JOURNAL OF THE Vol. 41, No.

S2
WORLD AQUACULTURE SOCIETY May, 2010

Ascorbic Acid (Vitamin C) and Iron Concentration in Tambaqui,

Colossoma macropomum, Iron Absorption

Paulo Henrique Rocha Aride1

University Center Nilton Lins, Laboratory of Environmental Toxicology, Manaus, Amazon
State, Brazil

Marcio Soares Ferreira, Rafael Mendonca Duarte,

and Alzira Miranda de Oliveira
National Institute of Amazon Research, Laboratory of Ecophysiology and Molecular Evolution,
Manaus, Amazon State, Brazil

Danival Vieira de Freitas, Andre Luis Wendt dos Santos,

and Sergio Ricardo Nozawa
University Center Nilton Lins, Laboratory of Environmental Toxicology, Manaus, Amazon
State, Brazil

Adalberto Luis Val

National Institute of Amazon Research, Laboratory of Ecophysiology and Molecular Evolution,
Manaus, Amazon State, Brazil

Abstract. Tambaqui, Colossoma macropomum, were
fed with three vitamin C levels and two iron levels for
21 d: Diet 1 without vitamin C and with 30 mg of iron/kg;
Diet 2 with 100 mg of vitamin C and 30 mg of iron/kg; and
Diet 3 with 100 mg of vitamin C and 300 mg of iron/kg.
Mortality was not observed during the feeding experiment.
There were no changes in hematological parameters in fish
that were fed the different diets. Iron contents in liver,
muscle, and plasma were significantly different in fish fed
with different iron content in the diets; when associated
with vitamin C, changes were observed only for plasma
iron. Therefore, tambaqui exhibits varied iron absorption
in response to different levels of iron with and without
vitamin C, a conclusion consistent with findings for other
species of fish.
Fish, like other animals, absorb required
minerals through feeding and from the aquatic
environment (NRC 1993). Elements such as
calcium, magnesium, sodium, potassium, iron,
zinc, copper, and selenium are found in water,
but not always in sufficient concentrations to
1
Corresponding author.
Copyright by the World Aquaculture Society 2010
satisfy the requirements of fish. These elements
are necessary to promote the formation of
bone structure, electron transfer, acidbase
equilibrium, and osmoregulation (NRC 1993;
Canli and Atli 2003). In addition, the Amazon
water types present low levels of these metals
(Aride et al. 2007).
Metals are introduced into the aquatic system
by leaching from rocks and soil and by volcanic
eruptions, but mainly by human activities such
as mining and industrial processes (Gutenmann
et al. 1988; Zhou et al. 2008). Recently, the use
of fertilizers has also contributed to this process
(Adeyeye et al. 1996). Iron has been used
widely in many countries to reduce available
phosphate in freshwaters. Typically, iron is
added to reservoirs in the form of acidic
ferric sulfate liquor that contains other trace
metals, including titanium, manganese, zinc,
and nickel (Dalzell and Macfarlane 1999; Duis
and Oberemm 2000).
Iron is essential for the proper functioning
of organs and tissues of animals, including

291
292 ARIDE ET AL.
fish. It plays a critical role in oxygen trans-
port and cellular respiration (Lim et al. 2000),
and is one of the most important micronutrients
with an active function in the immune system
(Bhaskaram 1988; Lim et al. 2000). Character-
istically, the different valences of iron allow it
to accept or donate electrons and participate in
redox reactions (Baker et al. 1997).
Fish absorbs the soluble iron in water through
the intestinal membrane (NRC 1993; Cooper
et al. 2006). Feeding is considered the major
source of iron uptake, although absorption
through the gills does occur, but only to a
limited extent, as iron is present only in small
quantities in natural bodies of water (Halver
1988).
In biological systems, iron is found in com-
plex forms such as hemoglobin, myoglobin,
heme enzymes (mitochondrial and microsomal
cytochrome, catalysts and peroxides), and non-
heme compounds such as transferrin, ferritin,
and flavoproteins (Halver 1988; NRC 1993). In
vertebrates, ferritin is present in tissues such
as liver, spleen, bone marrow, heart, kidney,
intestinal mucosa, and blood (Geetha and Desh-
pande 1999). Iron deficiency has been described
in some fish species, including brook trout
(Salvelinus fontinalis Mitchill 1814), carp
(Cyprinus carpio Linnaeus 1758), and eel
(Anguilla japonica Temminck and Schlegel
1847). The effects of iron deficiency may
involve microcytic anemia and a decreased red
blood cell count and hemoglobin. In most cases,
however, iron deficiency does not affect the
growth rate of fish (Halver 1988). Like iron
deficiency, excess iron also causes problems in
fish because of its toxic effects which include
reduced growth rate, increased mortality, diar-
rhea, and decreased hepatic function (NRC
1993).
Ascorbic acid (AA) is essential for most
teleost fish species, both as a cofactor in
hydroxylation reactions in living tissues and
as important enzymes to involve in redox
reactions (Franke et al. 2005). AA is not
synthesized by fish because of the absence
of l-gulonolactone which is necessary for AA
oxidization (Lovell 1998). AA is one of the
most labile vitamins; it is easily oxidized by
air, heat, oxidizing enzymes, and multivalent
cations (Andersen et al. 1998). AA prevents
the formation of insoluble Fe composites and
assists in the absorption of iron that becomes
absorbable after reduction to its ferrous state in
the stomach. AA also participates in releasing
iron from transferrin and ferritin, which is later
incorporated into hemoglobin or other essential
composites (Affonso et al. 2007), and may
function as a Fe-chelating agent (Halliwell and
Gutteridge 2000).
The aim of the present study is to evaluate
the effects of dietary AA associated with iron
and the influence of iron levels in tambaqui
iron absorption and hematological parameters
(Fig. 1).
Materials and Methods
Juvenile tambaqui (62 4 g, mean SD,
for 90 [n] animals) were obtained from a local
fish farm. For the experimental feeding period
of 21 d, 10 fish were placed in each of the nine
(3 treatments, 3 replicates) continuously aer-
ated experimental units, 60 L glass aquaria. The
water temperature was maintained at 26 C, pH
was maintained at 5.8 0.5 (mean SD), the
level of dissolved oxygen was 5.6 mg/L, and
total ammonia level was 0.2 mg/L throughout
all the experiments. Fish were fed a commercial
diet for 6 d prior to the onset of the experimen-
tal diet.
Three diets were formulated with or without
vitamin C (Sigma Chemical Co., St Louis, MO,
USA) and with different iron (Sigma Chemical
Co.) concentrations (Table 1). The methodol-
ogy used to determine the diet composition has
been described by Asca (1985). The diets were
prepared in the Laboratory of Food Technology
at INPA. All diets contained 80 UI of vita-
min E per kilogram of feed, as vitamin E is
an important substrate for synergism with AA.
Experimental diets were stored in a freezer until
15 min before administration.
After feeding on the experimental diets
for 21 d, fish were anesthetized in a solu-
tion containing 100 mg/L of anesthetic (MS-
222, Syndel Laboratories Ltd, Vancouver, BC,
Canada). Hematocrit, red blood cell count, and
 ASCORBIC ACID AND IRON CONCENTRATION IN TAMBAQUI 293

diet 1
10 diet 2
diet 3

Muscle iron (g/g wet sample) 8 A

B
B
4

260
diet 1
diet 2
240
diet 3
Liver iron (g/g wet sample)

A A
220

200

180

160 B

140

120

diet 1
diet 2
6
diet 3
Plasma iron (g/g wet sample)

4
B

2
C

Figure 1. Iron levels in blood plasma, liver, and muscle of tambaqui fed experimental diets for 21 d. Diet 1 without
vitamin C and with 30 mg of iron/kg of diet; Diet 2 with 100 mg of vitamin C and 30 mg of iron/kg of diet; and Diet 3
with 100 mg of vitamin C and 300 mg of iron/kg. Bars with different superscripts are significantly different (P < 0 .05).
294
Table 1. Proximate composition, iron and vitamin C
concentration in experimental diets.
Diet 1 (%) Diet 2 (%) Diet 3 (%)
Moisture 10.2 10.4
Crude protein 29.6 28.5
Crude lipid 10.3 9.8
Fiber 1.6 1.5
Ash 7.1 6.6
Carbohydrate 41.2 43.2
Fe 30 mg 30 mg
AA 0 100 mg
AA = ascorbic acid; Fe = ferric chloride.
hemoglobin were immediately determined in
blood samples drawn from the caudal vein
using heparinized syringes. The animals were
sacrificed to collect white muscle and liver
samples.
The white muscle and liver tissue sam-
ples were weighted in sterile plastic tubes
and digested with 10% HNO3 at the ratio
of 5:1 (volume : tissue weight) for 48 h at
80 C. The samples were then centrifuged at
10,000 g/10 min and 50 L of the supernatant
diluted in 1% HNO3 . The levels of iron in tis-
sues were measured using a graphite furnace
atomic absorption spectrophotometer (Perkin
Elmer, AAnalyst 800) (Lemly 1997; Medina
et al. 1998; Campillo et al. 2000).
Hematological results (hematocrit, red blood
cell count, and hemoglobin) were used to
determinate corpuscular constants (mean cor-
puscular volume MCV, mean corpuscular
hemoglobin MCH, and mean corpuscular
hemoglobin concentration MCHC) according
to the methodology described by Brow (1976).
Plasma glucose levels were determined using a
portable reader (Advance).
The results are shown as mean SEM. Sta-
tistical differences among means were analyzed
using one way analysis of variance (ANOVA,
all pairwise) with Tukey test, assuming a 95%
confidence interval (P < 0.05) (Zar 1996).
Results and Discussion
The effect of AA in hematological parame-
ters of fish varies because of water chemistry
and dietary factors. Our results demonstrate
ARIDE ET AL.
that the presence of vitamin C improves tissue
absorption of iron, and this finding corroborates
previous findings (Dabrowski and Kock 1989;
Lygren et al. 1999). There were no significant
9.8
29.9
differences in either the hematological parame-
9.7 ters or plasma glucose levels in fish that were
1.5 fed the studied range of diet compositions. No
6.8 mortality was observed during the experimen-
42.3 tal period. Increases in hematocrit, red blood
300 mg
100 mg
cell count, and hemoglobin were observed in
tambaqui that were fed diets supplemented
with vitamin C. Similar effects have been
observed in channel catfish (Ictalurus puncta-
tus Rafinesque 1818) (Lim et al. 2000) and
the Atlantic salmon (Salmo salar Linnaeus
1758) (Sandnes et al. 1990). The increase in
the number of red blood cells in tambaqui was
probably because of the effect of AA on ery-
thropoiesis, first suggested by Dinning (1962)
and Cox (1968) (Table 2). AA is necessary for
the release of ferritin-bound iron from the liver
for erythropoiesis (Bhaskaram 1988).
The absorption of iron by the liver and
white muscle in animals that were fed Diet 2
(30 mg of iron and 100 mg of vitamin C/kg
of diet) increased significantly. This represents
about three times the amount observed for
channel catfish, I. punctatus, that were fed
a diet containing 3000 mg of vitamin C and
30 mg of iron/kg of diet (Lim et al. 2000). High
levels of AA have been reported to interact
with metallothionein, a copper-binding protein,
and to alter the metabolism of copper in the
liver (Hsu and Shiau, 1999). The increase
in the iron concentration in tambaqui muscle
was similar to that described by Lorentzen
(1998) in Atlantic salmon (Maage et al. 1990).
However, this increase suggests that dietary
supplementation with iron is not reflected in
muscle iron concentration. Although we did
not assess iron toxicity in the present study,
consumption of diets high in iron may result in
poor growth rates and decreased food utilization
efficiency (Lim et al. 2000). The early stages of
iron deficiency may induce changes in various
enzyme systems for which iron is essential
(Franke et al. 2005). Excess iron has also
been shown to increase the generation of lipid
peroxidation products (Galan et al. 1984).
 ASCORBIC ACID AND IRON CONCENTRATION IN TAMBAQUI 295

Table 2. Blood hemoglobin concentration (Hb), hematocrit (Ht) and red blood cell count (Rbc), mean corpuscular
volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and
glucose of tambaqui which were fed a diet supplemented with either 30 mg/kg of iron without vitamin C, 30 mg/kg of iron
and 100 mg/kg of vitamin C, or 300 mg/kg of iron and 100 mg/kg of vitamin C.

Diet treatment 0 mg AA/30 mg Fe 100 mg AA/30 mg Fe 100 mg AA/300 mg Fe

Hb (g/dL) 7.5 0.72 8.3 0.98 8.0 0.88
Ht (%) 27.3 0.71a 30.3 0.56b 29.7 0.72b
Rbc (106 mm3 ) 1.5 0.32 1.6 0.25 1.8 0.25
MCV (m3 ) 179.8 3.23a 188.7 3.82b 165.0 4.09c
HCM (pg) 49.6 1.63a 51.8 1.01b 44.5 0.99c
CHCM (%) 27.6 0.48 27.4 0.46 26.9 0.44
Glucose (mg/dL) 51.2 2.50ab 54.1 2.22a 48.8 1.56b
AA = ascorbic acid; Fe = ferric chloride.
Values with different superscripts within rows are significantly different (P < 0.005).

A decrease in iron may occur in the tissues
of fish that are fed diets low in iron, but sup-
plemented with vitamin C. This effect may
be because of vitamin Cs important role in
the release of iron from ferritin in the liver,
as well as in the transport of iron from the
plasma to the liver and its subsequent incor-
poration into tissues (Franke et al. 2005). For
Atlantic salmon, S. salar, a hepatic iron con-
centration of 30 g/g of wet tissue is insuffi-
cient to maintain erythropoiesis and the stability
of some hematological parameters (Andersen
et al. 1998). Our results suggest that tambaqui
liver absorbs, with high efficiency, the iron
present in food when associated with vitamin C;
otherwise, this capacity is decreased. The con-
centration of iron in plasma decreased signifi-
cantly in animals that were fed diets containing
vitamin C and iron. This decrease may suggest a
faster iron clearance from plasma with redistri-
bution to other tissues. Alternatively, it may be
related to the capacity of vitamin C chelation
of Fe (Halliwell and Gutteridge 2000). Baker
et al. (1997) also reported only minor increases
in plasma iron concentration in African catfish
(Clarias gariepinus Burchell 1822) that were
fed a diet high in iron (6300 mg of iron/kg
wet diet).
AA is reportedly involved in the metabolism
of iron in higher animals, including fish (Maage
et al. 1990; Andersen et al. 1998). No gross
signs of vitamin C deficiency were observed
in tambaqui that were fed vitamin C-deficient
diets for 21 d.
Increased iron concentration in the diet did
not result in significant changes in hematologi-
cal parameters, glucose levels (Table 2), or iron
absorption by the liver or muscle tissue. This
is consistent with results from previous studies
from other fish species, such as Atlantic salmon
(Bjrnevic and Maage 1993; Lim et al. 2000).
Reduced hematological parameters under iron
deficiency signs are well documented in human
beings and several animals besides fish (Sayers
et al. 1994; Ye et al. 2007). An observed effect
of increased dietary iron concentration associ-
ated with vitamin C in tambaqui was a signifi-
cant decrease in plasma iron concentration. This
decrease can be attributed to a small increase in
iron in the liver of tambaqui that were fed an
iron-supplemented diet. Consumption of diets
high in iron decreased the efficiency of food uti-
lization, which implies some degree of iron tox-
icity. High levels of iron may lead to oxidative
damage to cells as a result of increased cata-
lase activity. Alternatively, iron-deficient diets
may induce changes in various enzyme systems
where iron is an essential element (Andersen
et al. 1996).
Animals that were fed a diet supplemented
with 300 mg of iron and 100 mg of vitamin C
presented similar levels of hepatic iron as those
of animals fed diets with 100 mg vitamin C
and 30 mg of iron, corroborating the findings
of Andersen et al. (1998) for Atlantic salmon,
S. salar, and of Baker et al. (1997) for African
catfish, C. gariepinus. In short, iron supplemen-
tation of diets with sufficient vitamin C does
296 ARIDE ET AL.
not result in increased hepatic iron levels in
tambaqui. The maintenance of the hepatic iron
level suggests the action of a regulating mech-
anism in tambaqui similar to that observed in
other fish species, domestic animals, and human
beings (Sayers et al. 1994).
The levels of iron in tambaqui white muscle
and liver (4 g of iron/g and 200 g of iron/g,
respectively) were low when compared to those
found in African catfish, C. gariepinus, which
were fed diets containing more than 600 mg
of iron for 5 wk. The study reported an iron
concentration of over 700 g/g in the liver, and
over 200 g/g in muscle (Baker et al. 1997).
Our results for the liver of tambaqui were
similar to those described for Atlantic salmon,
S. salar, that were fed a diet with 150 mg of
AA and 400 mg of iron (160 g of iron/g of wet
tissue) (Andersen et al. 1998). Both deficiency
and excess of iron can depress the immune
system in fish as reported elsewhere (Barros
et al. 2002), a condition not observed in the
present study.
Results indicate that iron deficiency results
in microcytic anemia in tambaqui. In the
presence of vitamin C, hepatic iron absorption
increases. However, iron absorption does not
increase in the liver with a 10-fold dietary iron
supplementation. More studies are necessary to
better understand the relationship between iron
and AA in iron metabolism.
Acknowledgments
This research was supported by CNPq
(ALTALI 557094/2005-4), INPA (PPI #3510),
and PNOPG #400030/99-3. All the fish were
donated by Project Tambaqui (PPD 1139-99).
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