Physiological modulation of iron metabolism in rainbow trout (Oncorhynchus mykiss) fed low and high iron diets
1 Environmental Research Centre, National University of La Plata-CONICET, La
Plata, Bs. As., Argentina
2 School of Biological Sciences, University of Plymouth, Drake Circus,
Plymouth PL4 8AA, UK
* Author for correspondence (e-mail: rhandy{at}plymouth.ac.uk)
Accepted 9 September 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: rainbow trout, Oncorhynchus mykiss, dietary iron, transferrin, ferrireductase, intestine
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fish acquire iron predominantly from the diet, and with negligible iron
uptake at the gills compared with the gut
(Andersen, 1997;
Bury et al., 2001
), teleost
fish have a dietary iron requirement of
30-200 mg kg-1 dry
mass (d.m.) of food (for reviews, see
Davis and Gatlin, 1991
;
Watanabe et al., 1997
). There
are only a few reports of dietary iron deficiency in fish, and these have
focused on defining the nutritional requirements to avoid anaemia and growth
retardation in aquaculture (Kawatsu,
1972
; Sakamoto and Yone,
1978
; Davis and Gatlin,
1991
; Watanabe et al.,
1997
). A precise iron requirement for most fish species, including
rainbow trout, remains to be determined. However, normal dietary levels of
100-250 mg Fe kg-1 d.m. food have been suggested for salmonids
(Desjardins et al., 1987
;
Andersen et al., 1996
). A few
studies have used large excesses of dietary iron to explore the role of iron
in oxidative stress in fish, as indicated by lipid peroxidation products in
the liver (e.g. 6.3 g Fe kg-1 d.m. of food, African catfish;
Baker et al., 1997
). Despite
this information on the nutritional requirement and toxic effects of iron, few
attempts have been made to explore physiological regulation and mechanisms of
iron metabolism in fish. However, two early studies using injected
59Fe suggest the liver is the main storage pool for iron in fish
(in tench, Tinca tinca L.; Van
Dijk et al., 1975
; rainbow trout, Oncorhynchus mykiss;
Walker and Fromm, 1976
).
Iron forms insoluble ferric (hydro)oxides at neutral pH
(Aisen et al., 2001) and
molecular evidence suggests that the small fraction of Fe3+
presumably present in the gut lumen will be reduced to Fe2+ prior
to import into the gut enterocytes of fish
(Bury et al., 2003
). In
mammals, ferrireductase activity in the brush border of the intestinal mucosa
facilitates the reduction of Fe3+ to Fe2+
(Riedel et al., 1995
;
McKie et al., 2001
) prior to
Fe2+ import on divalent metal ion transporter 1 (DMT 1,
Gunshin et al., 1997
;
Trinder et al., 2000
).
Although intestinal ferric reductase activity has not been measured in rainbow
trout, in the European flounder at least
(Bury et al., 2001
),
Fe2+ is absorbed three times faster than Fe3+. DMT 1
genes are also expressed in fish intestine, for example, rainbow trout
(Dorschner and Phillips, 1999
)
and zebrafish (Donovan et al.,
2002
).
Intracellular Fe is stored as Fe3+ by ferritin, a 450 kDa
protein with a spherical cavity capable of carrying 4500 iron atoms
(Aisen et al., 2001). Ferritins
are an ancient group of proteins conserved in bacteria, plants and man
(Aisen et al., 2001
), and have
also been found in fish (Andersen,
1997
). The precise mechanism of how imported Fe2+ is
re-oxidised to Fe3+ by cytoplasmic ferritin, or how the
Fe3+ is subsequently reduced to Fe2+ for export from the
cell to the blood, remains controversial in mammals
(Reilly and Aust, 1998
;
Winzerling and Law, 1997
;
Aisen et al., 2001
) and unknown
in fish. In mammals basolateral export of Fe2+ from the cell to the
blood is against the electrochemical gradient, and probably mediated by iron
regulated transporter (IREG 1, also called MTP 1 or ferroportin), and recent
evidence from the zebrafish genome suggests IREG 1 genes are present in fish
(Donovan et al., 2000
;
Bury et al., 2003
). In
mammals, exported Fe2+ is oxidised on the extracellular surface of
the serosal membrane by a membrane bound copper oxidase (a ceruloplasmin
homologue, hephaestin), and the resulting Fe3+ binds rapidly to
extracellular transferrin to facilitate bulk iron transport in the blood
(Winzerling and Law, 1997
;
Aisen et al., 2001
).
Circumstantial evidence argues for a similar iron export process in fish cells
(Bury et al., 2003
). Fish have
long been known to have transferrin for bulk iron transport in the blood
(Hershberger, 1970
;
Ikeda et al., 1972
), and in
the hagfish (Myxine glutinosa) at least, the transferrin has a
similar structure to that in humans (Aisen
and Leibman, 1972
).
In this paper, we give a detailed account of dietary iron accumulation and
distribution in rainbow trout, and for the first time demonstrate that rainbow
trout modulate ferrireductase activity in the intestine, the pool of
transferrin in the blood and Fe storage in the liver, to control whole body
iron status. We also present details of tissue Cu, Zn and Mn levels in
relation to iron status because of apparent promiscuity of the iron import
mechanisms (DMT 1; Gunshin et al.,
1997) and the involvement of Cu-dependent oxidases in facilitating
iron transport across cell membranes
(Winzerling and Law,
1997
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental design
Fish were randomly allocated to one of three dietary iron treatments (see
below) containing no added iron, a normal level of iron (+100 mg Fe
kg-1 dry mass; d.m.), or an elevated level of iron (+1500 mg Fe
kg-1 d.m.), for up to 8 weeks. These iron concentrations and
treatment times were chosen to generate total iron doses that would stimulate
a physiological response, whilst avoiding toxic effects such as reduced growth
and anaemia. Each treatment was performed in triplicate (three
tanks/treatment, nine tanks in all) with 44 fish/tank (132 fish/treatment).
Fish were starved for 48 h to clear the gut contents before starting the
experiment, and were then fed twice daily on one of the iron treatments for 8
weeks, using a measured food ration of 1.5% body mass per day. Fish biomass
per tank was measured fortnightly so that the ration could be maintained at
1.5% throughout the experiment. All food was eaten, and faecal waste from the
fish was flushed by the self-cleaning design of the tanks (99% replacement of
water in 12 h) and routine siphoning after feeding. Nonetheless, water quality
was monitored before and after feeding and aqueous iron levels remained at or
below the detection limit. The water quality was identical to stock conditions
(mean ± S.E.M., N=8, in mmol l-1), Na,
0.36±0.01; K, 0.17±0.02; Ca, 0.41±0.01; Cu, <0.0001;
Zn, 0.0010±0.0001; Fe, <0.0005 at pH 7.7±0.07 and
15±1°C. Fish were collected (before the daily feeding) at
fortnightly intervals for tissue biochemistry, haematology, nutritional
performance and trace metal composition (see below).
Diet formulation
Diets were formulated using purified casein (Natural Adhesive Co.,
Daventry, UK) that was low in iron compared with fish meal-derived protein
sources, and thus enabled modification of the iron content of the diet by the
addition of FeSO4.7H2O. The formulation was (in g
kg-1 d.m. of food) casein, 434.6; corn starch, 205.5; cod liver
oil, 18; mineral premix, 100; fish meal, 50; vitamin premix, 20; carboxymethyl
cellulose, 10; astaxanthin, 0.5. Measured proximate composition of the diet
was (% dry matter) 41% protein, 17% lipid and 14% ash. The vitamin premix
(Trouw, standard salmonid premix) contained ascorbyl polyphosphate (100 mg
kg-1 food; Rovimix StayC-vit, Ròche Ltd, Basle, Switzerland)
to prevent auto-oxidation of the food. The mineral premix was formula MIN-101
of Cho et al. (1985). Diets
were prepared by mixing 1 kg of dry ingredients with either zero, 0.49 or 7.44
g of FeSO4.7H2O powder to give nominal added iron premix
to the food of 0, 100 and 1500 mg Fe kg-1 dry matter. Measured Fe
contents of diets were (mean ± S.E.M., N=5)
33.0±1.5, 174.5±9.5 and 1974.8±145 mgFe kg-1
dry matter and hereafter are called low-, normal- and high-iron diets
respectively. The low-Fe diet was the lowest Fe content of food possible based
on available feed ingredients, whilst the normal diet was selected to be in
the normal range suggested for salmonids, of between 100-250 mg Fe
kg-1 dry matter (Andersen et
al., 1996
; Desjardins et al.,
1987
). The high-Fe diet was selected to be above the normal range,
but below doses known to cause overtly depressed growth and toxicity in fish
(Baker et al., 1997
).
Growth and nutritional performance
Specific growth rate (SGR, % gain in body mass per day) was calculated from
the mean weights of fish at time zero and week 8 for each treatment. Food
conversion ratio (FCR) was calculated from the food intake per fish (1.5%
ration) divided by the mean body weight gain of fish in each treatment over 8
weeks. Condition factor (K=100 x mass/standard
length3) was calculated from fish weights at the end of the
experiment. The proximate composition of the whole body (including the washed
gut) was determined (Baker and Davies,
1996) for seven fish taken at random from the tanks at time zero
(initial fish) and for a further seven fish per treatment at week 8.
Haematology and trace metal analysis
Fish (nine/treatment, three from each tank) were anaesthetised with
benzocaine, weighed and standard length recorded. Blood was collected by
caudal pucture into a nonheparinised syringe and divided into heparinised (for
haematology) and non-heparinised (for iron assays) Eppendorf tubes.
Haematology was performed immediately
(Handy and Depledge, 1999) and
an aliquot of plasma collected for Cu analysis
(Handy et al., 1999
).
Nonheparinised blood samples were allowed to clot for 2-3 h in a refrigerator
before iron assays. Total iron in the serum (TI), unsaturated iron-binding
capacity (UIBC) and total iron-binding capacity (TIBC) were measured using a
diagnostic kit (Sigma Diagnostics, kit N°. 565, manual procedure). The
iron assays were adapted for microplates by using 50 µl of serum in 250
µl of iron buffer reagent plus 5 µl of colour reagent (total serum iron
assay), and 50 µl serum with 200 µl of UIBC buffer and 50 µl standard
plus 5 µl colour reagent (UIBC assay). The absorbance of samples was read
at 550 nm on a Dynex MRX plate reader. TIBC was calculated from total iron
plus UIBC. The percentage of transferrin saturated with Fe was calculated as
(100-(UBIC/TIBC))x100%, according to the Sigma protocol.
Fish were immediately dissected after blood sampling to collect gill,
liver, stomach, intestine (minus pyloric caecae) and muscle for metal analysis
according to the method of Handy et al.
(1999) with modifications
(Handy et al., 2000
). Briefly,
tissues were digested in 5 ml of concentrated nitric acid at 60°C for 4 h,
diluted to 20 ml and analysed by inductively coupled plasma atomic emission
spectrophotometry (ICP-AES; Varian Liberty 200). Whole body Fe concentration
was also measured in dry ground carcass prepared for proximate composition.
Working detection limits of matrix matched standards were (in µmol
l-1): Na, 0.27, Fe, 0.04; Zn, 0.01; Cu, 0.03; K, 0.27; Mn, 0.01.
Serum samples were also analysed for Cu (100 µl diluted to 1.5 ml in
deionised water). Serum chloride was determined by automated titration
(Corning Chloride Meter 920).
Biochemistry and histology
A further seven to nine fish per treatment were collected every 2 weeks for
biochemistry. Fish were anaesthetised and weight/length measured as above.
Whole gill filaments, liver and intestine (posterior to the pyloric caecae)
were carefully dissected, rinsed in distilled water and snap frozen in liquid
nitrogen, then stored at -80°C for subsequent analysis. At weeks 0, 4 and
8 samples of these tissues were also fixed in buffered formal saline, prepared
for routine wax histology, and stained with Mallory's trichrome
(Handy et al., 1999).
Tissue homogenates for biochemistry were prepared by adding 0.5 g of
defrosted tissue to 2 ml of ice cold Dulbecco's phosphate-buffered saline
(PBS, pH 7.4, + 1 mmol l-1 EDTA) and homogenised (Ultra Turrax T8,
IKA Labortechnik, 4 mm dia. shaft, 2x15 s bursts with a 30 s rest in
ice, maximum speed). Homogenates were aliquoted into Eppendorf tubes and
frozen at -80°C until required for protein determination
(Handy and Depledge, 1999),
thiobarbituric acid reactive substances (TBARS;
Camejo et al., 1998
), and
NADH-dependent ferrireductase activity (Schulte and Weiss, 1995).
Calculations and statistics
Data were analysed on Statgraphics Plus for Windows version 4.0 or
Statistica version 6.0. as described by Handy et al.
(1999) using 2-way ANOVA with
LSD multiple range test. The ANOVA used dietary Fe treatment as the first
factor, and time as the second factor, to compare the effects of the
experimental diets from weeks 2 to 8. Non-parametric data were transformed
(log or square root) as appropriate prior to ANOVA and the LSD test.
Comparisons between initial fish on commercial trout food and the normal diet
controls on the casein-based diet were made using the Student's
t-test. A rejection level of P=0.05 was used for all
analysis. The percentage distribution of iron in organs was calculated from
the ratio of absolute metal content of organ:whole body. Absolute metal
content for the whole body was calculated from whole body metal concentration
multiplied by fish weight. Absolute metal content of organs was calculated
from whole organ weights (derived from
Barron et al., 1987
; except
liver which was directly measured) multiplied by metal concentration of the
tissue. For blood, absolute Fe content was calculated assuming a plasma volume
of 2.27 ml 100 g-1 (Gingerich
and Pityer, 1989
). Apparent net retention of metals was calculated
from cumulative food intake and absolute change in metal content of the whole
body during the experiment (Baker et al.,
1998
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Fish on the low-Fe diet, were able to maintain whole body Fe levels comparable with those of controls, but fish on the high-Fe diet showed a 3.7-fold increase in whole body Fe compared with controls (Student's t-test, P>0.05). This was reflected by an apparent redistribution of body Fe to the serum and intestine of fish fed the high-Fe diet (Table 1). Theintestine of the high-Fe fed group supporting 37% of the body burden by the end of the experiment compared with only 1% at the start (Table 1). Similarly, the percentage of body burden in the serum increased 15 fold over the entire experiment. The proportion of whole body Fe stored in the gills of fish fed the high-Fe diets were about 10% lower than either the control or low-Fe diet treatments. However, the low-Fe diet did not significantly reduce the proportion of whole body Fe stored in the serum or intestine. The percentage body burden of Fe in the liver remained constant at about 10% of the whole body Fe for all treatments (Table 1).
Iron binding in the blood
Dietary Fe treatment did not affect Hct or MEV, and there was only a small
(but statistically significant, ANOVA and LSD, P<0.05) decrease in
red cell haemoglobin content in the low-Fe fed fish compared with the other
treatments at the end of the experiment
(Table 2). Serum Cl-
remained in the normal range throughout the experiment (in mmol
l-1; mean ± S.E.M., N=27 fish per
treatment; low Fe, 113±3; normal Fe, 114±3; high Fe,
114±2). However, there were major changes in the iron storage pattern
of the serum (Fig. 2).
Elevation of total serum Fe concentration in response to the high-Fe diet
(Fig. 1) was reflected in an
increase in the TIBC of the serum, and a simultaneous decrease in UIBC,
leading to a 1.5-fold increase in the saturation of transferrin in the serum
(Fig. 2). At the end of the
experiment, fish on the high-Fe diet showed a 37% saturation of the
transferrin pool in the blood, compared with only 15% in the initial fish at
the start of the experiment (Fig.
2). The Fe binding characteristics of the serum in fish fed the
low-Fe diet were not different from fish on the normal-Fe diet, except at the
end of the experiment where saturation of transferrin decreased compared with
that in fish fed the normal diet (Fig.
2).
|
|
Although treatment-dependent effects on total serum Fe (Fig. 1), and saturation of transferrin in the serum occurred from week 4 onwards (Fig. 2), there were also some effects of switching from the commercial trout food to the casein-based experimental diets. Serum total Fe concentration in the initial fish at the start of the experiment was higher than in all treatments at week 2 (Student's t-test, P<0.05). Furthermore, TIBC and UIBC were significantly lower (Student's t-test, P<0.05) in the initial fish compared with fish fed the normal-Fe experimental diet (Fig. 2) and also the high- or low-Fe diets, suggesting that the casein diet stimulates the appearance of new (unsaturated) transferrin in the blood to increase the UIBC relative to that in the initial fish.
NADH-dependent ferrireductase activity
NADH-dependent ferrireductase activity was measured in gill, intestine and
liver (Fig. 3). NADH-dependent
ferrireductase activity was significantly affected by the dietary Fe
treatments (ANOVA, P>0.05) in liver and intestine. There was a
marked and progressive stimulation of ferrireductase activity in the intestine
of fish fed the low-Fe diet over the experiment (ANOVA, P<0.05).
Conversely, by week 8 the high-Fe diet caused a significant increase (ANOVA
and LSD, P<0.05) in ferrireductase in the liver compared with the
other treatments (Fig. 3).
Switching from the commercial trout food to the casein based diet (initial
fish compared with controls at week 2) had no effect on enzyme activities
(Student's t-test, P>0.05).
|
Ferrireductase activity was also correlated with tissue Fe concentration. In the intestine, ferrireductase activity was inversely related to the log of tissue Fe concentration, with fish fed either the high or low-Fe diets fitting on the same trend line. Interestingly, values from fish on the normal-Fe diet clustered together away from the trend line suggesting that there may be endogenous and inducible forms of intestinal ferrireductase (Fig. 3B inset). Ferrireductase activity in the liver showed the opposite trend to the intestine, with hepatic ferrireductase increasing with the log of liver Fe concentration, but only in fish on the normal and high-Fe diets (Fig. 3C inset). Hepatic Fe concentration and ferrireductase activity were also correlated with the percentage saturation of transferrin in the serum (Fig. 4), indicating that increased Fe loading of the liver in response to Fe overload in the blood is partly facilitated by increased ferrireductase activity. Hepatic ferrireductase did not correlate with liver Fe concentration or serum saturation in fish from the low-Fe diet (data not shown).
|
Thiobarbituric acid reactive substances (TBARS)
TBARS broadly indicates the level of oxidative stress in the tissue
(Fig. 5). There was a
significant effect of dietary Fe on TBARS in the intestine and liver (ANOVA,
P<0.05), but no effect on the gills. Notably, TBARS in the
intestine of fish fed the high-Fe diet was significantly elevated by week 4
and remained above normal for the entire experiment
(Fig. 5B). Hepatic TBARS were
also elevated in the high-Fe fed fish by week 8. TBARS in the intestine were
also linearly related to the log of intestinal Fe concentration for all fish
(inset, Fig. 5B), and similarly
in the liver of fish on normal and high Fe-diets (inset,
Fig. 5B). Switching from the
commercial food to the casein-based diet (initial fish compared with 2 week
controls) had no statistically significant effect (Student's t-test,
P>0.1).
|
Nutritional performance
The different dietary iron treatments had no effect on growth, FCR, or
proximate composition of the fish (Table
3). However, faecal Fe concentration increased significantly with
dietary Fe intake (Student's t-test, P<0.001 for all
comparisons) and this was also reflected in a fall in apparent net Fe
retention with increasing dietary Fe concentration
(Table 3).
|
Histology
There were no pathological changes in the gross anatomy of the gills,
intestine or liver associated with the dietary iron treatments by the end of
the experiment (week 8). The gills of all fish were normal (not shown) with no
signs of oedema or mucocyte proliferation. The intestinal mucosa of fish were
also intact and healthy. However, the mucous epithelium of the fish fed the
low-Fe diet had fewer goblet cells, and enterocytes contained fewer absorptive
vacuoles, than the intestines of fish from either the normal or high-Fe diet
(not shown). The livers of all fish were also normal and without overt
pathology, although there were some subtle changes in intracellular glycogen
storage and sinusoid space between treatments (see
Fig. 6 for details).
|
Trace element composition
Fish tissues were also analysed for Cu, Zn and Mn, in addition Na and K
were also measured in the gills and intestines. Dietary Fe status had no
effect on gill or intestine Na concentration, and the intestine from fish on
the low Fe-diet showed a persistent trend of K depletion (not statistically
significant, data not shown).
Dietary Fe had no clear treatment-dependent effect on tissue Cu levels that persisted over time during the experiment. However, there were some transient changes in Cu in some tissues. In the early stages of the experiment (weeks 2), fish fed the low-Fe diet had a significantly higher muscle Cu concentration (ANOVA and LSD, P<0.05) compared with either normal- or high-Fe treatments (Cu in µmol g-1 d.m.; mean ± S.E.M., N=9; low Fe, 0.023±0.001; normal Fe, 0.015±0.002; high Fe, 0.016±0.002). This treatment difference in muscle Cu was absent by week 6. At the end of the experiment Cu concentrations in the intestine of fish fed the low-Fe diet were significantly higher (ANOVA and LSD, P<0.05) that the other treatments (Cu in µmol g-1 d.m., mean ± S.E., N=9; low Fe, 0.521±0.067; normal Fe, 0.299±0.094; high Fe, 0.194±0.035). A correlation analysis of intestinal Fe versus Cu concentration (regardless of treatment) at week 8 yielded a negative correlation (correlation coefficient=-0.27, P<0.05), indicating that reductions in intestinal tissue Fe levels was partly responsible for increases in intestinal Cu concentration. No notable treatment effects of dietary Fe on tissue Cu status were apparent for stomach, gill or liver (data not shown).
Dietary Fe alone had no effects on the Zn status of the stomach, intestine, liver or gill of the fish (data not shown). There was, however, a statistically significant transient rise in Zn concentration in the muscle of fish fed the low-Fe diet at week 4 compared with the other treatments (Zn in µmol g-1 d.m.; mean ± S.E.M., N=9; low Fe, 0.284±0.017; normal Fe, 0.155±0.008; high Fe, 0.195±0.001).
Dietary iron level was generally inversely related to tissue Mn, indicating that elevation of dietary Fe caused tissue Mn depletion, and reduction of dietary Fe caused Mn overload. This effect was most notable (ANOVA and LSD, P<0.05) in the intestine at week 2 (Mn in µmol g-1 d.m., mean ± S.E.M., N=9; low Fe, 0.320±0.031; normal Fe, 0.205±0.048; high Fe, 0.123±0.030). Similar statistically significant effects were noted in the liver at week 4 and the gill at week 6 (data not shown).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Normal iron metabolism in rainbow trout
In this study normal whole body Fe concentrations were around 0.2 µmol
g-1 d.m. (11.2 µgg-1 d.m.;
Table 1), which are comparable
to the few other studies on rainbow trout, for example, using casein-based
diets (0.9 µmol g-1 d.m.;
Desjardins et al., 1987), and
to those in Atlantic salmon fed commercial diets (Salmo salar, 0.44
µmol g-1 d.m., calculated from
Andersen et al., 1996
). Normal
hepatic Fe levels in this study (1.3-2.0 µmol g-1 d.m.;
Fig. 1) were also similar to
previous reports for rainbow trout (e.g. 2.5 µmol g-1 d.m.;
Lanno et al., 1985
). Dietary
Fe levels between 100 and 400 mg kg-1 (1.8-7.2 mmol kg-1
d.m. food) produced steady state hepatic Fe levels and blood haemoglobin
concentrations in Atlantic salmon, suggesting the normal dietary Fe
requirement for salmon is between 100 and 400 mg Fe kg-1 d.m. food
(Andersen et al., 1996
).
Steady-state Fe concentrations in most organs
(Fig. 1), normal haematology
(Table 2) and hepatic
morphology (Fig. 6), of control
fish suggest the 'normal' control diet used in this study was within the
physiologically normal range for rainbow trout. Unlike other studies on fish
we also examined the body distribution of Fe
(Table 1). The visceral organs
of both initial and control fish support a small proportion (about 15%) of the
whole body Fe load (as in mammals; Van
Campen and Mitchell, 1965
). This is also consistent with an early
study in rainbow trout, where intraperitoneal injections of 59Fe
resulted in 14% of the radiotracer initially appearing in the liver and
intestine (Walker and Fromm,
1976
).
There are only a few measurements of transferrin concentration and
percentage saturation of transferrin with Fe in healthy salmonid fish, and
these are taken from wild populations or farmed fish
(Ikeda et al., 1972;
Hershberger and Pratschner,
1981
). They suggest TIBC is around 65 µmol l-1 with
about 23% saturation of transferrin with Fe
(Hershberger and Pratschner,
1981
), although this may vary with season and nutritional quality
of the food (Ikeda et al.,
1972
). In trout on the normal diet, transferrin levels were around
85 µmol l-1 (TIBC Fe equivalence;
Fig. 2A), and between 10 and
21% of the transferrin was saturated with Fe when the total serum Fe
concentration was about 10 µmol l-1. The latter is consistent
with the results of Walker and Fromm
(1976
) who recorded a similar
value (total plasma Fe of 9.8 µmol l-1) in controlled laboratory
conditions. In humans, the normal clinical range for total serum Fe is 6-25
µmol l-1 (35-140 µg dl-1; Sigma data sheet with Fe
kit). Thus total Fe levels in trout serum are about the same as in humans.
However, trout have less than half the circulating apotransferrin (iron-free
transferrin) levels of humans (clinical range UIBS, 23.2-67.1 µmol
l-1), and a smaller proportion of the transferrin is saturated
(trout 10-21%, Fig. 2C, and
humans 13-45%). The TIBC of normal trout serum (80-90 µmol l-1;
Fig. 2A) was also higher than
in humans (clinical range TIBC, 43-71 µmol l-1). This may imply
that trout normally use a smaller proportion of the circulating transferrin
than humans, despite having higher circulating Fe levels than humans.
Alternatively, other blood proteins (e.g. ferritin) in the blood of trout
might contribute more to TIBC than in humans. Nonetheless, the rainbow trout
model for Fe metabolism also seems much closer to that of humans than the
rodent model. Rats have serum Fe concentration that are 2 fold or more higher
than humans (about 35 µmol l-1 or 200 µg dl-1;
Horne et al., 1997
), and
normal transferrin saturation in rats is 50% or more
(Horne et al., 1997
).
Intestinal absorption of Fe in the mammalian involves the reduction of
Fe3+ to Fe2+ in the brush border of the mucosal
membrane, and this is achieved by a ferrireductase
(Riedel et al., 1995;
McKie et al., 2001
).
Ferrireductase activity has not been previously reported in fish, but its
involvement in intestinal Fe absorption has been implicated by the relatively
slow absorption of Fe3+ compared with Fe2+ by the gut of
European flounder, Platichthys flesus
(Bury et al., 2001
).
Ferrireductase activity in crude intestinal homogenates from normal trout was
between 11.2 and 21.9 nmol mg-1 protein min-1
(Fig. 3B), and this is similar
to that in homogenates of human intestinal cells (27 nmol mg-1
protein min-1; Riedel et al.,
1995
). Furthermore, we also find ferrireductase activity in the
gills and liver of normal trout (Fig.
3). The latter being increased when serum transferrin becomes
saturated (see below and Fig.
4).
Iron metabolism in response to high dietary Fe
Increasing dietary Fe concentration from 175 to 1975 mg Fe kg-1
d.m. food caused Fe concentrations in the stomach, intestine, liver and serum
to rise (Fig. 1). This was
accompanied by a redistribution of whole body Fe, so that the proportion of
whole body Fe held in the intestine and serum both increased 13 fold
(Table 1). However on a body
burden basis, the percentage of Fe in the liver did not increase suggesting
that Fe storage in the liver keeps pace with Fe loading of the whole body.
These changes in the distribution of whole body Fe occurred without alteration
in haematological (Table 2) or
nutritional performance (Table
3), and therefore suggest a physiological adjustment of Fe
metabolism. The slight enlargement of liver cells of fish on the high Fe diet
without pathology (Fig. 6) also
suggests increased metabolic activity in the liver. This physiological
response to elevated dietary Fe did not involve the prevention of Fe
absorption at the intestine because tissue Fe levels generally increased
(Fig. 1), and intestinal
ferrireductase activity did not decline (remained normal;
Fig. 3B). A lack of down
regulation of ferrireductase in the presence of high dietary Fe is consistent
with observations in mice, where levels of putative ferrireductase mRNA remain
unaltered from control levels in mice fed Fe replete diets
(McKie et al., 2001).
Instead, trout appear to regulate whole body Fe status during periods of high dietary Fe intake by modulating the labile Fe pool in the blood, which may subsequently enable transfer of Fe to the liver for storage. The total transferrin pool in the blood increased in response to elevation of serum Fe during high dietary Fe uptake (Figs 1D, 2). This resulted in a 4-fold increase in saturated transferrin (to 37% saturation; Fig. 2C). The presence of unsaturated transferrin (Fig. 2B) indicated ample spare capacity for Fe binding in the blood of fish fed the high-Fe diet, even though Fe accumulation by the intestine had increased. However, fish fed the high-Fe diet did increase Fe accumulation in the liver. Both hepatic Fe levels and ferrireductase activity were positively correlated with the percentage saturation of transferrin in the blood in fish fed normal and high-Fe diets (Fig. 4). Hepatic ferrireductase was also positively correlated with hepatic Fe concentrations (Fig. 3C). Together these observations suggest that hepatic ferrireductase enables Fe removal from the blood, and facilitates the accumulation of Fe2+ in the liver when serum Fe levels are normal or higher.
The precise mechanism of intracellular Fe handling in the liver of trout
remains to be investigated, but the ubiquitous nature of ferritin in animal
cells suggests that intracellular Fe will be stored as Fe3+ inside
the core of cytoplasmic ferritin (Aisen et
al., 2001). It could therefore be argued that any intracellular
mechanism that promotes the oxidation of intracellular Fe2+ to
Fe3+ would promote Fe storage by ferritin in the liver.
We also measured TBARS in the liver to monitor the general level of
oxidation in the tissue. The TBARS values we report (<1 nmol mg
protein-1; Fig. 5)
are at least an order of magnitude less than measurements made during
sub-lethal toxicity in fish (as malondialdehyde equivalence;
Baker et al., 1997;
Baker et al., 1998
), and
therefore represent subtle physiological changes in oxidative status of the
tissue during Fe metabolism. This notion is supported by the absence of
oxidative damage in the livers of all fish
(Fig. 6). Hepatic TBARS were
positively correlated with Fe concentration in the liver at the end of the
experiment (insert, Fig. 5C).
However this is not evidence for spontaneous auto-oxidation of Fe2+
to Fe3+ to promote hepatic Fe storage by ferritin, because hepatic
Fe levels increased several weeks prior to changes in TBARS (Figs
1 and
5). Instead the slight
elevation of hepatic TBARS is a consequence of excess Fe in the liver. Normal
HSI (Table 3), hepatic
ferrireductase (Fig. 3) and
gross histology (Fig. 6) also
argues against spontaneous oxidation of Fe2+ to Fe3+ in
the liver. Similar arguments apply to the intestine where a small but
persistent elevation of intestinal TBARS were positively correlated with
increasing Fe levels in the gut (Fig.
5B), but did not undermine nutritional performance
(Table 3).
Several authors argue against the involvement of the gills in Fe uptake
(see Bury et al., 2003), and
evidence here suggests the gills do not have a primary role in Fe storage and
excretion during high dietary-Fe intake. The absence of clear changes in
branchial Fe levels, ferrireductase or TBARS (Figs
1,
3,
5)implies that the gill were
not involved in the regulation of excess Fe absorption. Indeed, the proportion
of the body burden held by the gills decreased in response to the high-Fe diet
(Table 1).
Iron metabolism in response to low dietary Fe
The low-Fe diet generally had no effect on tissue Fe levels
(Fig. 1) or Fe distribution
(Table 1) compared with the
normal diet, except for a 50% reduction in Fe distribution to the stomach.
However, there were some changes in Fe handling in the serum. The percentage
saturation of transferrin in the blood of fish on the low-Fe diet was less
than controls by the end of the experiment
(Fig. 2C). The total Fe content
in the serum of fish fed the low-Fe diet was marginally (and consistently)
lower than that in fish fed the normal diet
(Fig. 1). Similar observations
were made by Walker and Fromm
(1976), when, despite repeated
bleeding to reduce whole body iron levels over 30 days, the serum of
Fe-deficient fish remained only marginally lower than controls. In the present
study, fish fed the low-Fe diet also showed increased intestinal
ferrireductase activity over both controls and fish on the high-Fe diet
(Fig. 3B). Together these data
suggest that trout maintained tissue Fe status by increasing Fe acquisition,
probably facilitated by the intestinal ferrireductase (as in mice,
McKie et al., 2001
). This
notion would also explain why intestinal ferrireductase was inversely
correlated with intestinal Fe concentration in fish on the low and high-Fe
diets (inset, Fig. 3B). The
reduction in saturation of transferrin in fish fed the low-Fe diet compared
with controls (Fig. 2C) might
also suggest that the labile pool of Fe in the serum (although small) was also
used to partly maintain tissues Fe levels.
However, we may have also revealed different functional isoforms of the
intestinal ferrireductase in trout. Data from control fish were clustered away
from the regression line for the high- and low-Fe fish (inset,
Fig. 3B). This suggests that
the inducible ferrireductase in the intestine may not be the same as the
apparently normal isoform of the enzyme in control fish, or alternatively the
control fish normally use an Fe absorption pathway that does not require
ferrireductase. The former seems more likely given the expression of three
transcripts of ferrireductase-related mRNAs in the duodenum of mice
(McKie et al., 2001) and the
presence of NADH-dependent and independent ferrireductase in human intestinal
cells (63% NADH dependent; Riedel et al.,
1995
). We also incidentally noted an NADH-independent component in
trout intestine (data not shown). A 5-fold higher net Fe retention in fish fed
the low-Fe diet than in controls (Table
3) also supports the notion of improved Fe absorption across the
gut. The significance of slight reductions in glycogen stores and increased
sinusoid space in the livers of fish on the low-Fe diet compared with controls
is unclear given the normal nutritional performance of all fish
(Table 3). There were no
obvious trends in branchial Fe levels, ferrireductase, or TBARS in the gills
of fish fed low-Fe diets compared with controls (Figs
1,
3,
5), indicating that fish were
not making good dietary deficiency by Fe absorption at the gills (in agreement
with Bury et al., 2003
).
Trace element interactions with Fe metabolism
There have been a number of nutritional studies in which interactions
between Fe, Zn, Cu and Mn metabolism have been suspected (e.g.
Winzerling and Law, 1997;
Knox et al., 1984
;
Lanno et al., 1985
;
Andersen et al., 1996
).
Elevation of dietary Cu intake causes increased Fe accumulation in the liver
of trout (Lanno et al., 1985
)
without appreciable changes in total plasma Fe
(Knox et al., 1984
), and this
might be explained by the presence of an intracellular ceruloplasmin-like
ferroxidase, which enables Fe loading into hepatic ferritin
(Reilly and Aust, 1998
). Low
dietary-Fe also alters Cu metabolism. In this study we found that intestinal
Cu concentrations increased by 174% by the end of the experiment. This may
reflect the ability of the intestinal Fe carrier, DMT1, to import Cu in to the
gut enterocytes in absence of Fe (Gunshin
et al., 1997
). Similarly, DMT1 will also import Mn in the absence
of Fe (Gunshin et al., 1997
)
and might explain the observed increases in intestinal Mn (increased by 65%,
week 8) in fish fed the low-Fe diet. Andersen et al.
(1996
) also noted an inverse
relationship between dietary Fe level and tissue Mn. This relationship can be
partly explained by the characteristics of DMT1, but transferrin also binds
Mn2+ (Nelson,
1999
), and increased UIBC in fish on the high-Fe diet
(Fig. 2) may facilitate Mn
accumulation.
In conclusion, rainbow trout closely regulate Fe status by increasing
Fe-binding to transferrin in the blood and promoting hepatic Fe accumulation,
probably facilitated by hepatic ferrireductase activity when dietary Fe intake
is high. Trout resist iron depletion when dietary Fe intake is low by up
regulating intestinal ferrireductase which probably results in improved Fe
accumulation in the intestine. The characteristics of Fe storage in the blood
of trout and patterns of tissue Fe accumulation, coupled with the potential
utility of rainbow trout for molecular studies of Fe metabolism
(Bury et al., 2003), suggest
the rainbow trout is a good model for future studies on vertebrate Fe
metabolism.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aisen, P. and Leibman, A. (1972). Molecular weight and subunit structure of hagfish transferrin. C. R. Sia. Biochem. (Wash.) 11,3461 -3464.
Aisen, P., Enns, C. and Wessling-Resnick, M. (2001). Chemistry and biology of eukaryotic iron metabolism. Int. J. Biochem. Cell Biol. 33,940 -959.[CrossRef][Medline]
Andersen, F., Maage, A. and Julshamn, K. (1996). An estimation of dietary iron requirement of Atlantic salmon, Salmo salar L., parr. Aquaculture Nutrition, 2,41 -47.
Andersen, F., Lygren, B., Maage, A. and Waagbø, R. (1998). Interactions between two dietary levels of iron and two forms of ascorbic acid and the effect on growth, antioxidant status and some non-specific immune parameters in Atlantic salmon (Salmo salar) smolts. Aquaculture 161,437 -451.[CrossRef]
Andersen, O. (1997). Accumulation of waterborne iron and expression of ferritin and transferrin in early developmental stages of brown trout (Salmo trutta). Fish Physiol. Biochem. 16,223 -231.[CrossRef]
Baker, R. T. M. and Davies, S. J. (1996).
Changes in tissue -tocopherol status and degree of lipid peroxidation
with varying
-tocopherol acetate inclusions in the diets for the
African catfish. Aquaculture Nutrition,
2, 71-79.
Baker, R. T. M., Martin, P. and Davies, S. J. (1997). Ingestion of sub-lethal levels of iron sulphate by African catfish affects growth and tissue lipid peroxidation. Aquat. Toxicol. 40,51 -61.[CrossRef]
Baker, R. T. M., Handy, R. D., Davies, S. J. and Snook, J. C. (1998). Chronic dietary exposure to copper affects growth, tissue lipid peroxidation, and metal composition of the grey mullet, Chelon labrosus. Mar. Envir. Res. 45,357 -365.[CrossRef]
Barron, M. G., Tarr, B. D. and Hayton, W. L. (1987). Temperature-dependence of cardiac output and regional blood flow in rainbow trout, Salmo gairdneri, Richardson. J. Fish. Biol. 31,735 -744.
Bury, N. R., Grosell, M., Wood, C. M., Hogstrand, C., Wilson, R.
W., Rankin, J. C., Busk, M., Lecklin, T. and Jensen, F. B.
(2001). Intestinal iron uptake in the European flounder
(Platichthys flesus). J. Exp. Biol.
204,3779
-3787.
Bury, N. R., Walker, P. A. and Glover, C. N.
(2003). Nutritive metal uptake in teleost fish. J.
Exp. Biol. 206,11
-23.
Camejo, G., Wallin, B. and Enojärvi, M. (1998). Analysis of oxidation and antioxidants using microtiter plates. In Free Radical and Antioxidant Protocols, Methods in Molecular Biology vol. 108 (ed. D. Armstrong), pp. 377-386. Totawa, NJ: Humana Press Inc.
Cho, C. Y. Cowey, C. B. and Watanabe, T. (1985). Finfish Nutrition in Asia. Part I. Methodological Approaches to Research and Development, pp.1 -80. Ottawa, Ontario, Canada: International Development Research Centre (IDRC vol.134 ).
Davis, D. A. and Gatlin, D. M. (1991). Dietary mineral requirements of fish and shrimp. In Proceeding of the Aquaculture Feed Processing and Nutrition Workshop (ed. D. M. Akiyama and R. K. H. Tan), pp. 49-67. Singapore: American Soybean Association.
Donovan, A., Brownlie, A., Dorschner, M. O., Zhou, Y., Pratt, S.
J., Paw, B. H., Phillips, R. B., Thisse, C., Thisse, B. and Zon, L. I.
(2002). The zebrafish mutant gene chardonnay
(cdy) encodes divalent metal transporter 1 (DMT1).
Blood 100,4655
-4659.
Donovan, A., Brownlie, A., Zhou, Y., Shepard J., Pratt, S. J., Moynihan, J., Paw, B. H., Drejer, A., Barut, B., Zapata, A., Law, T. C., Brugnara, C., Kingsley, P. D., Palis, J., Fleming, M. D., Andrews, N. C. and Zon, L. I. (2000). Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 403,776 -781.[CrossRef][Medline]
De Silva, D. M., Askwith, C. C. and Kaplan, J.
(1996). Molecular mechanisms of iron uptake in eukaryotes.
Physiol. Rev. 76,31
-47.
Desjardins, L. M., Hicks, B. D. and Hilton, J. W. (1987). Iron catalysed oxidation of trout diets and its effect on growth and physiological response of rainbow trout. Fish Physiol. Biochem. 3,173 -182.
Dorschner, M. O. and Phillips, R. B. (1999). Comparative analysis of two Nramp loci from rainbow trout. DNA Cell Biol. 18,573 -583.[CrossRef][Medline]
Gingerich, W. H. and Pityer, R. A. (1989). Comparison of whole body and tissue blood volumes in rainbow trout (Salmo gairdneri) with 125I bovine serum albumin and 51Cr-erythrocyte tracers. Fish Physiol. Biochem. 6,39 -47.
Gunshin, H., McKenzie, B., Berger, U. V., Gunshin, Y., Romero, M. F., Boron, W. F., Nussberger, S., Gollan, J. L. and Hediger, M. A. (1997). Cloning and characterisation of a mammalian proton-coupled metal-ion transporter. Nature 388,482 -488.[CrossRef][Medline]
Handy, R. D. and Depledge, M. H. (1999). Physiological responses: Their measurement and use as environmental biomarkers in ecotoxicology. Ecotoxicology 8, 329-349.[CrossRef]
Handy, R. D., Sims, D. W., Giles, A., Campbell, H. A. and Musonda, M. M. (1999). Metabolic trade-off between locomotion and detoxification for maintenance of blood chemistry and growth parameters by rainbow trout (Oncorhynchus mykiss) during chronic dietary exposure to copper. Aquat. Toxicol. 47, 23-41.[CrossRef]
Handy, R. D., Musonda, M. M., Phillips, C. and Falla, S. J.
(2000). Mechanisms of gastro-intestinal copper absorption in the
African walking catfish: copper dose-effects and a novel anion-dependent
pathway in the intestine. J. Exp. Biol.
203,2365
-2377.
Hershberger, W. K. (1970). Some physicochemical properties of transferrins in brook trout. Trans. Am. Fish. Soc. 99,207 -218.
Hershberger, W. K. and Pratschner, G. A. (1981). Iron binding capacities of six transferrin phenotypes of Coho salmon and potential fish health applications. Prog. Fish-Cult. 43,27 -31.
Horne, W. I., Tandler, B., Dubick, M. A., Niemelä, O., Brittenham, G. M. and Tsukamoto, H. (1997). Iron overload in the rat pancreas following portacaval shunting and dietary iron supplementation. Exp. Mol. Path. 64, 90-102.[CrossRef][Medline]
Ikeda, Y., Ozaki, H. and Uematsu, K. (1972). Serum iron level and total iron binding capacity in cultured fish. J. Tokyo Univ. Fish. 59,43 -53.
Kawatsu, H. (1972). Studies on the anemia of fish V. Dietary iron deficient anaemia in brook trout, Salvelinus fontinalis. Bull. Freshwater Fish. Res. Lab. (Tokyo) 22, 59-67.
Knox, D., Cowey, C. B. and Adron, J. W. (1984). Effects of dietary zinc intake upon copper metabolism in rainbow trout (Salmo gairdneri). Aquaculture 40,199 -207.[CrossRef]
Lanno, R. P., Slinger, S. J. and Hilton, J. W. (1985). Maximum tolerable and toxicity levels of dietary copper in rainbow trout (Salmo gairdneri Richardson). Aquaculture 49,257 -268.[CrossRef]
Lorentzen, M. and Maage, A. (1999). Trace element status of juvenile Atlantic salmon Salmo salar L. fed a fish-meal based diet with or without supplementation of zinc, iron, manganese and copper from first feeding. Aquaculture Nutrition, 5, 163-171.[CrossRef]
McKie, A. T., Barrow, D., Latunde-dada, G. O., Rolfs, A., Sager,
G., Mudaly, E., Mudaly, M., Richardson., C., Barlow, D., Bomford, A., peters,
T. J., Raja, K. B., Shirali, S., Hediger, M. A., Farzeneh, F. and Simpson, R.
J. (2001). An iron-regulated ferric reductase associated with
absorption of dietary iron. Science
291,1755
-1759.
Nelson, N. (1999). Metal ion transporters and
homeostasis. EMBO J. 18,4361
-4371.
Reilly, C. A. and Aust, S. D. (1998). Iron loading into ferritin by an intracellular ferroxidase. Arch. Biochem. Biophys. 359,69 -76.[CrossRef][Medline]
Riedel, H. D., Remus, A. J., Fitscher, B. A. and Stremmel, W. (1995). Characterisation and partial purification of a ferrireductase from human duodenal microvillus membranes. Biochem. J. 309,745 -748.[Medline]
Sakamoto, S. and Yone, Y. (1978). Iron deficiency symptoms in carp. Bull. Jap. Soc. Scient. Fish., 44,1157 -1160.
Shulte, U. and Weiss, H. (1995). Generation and characterisation of NADH: Ubiquinone oxidoreductase mutants in Neurospora crassa. Meth. Enzymol. 260,3 -14.[Medline]
Trinder, D., Oates, P. S., Thomas, C., Sadlier, J. and Morgan,
E. H. (2000). Localisation of divalent metal transporter 1
(DMT1) to the microvillus membrane of rat duodenal enterocytes in iron
deficiency, but to hepatocytes in iron overload. Gut
46,270
-276.
Van Dijk, J. P., Lagerwerf, A. J., van Eijk, H. G. and Leijnse, B. (1975). Iron metabolism in the tench (Tinca tinca L.). Studies by means of intravascular administration of 59Fe(III) bound to plasma. J. Comp. Physiol. 99,321 -330.
Van Campen, D. R. and Mitchell, E. A. (1965). Absorption of Cu64, Zn65, Mo99, and Fe59 from ligated segments of the rat gastrointestinal tract. J. Nutr. 86,120 -124.
Walker, R. L. and Fromm, P. O. (1976). Metabolism of iron by normal and iron deficient rainbow trout. Comp. Biochem. Physiol. 55A,311 -318.[CrossRef]
Walker, R. L. and Fromm, P. O. (1978).
Watanabe, T., Kiron, V. and Satoh, S. (1997). Trace minerals in fish nutrition. Aquaculture 151,185 -207.[CrossRef]
Winzerling, J. J. and Law, J. H. (1997). Comparative nutrition of iron and copper. Ann. Rev. Nutr. 17,501 -526.[CrossRef][Medline]