)-1.2.4-triazine ImM).
a water-soluble, specific Fe(ll) chelator 151. was included in the
incubation medium (0.4+0.1 ( 4 ) vs. 4.1+1 .O pniol/rng/rnin (3).
p < O . O I ) . These data support the view that reduction is required
prior to uptake.
In order to quantitate the rate of formation of ferrous iron
species in the medium, the reduction process was monitored
spectrophotometrlcally using ferrozine. which forms a stable.
coloured complex with Fe(II). Reduction of niediuni Fe(lII) only
occured when tissue was present in the niedium. suggesting the
presence of a cellular Fe(III) reducing activity. N o evidence for
significant release of reductant from mucosal fragments was
demonstrated. Moreover, formation of Fell11 increased in a
linear-fashion with time. Regional studies demonstrated
reducing activity to be confined mainly to the proximal intesliiial
region. Inside-out (i.e everted) duodenal segments displayed
slmllar rates for reduction of mediuni Fe(II1) as duodenal
fragments. whilst serosal side-out showed little activity. The
ability of mouse duodenum to reduce medium Fe(lII) was fouiitl
to be reduced by over 40% by the omission of medium glucose
or oxygen. Further studies demonstrated decreases in reduction
rates of upto 40% by inclusion of either sodium fluoride ( I OmM)
or dinitrophenol (0.ImM). or by upto 65% when both metabolic
inhibitors were present. The replacement of medium N a + by K+.
a manoeuvre associated with membrane depolarization 161. or
the presence of fenicyanide also markedly reduced the ability 01
duodenal Ussue to reduce Fe(III). The above alterations in Fe(1II)
reduction activity parallels the changes in duodenal 59Fe
uptake capacity 17.81. Studies carried out in hypoxic animals ( 3
days at 0.5atml. a model of enhanced iron absorption [ I ] .
demonstrated a 3-fold increase in the rnucosal uptake rate 01
59Fe as compared to control values (Table I ) . A parallel 2-3-foId
increase in the Fe(IlI) reduction rate was also observed in this
experimental group. It is noteworthy that about 15-20% of the
overall duodenal uptake of 59Fe in control and hypoxic mice is
accounted for by a non-regulated.
Fe(II1-independent
mechanism. The presence of more than one pathway for uptake
Table 1. Mucosal reduction and duodenal uptake e F c ( l l l ]
Duodenal fragments obtained froni nomial (CD 1 straiii) or
chronically hypoxic mice were incubated lor 5niin at 370(‘ i t i
physiological medium. containing 59Fe(lIIlNTA2 with. or witliotlt
ferrozine (1niM). The reduction of ferric iron was nioliitowtl
using a spectrophotometer set at 562nni. The \values h ; i \ ~t)c(,ii
corrected for serosal entry/reduction. Medium Fe(lI1): 2 5 0 1 1 h l :
NTA 500uM. Results: mean+SEM for three scpr.r;i(c.
experinients. Statistical analysis by students 1-test.
~~
Fe(Ill1
reduction rate
59Fe uptake
+ Ferrozine
- Ferrozine
Controls
Hypoxir
(iiniol/ni~niinl
~
3.2220.24
7.20+0.80
0 001)
0.72+0.53
3.5250.65
1.6320.42
10.12+0.92
0.25
0.004
a
l
w
.
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