Fish Physiology and Biochemistry 25: 209219, 2001.

2002 Kluwer Academic Publishers. Printed in the Netherlands.

209

Feed intake and absorption of lipid oxidation products in Atlantic salmon

(Salmo salar) fed diets coated with oxidised fish oil
Kristin Hamre1 , Kjersti Kols1, Kjartan Sandnes2 , Kre Julshamn1 and Anders Kiessling3
1 Institute

of Nutrition, Directorate of Fisheries, P.O. Box 185, N-5804 Bergen, Norway (Phone:
+47 55 90 51 30; Fax: +47 55 90 52 99; E-mail: kristin.hamre@nutr.fiskeridir.no); 2 Sea Grain
AS, 5300 Kleppest, Norway; 3 Institute of Marine Research, Matre Aquaculture Research Station,
5984 Matredal, Norway
Accepted: September 4, 2002

Abstract
Two questions were asked in the present study; does Atlantic salmon taste and discriminate against oxidised feed
and are lipid oxidation products absorbed from the intestine. In Experiment 1, a control diet, a medium oxidised
diet and a highly oxidised diet were prepared (TBARS levels: 345, 614 and 762 nmol g1 , respectively). The
control diet was marked with holmium and the experimental diets with europium. Each of the experimental diets
were fed together with the control diet (1:1) to Atlantic salmon in duplicate tanks in one meal to satiation and the
stomach contents were analysed for the lanthanides. The fish discriminated slightly against the highly oxidised diet,
but not against the medium oxidised diet. In Experiment 2, Atlantic salmon cannulated through the dorsal aorta
were fed control and oxidised feed (TBARS: 71 5 and 204 18 nmol g1 ) and blood samples were collected
regularly over a nine days period. On day 9, the fish were sacrificed, and samples of muscle, liver, intestinal tissue
and contents were taken and analysed for oxidation products, and vitamins C and E. Plasma TBARS increased
3-4 fold in response to the oxidised diet and there was a slight increase in muscle and liver TBARS. The data on
peroxide value (PV) in the tissues showed large variation, but no differences between the groups were detected. PV
and TBARS in the contents of the large intestine increased 7- and 1.6-fold, respectively, in response to oxidised
feed. There were no differences in levels of vitamins C and E between the groups. It seems that Atlantic salmon
feed quite well on oxidised feed and that aldehydes, here represented by MDA and measured as TBARS, are
absorbed from the intestine. The question as to whether lipid hydroperoxides are absorbed is unanswered due to
the large variation in tissue PV. On the other hand, animals in general seem to be protected from absorption of lipid
hydroperoxides and we hypothesise that similar mechanisms are active in Atlantic salmon.

Introduction
Fish feed is rich in n-3 polyunsaturated fatty acids
(PUFAs) which are very susceptible to peroxidation,
where the double bonds of the fatty acids react with
molecular oxygen through a free radical chain reaction, to form lipid hydroperoxides (Frankel 1998). The
lipid hydroperoxydes are toxic compounds. They are
unstable and decompose readily to fatty acid alkoxy
radicals, which react strongly with various biomolecules, such as protein and DNA. They also evoke new
chain reactions of lipid oxidation. The lipid hydroperoxides decompose further to shorter chain compounds
such as aldehydes, ketones, alcohols, alkanes and

alkenes. Many of these compounds are volatile and are

the primary source of rancid odour and taste (Frankel
1998). Some of them, especially the aldehydes, are
toxic due to their ability to bind to the amino groups
of protein, nucleic acid bases, the N bases of phospholipid and the SH groups of sulfhydryl compounds
(Nair et al. 1986; Draper et al. 1986; Estrbauer 1993;
Mahmoodi et al. 1995). In mammals, an important
mechanism of protection against rancidity is avoidance due to unpleasant odour and taste. We do not
know to what extent fish are able to taste rancidity
and one of the experiments in this study is designed
to answer this question.

210
The next question asked was whether oxidation
products from the feed are taken up into the fish body.
It is well known that oxidised feed, especially when
combined with low levels of vitamin E, cause pathologies in fish (Tacon 1996). Oxidised oil combined with
low vitamin E cause reduced growth and increased
mortality in a large number of fish species. These
effects are reversed when the vitamin E supplementation is increased, and to a lesser extent with increased
ethoxyquin supplementation (Hung et al. 1981; Tacon
1996; Baker and Davis 1997). Further, oxidised oil often leads to reduced vitamin E levels in the fish tissues
(Hung et al. 1981; Baker and Davis 1997). Similar
results have also been shown for rats (Liu and Hung
1995, 1996). However, based on these experiments
it is difficult to say whether the oxidised oil leads to
decreased available vitamin E in the gut and therefore causes a secondary vitamin E deficiency, or if
oxidation products are taken up into the body where
they initiate lipid oxidation and thereby an increased
consumption of vitamin E in vivo.
Malondialdehyde (MDA; Draper et al., 1986;
Draper and Hadley 1990) and trans-2-alkenals
(Grootveld et al. 1998) seem to be readily absorbed
from the gastrointestine in mammals. On the other
hand, there is a question as to the absorption of
lipid hydroperoxides. A large fraction of linoleic acid
hydroperoxides or trilineoylglycerol hydroperoxides
administered intragastricly to rats, decomposed to
aldehydes before they reached the intestine (Kanazawa
and Ashida 1998a, b). Only at the larger doses were
small amounts of hydroperoxides detected in the intestine. Further, vertebrates are protected from uptake
of lipid hydroperoxides by gastrointestinal glutathione
peroxidase (GI-GPX) which reduces fatty acid hydroperoxides to hydroxy fatty acids at the expense
of reduced glutathione, thereby preventing the decomposition to fatty acid alkoxy radicals (Aw 1992;
Esworthy et al. 1998). It is therefore possible that, at
least at low doses, fatty acid hydroperoxides are transformed to hydroxy fatty acids in the enterocyte before
transport into the blood.
In the present study, we fed cannulated Atlantic
salmon with control and oxidised feed. We measured
uptake of MDA, a representative of the aldehydes,
as thiobarbituric acid reactive substances (TBARS) in
blood and tissues. We also measured peroxide value
(PV) in tissues to identify uptake of lipid hydroperoxides.

Table 1. Dietary ingredients (g kg1 wet wt.)

Ingredient

Content

SUPREX wheata
Whole herringb
Whole saitheb
Vitaminsc
Mineralsc
SUM

20.8
538.5
434.5
3.1
3.1
1000.0

a From Condroco B.V., Rotterdam, the Nether-

lands.
b From local suppliers.
c Vitamins and minerals were added according to

NRC (1993). The vitamins were from Hoffman La

Roche, Basel, Switzerland and the minerals from
Merch, Darmstadt, Germany.

Materials and methods

Diets
The dietary ingredients (Table 1) were minced and
blended thoroughly. The blend was processed to diet
strings that were heated to 80 C using electromagnetic energy at 2450 MHz. The strings were pelleted
and dried in warm air. Dry matter was 98.3% and lipid
31.7 0.8% of dry matter. The diets for the taste
experiment were supplemented with 16.9 mg kg1
europium (Eu) or holmium (Ho) as Eu2O3 and Ho2 O3 .
The oxidised oil was prepared by the following
procedure: About 150 ml of herring oil (local suppliers) was continuously stirred and aerated at room
temperature. To control for the degree of oxidation,
aliquots of aerated oil were taken periodically and
peroxide values (PV) and concentrations of thiobarbituric acid-reactive substances (TBARS) were determined. After 670 h the peroxidation procedure was
terminated. This treatment caused a marked loss of tocopherol, and a marked elevation of PV and TBARS.
The oxidised oil was adjusted to a vitamin E level of
355 mg kg1 free RRR--tocopherol, as found in the
fresh herring oil.
The experimental diets for the taste experiment
were marked with europium and were termed highly
oxidised (HO) and medium oxidised (MO). The HOdiet was coated with 10% (w/w) oxidised oil and
the MO-diet was coated with 10% (w/w) oxidised
oil and fresh herring oil (1:1). The control diet was
marked with holmium and coated with 10% (w/w)
fresh herring oil.
Two diets were prepared for the experiment with
uptake of oxidation products, one was coated with

211
fresh oil and the other with oxidised oil, both at 10%
(w/w). The dry matter in the diets was 99.0% and lipid
was 38.4 0.4% of dry matter.
Fish and sampling
The experiments were carried out at Matre Aquaculture Research Station, The Institute of Marine
Research, Bergen, Norway.
Experiment 1. For the taste experiment, Atlantic
salmon (Salmo salar, 622 118 g) were fed the control diet for one month prior the start of the study.
The fish were distributed to 4 tanks (40 fish per tank)
and bulk weighed. There were two replicate tanks
per dietary treatment. The fish were starved for 48 h
before they were fed control-diet and one of the experimental diets (1:1, w/w) in one meal to satiation.
Immediately after the meal ten fishes per tank were
sampled, anaesthetised in water with addition of a
saturated benzocaine-ethanol solution. The stomach
contents were sampled and stored at 20 C until the
levels of lanthanides were analysed by multi element
technique, ICP-MS.
Experiment 2. The experiment on uptake of oxidation products was performed using eight Atlantic
salmon weighing 837 122 g. The fish were accustomed to the experimental diet before they were
placed individually into eight experimental tanks. The
fishes were given some days to recommence feeding. When the fish were eating normally, they were
anaesthetised in accordance with Kreiberg and Powell
(1991), using 0.5 mg kg1 metomidate as pre anaesthesia sedative and 60 mg l1 Metacanium (MS 222,
tricaine methanesulfonate, Norwegian Medical Depot,
Bergen, Norway) as anaesthetics, and a cannulae was
fitted into the dorsal aorta as described by Soivio et al.
(1975). The cannula was filled with 300 IU heparine
(heparine aqua solution, Nycomed Pharma AS, Oslo,
Norway, injection quality) in 0.9% isotonic sodium
chloride solution (injection quality, Fresenius Kabi,
Uppsala, Sweden) before being resealed by heat. No
sedation was necessary during blood sampling from
fish held in individual tanks as lighting of the tank
and in the room was arranged to create a water surface mirror reducing the disturbance to the fish by
movements outside the tank. A more detailed description of anaesthetic and surgery procedures are given
in Kiessling et al. (1995). Once again the fishes were
given some days to recommence feeding. In the ex-

perimental period of nine days the fishes were fed

every day by hand from 8.00 a.m. to 12.00 a.m. and
from 8.00 p.m. to 12.00 p.m. Five of the fishes were
administered diets coated with oxidised oil and three
with fresh oil. The amount of food consumed was calculated as the difference between the weight of the
pellets fed and the pellets not eaten.
Blood samples were taken from each fish on day
1, 2, 5, 7, and 9 between 8.00 a.m. and 12.00 a.m.
A needle with the sharp point removed, was attached
to the cannula. The heparin lock and 0.1 ml of pure
blood was removed. A 0.8 ml blood sample was then
taken using a heparinized syringe. Subsequently the
cannula was refilled and resealed as described above
for surgery. Immediately after the blood had been
sampled haematocrit was determined (Sandnes et al.
1988). Plasma and red blood cells were separated by
centrifugation (2000 g for 10 min). The plasma was
frozen at 20 C and stored at 80 C until analyses. The fraction of red blood cells was washed three
times with PBS-solution before freezing at 20 C
and stored at 80 C.
On day nine the fishes were sedated with metomidate and killed by percussion. Samples of fillet, liver,
small intestine including pylorie cecae, large intestine
and gut contents were frozen on dry ice and stored at
80 C.
Analytical methods
Dietary dry matter was determined by freeze drying
and total lipid by the method of Lie et al. (1988).
PV was analysed using the ferric thiocyanate method
as described by Undeland et al. (1998). The results
were expressed as meq (milliequivalents) peroxide
kg1 lipid. Thiobarbituric acid reactive substances
(TBARS) were measured as described by Hamre et al.
(2001). -Tocopherol was analysed by HPLC by the
method of Lie et al. (1994) and vitamin C according
to Mland et al. (1999).
The control, medium and highly oxidised oils
used for coating the diets for the taste experiment
were analysed for levels of 2-pentenal, hexanal, 2,4heptadienal and nonanal by dynamic headspace. Approximately 0.2 g oil was purged with 50 ml min nitrogen at 70 C for 5 min. The volatiles were collected
with Tenax-GR, 6080 mesh, traps (Alltech Associates, Inc) and desorbed from the Tenax traps by use
of an automatic thermal desorber (ATD400, PerkinElmer). ATD400 thermally desorb volatiles by cryfocusing at 30 C in a Tenax TA 6080 mesh (Phase

212
separations Ltd) trap. By heating the trap to 300 C
at 40 C sec1 with helium flow of 30 ml min1
for 5 min, the volatiles were transferred via heated
(200 C) fused silica tubing into the column (DB5MS 30 m 0.25 mm 1.0 m J&W Scientific,
Folsom) of the gas chromatograph (GC 8000 Series,
Fisons Instruments). The aldehydes were identified
and quantified (arbitrary units) with a mass spectrometer (MD800, Fisons Instruments). The following temperature program was used to separate the volatiles:
40 C for 10 min, from 40 to 200 C at 2.5 C min1
and finally hold at 200 C for 5 min. The ionisation
energy of the mass spectrometer was set at 70 eV in
EI mode. A full scan (scan time 0.45 sec and interscan delay 0.05 sec) mass spectra over a mass range
from m/z 30 to 150 was used for identifying the analytes by comparison of spectra from standards and
with those of available libraries (National Institute
of Standards and Technology, NIST, USA). Relative
quantification was performed in SIM mode. The m/z
ion 55 of the 2-pentenal, m/z 81 of the 2,4-heptadienal
and m/z ion 44 of heptanal, hexanal and nonanal were
monitored. Quantitative sampling time for each channel was 0.80 ms and interchannel delay times were
20 ms. Areas from selected ions of the aldehydes can
only be compared within compounds and not between
compounds because different compounds in the same
amount do not produce the same peak area.
ICP-MS analysis
For analyses of europium (63 Eu) and holmium (67 Ho)
concentrations in feeds and stomach contents, samples
were digested by adding 2 ml nitric acid (HNO3 ) (extra pure quality) and 0.5 ml hydrogen peroxide (H2 O2 )
to the samples of about 0.2 grams and run the digestions in special bombs through a standardised temperature program in a microwave oven (Milestone MLS1200, Italy). After digestion, rhodium (45 Rh) was
added as internal standard to the digests, which were
then diluted to 25 ml. All measurements were made
using a Perkin Elmer SCIEX Elan 5000A ICP-MS
(Toronto, Canada) equipped with standardised configuration, with a single-channel mass flow controller.
The sample solutions were pumped by a peristaltic
pump from tubes arranged on a Perkin Elmer AS-90
autosampler and aspirated into the argon plasma. The
method of standard addition calibration was used for
quantification of europium and holmium at m/z 153
and 165, respectively. Three spikes of the elements
were added at 10, 20 and 40 ng ml to sample so-

lutions of each type of biological material, obtained

from the treatment of wet digestion. The correlation
coefficient obtained for both masses measured showed
values better than 0.99.
Statistics
The statistical analyses were performed using the software Statistica (version 4, Statsoft Inc., Tulsa, OK,
USA). The data were analysed by ANOVA and differences between means were identified with Scheffes
test, or Tukeys HSD test for uneaqual sample sizes
where appropriate. The data on feed intake and
TBARS in plasma and red blood cells in Experiment 2
were analysed by 2 way ANOVA, testing effects on
groups during repeated measurement. Differences and
effects were considered significant at P < 0.05.

Results
Experiment 1
In the taste experiment, the level of oxidation products,
measured as PV, TBARS, 2-pentenal, hexanal and 2,4heptadienal (Table 2, Figure 1) increased gradually
from the control diet via the medium oxidised diet to
the highly oxidised diet. Heptanal and nonanal showed
less variation in the diets (Figure 1), possibly because
they are less volatile than the shorter chain aldehydes and therefore more difficult to quantify with the
purge-trap method used.
Figure 2 shows how the fish chose between the
control diet and each of the two experimental diets. There was no discrimination between the control
diet and the medium oxidised diet, but the fish discriminated slightly against the highly oxidised diet
compared to the control diet.
Experiment 2
In the experiment on uptake of oxidation products, the
levels of PV and TBARS were approximately 3-fold
higher in the oxidised diet compared to the control diet
(Table 2). However, the level of oxidation products in
the control diet was similar to the highly oxidised diet
in Experiment 1. -Tocopherol was similar in the two
diets. Feed intake in fish fed the oxidised and control
diets was not significantly different (Figure 3), although there was a tendency towards slightly lowered
feed intake in fish fed the oxidised diet.

213
Table 2. Peroxide value (PV, meq kg1 lipid) thiobarbituric acid reactive substances
(TBARS, nmol g1 dry wt.) -tocopherol (mg kg1 dry wt.) and added lanthanides
in the experimental and control diets of Experiment 1 (taste of oxidation products)
and the oxidised and control diet in Experiment 2 (uptake of oxidation products). (-:
not determinated or not added)

Experiment 1
Control
Medium oxidised
Highly oxidised
Experiment 2
Control
Oxidised

PV
(meq kg1 )

TBARS
(nmol g1 )

11 3a
43 1bc
62 13c

34 5a
61 4b
76 2c

65 16
200 43

71 5
204 18

-Tocopherol
(mg kg1 )

Lanthanide

Holmium
Europium
Europium

153 3
154 1

Table 3. Peroxide value (PV, meq kg1 wet wt.), thiobarbituric acid reactive substances (TBARS, nmol g1 wet wt.) and
-tocopherol (mg kg1 wet wt.) in tissues and intestine contents of Atlantic salmon fed oxidized and control feed for 9 days
(meanSD; : not determinated)
PV
(meq kg1 )
Oxidised
Control
Liver
3.8 3.4
Fillet
1.2 0.4
Small intestine tissue
3.4 2.0
Large intestine tissue
9.5
Small intestine contents 9.0 11
Large intestine contents 48 22a

TBARS
(nmol g1 )
Oxidised
Control

4.7 1.1
8.9 1.1a
6.1 0.1b
a
0.8 0.4
3.3 0.5
2.3 0.4b
3.4 3.5
42 15
36 6
6.7 6.7 132 63
157 11
5.6 5.9 140 51
109 41
7.1 3.2b 233 38a 146 46b

Fish fed the oxidised diet had significantly higher

levels of TBARS in plasma than fish fed the control diet (Figure 4a), while there was no difference in
TBARS in red blood cells between the groups (Figure 4b). On day 9, TBARS in muscle and liver was
slightly higher than control in fish fed the oxidised
diet, while there were no differences in intestinal tissue (Table 3). There was a large variation in the data
on tissue PV, and no differences between the groups.
Also, there were no differences in tissue -tocopherol
or ascorbic acid between the groups. In the intestinal
contents there were large variations in the levels both
of PV and TBARS, but significantly higher levels of
oxidation products were found in the contents of the
large intestine in fish fed the oxidised diet (Table 3).

-Tocopherol
(mg kg1 )
Oxidised Control

Ascrobic acid
(mg kg1 )
Oxidised Control

46 23
8.0 1.4
30 23
31 25

52 8
31
30 8

34 19
9.3 0.5
37 22
41 30

52 10
31
20 4

Discussion
According to an overall view, both the medium and
highly oxidised diet in experiment 1 and the control diet in experiment 2 may be termed moderately
oxidised. This was unfortunate and may affect the interpretation of results, but in each experiment there
were significant differences between the control and
experimental diets.
In the taste experiment, the fish did not prefer the
control diet above the medium oxidised diet, and discriminated only slightly against the highly oxidised
diet. The levels of oxidation products in the control
diet were similar to those found in commercial diets, the upper limit being a TBARS of approximately
40 nmol g1 (own unpublished results). Both the
experimental diets were thus considerably higher in
oxidation products than what is the normal situation

214

Figure 1. Aldehydes collected by the purge trap method and measured by GC-MS from oils coated onto the diets in Experiment 1, taste of
oxidation products. Response is given as mV, letters indicate significant differences between diets and bars represent standard deviation.

in fish farming. Furthermore, feed intake in Experiment 2 was not significantly different between the
groups even though the oxidised diet had a TBARS
of 204 18 nmol g1 . Although in this latter case the
fish did not have a choice, but was only served control or oxidised feed. The results indicate that Atlantic
salmon tolerate quite high levels of oxidation products
before they avoid the diet due to unpleasant taste or
odour.
In the experiment on uptake of oxidation products, Atlantic salmon fed the oxidised diet showed
markedly higher levels of MDA in plasma, measured
as TBARS, than fish fed the control diet. Furthermore, slightly elevated TBARS levels were found in
muscle and liver of fish fed the oxidised diet. We interpret these data as an increased uptake of MDA from
the intestine. However, it could also be interpreted as
an uptake of fatty acid hydroperoxides that later decomposed to aldehydes. In rats, 14 C-labelled MDA
administered by stomach intubation was recovered as
14 CO (63%), urinary metabolites (13%) and in the
2
faeces (10%). MDA excretion was also responsive to
MDA administrtion as Na enol salt in the drinking water of rats and mice (Draper et al. 1986). Furthermore,
Grootweld et al. (1998) identified C-3 mercapturate
conjugates of trans-2 nonenal in the urine of rats after
oral administration. This shows that MDA and possibly other aldehydes are absorbed from the intestine
of rats and mice, suggesting that the increased MDA

measured in plasma of Atlantic salmon in the present

experiment was actually due to absorption of MDA
from the intestine.
The data on PV in the tissues show large variability and the reliability of these measurements can
be questioned. However, PV was similar in fish fed
the oxidised and control diets, which may indicate
that fatty acid hydroperoxides are not, or only to
a small extent, absorbed from the gastro-intestinal
tract. This is partly in accordance with the literature.
For example, orally administred methyl linoleate hydroperoxides at a single dose of 200 mg kg1 had
no effect on rats, but when 20 mg kg1 of the same
compound was injected intravenously, high mortality with severe lesions occurred (Cortesi and Privett
1992). Kanazawa and Ashida (1998a, b) found that
linoleic acid hydroperoxide or its triacylglycerol form
infused in the stomach of rats, were broken down to
the hydroxy fatty acid, epoxyketones and secondary
oxidation products such as hexanal, before reaching the intestine. At the higher doses, however, low
amounts of hydroperoxides were detected in the intestine. This is in agreement with the apparent defecation
of peroxides found in the present study.
The gastrointestinal glutathione peroxidase (GIGPX) is distinct from the ubiquitous GPX-1 (Esworthy et al. 1998) and may have a specialised function
in protecting vertebrates from absorption of lipid hydroperoxides, by reducing them to hydroxy fatty acids

215

Figure 2. Discrimination between the control diet and each of the medium and highly oxidised diets by Atlantic salmon (Experiment 1). The
control diet was marked with holmium and the experimental diets with europium. The control diet and one of the experimental diets were fed
simultaneously (1:1) in one meal to satiation and concentrations of lanthanides were measured in the stomach contents. Bars represent standard
deviations.

at the expense of reduced glutathione (GSH). Aw

et al. (1992) studied the role of mucosal GSH in intestinal metabolism of lipid hydroperoxides in rats.
Intestinal GSH concentration was lowered to approximately 50% of control, using drugs inhibiting the GSH
metabolism and appearance of TBARS in the lymph
was measured after duodenal infusion of oxidised oil.
They found that TBARS levels in the lymph increased
approximately 50% in response to the lowered GSH
concentration. They also found that supplementing
exogenous GSH to GSH depleted rats restored the protection against absorption of oxidation products, but
this effect was absent when uptake of GSH into the
enterocytes was inhibited (Aw and Williams 1992).
Although they measured TBARS in these studies, the
mechanism of protection of GI-GPX makes it probable
that fatty acid hydroperoxides were absorbed at low
mucosal GSH levels and that they later decomposed to
MDA.
On the contrary, Staprans et al. (1994) found that
feeding oxidised lipids to humans lead to higher levels
of TBARS and conjugated dienes in the chylomicrons.
They also found that when rat chylomicrons were
prepared by feeding oxidised 14 C-linoleic acid, and injected into new rats, the label appeared in the liver and
was excreted into very low density lipoprotein (VLDL;
Staprans et al. 1996). To these studies one could infer that the increased TBARS and conjugated dienes

in the chylomicrons could originate from aldehydes

formed in the gastro-intestine and from hydroxy fatty
acids formed in the mucosa, and that the label found
in VLDL could also be from transformed linoleic acid
hydroperoxides.
In the present study, fish fed the oxidised diet had
higher levels than controls, of both TBARS and PV
in the contents of the large intestine. The difference
was almost 7-fold for PV and 60% for TBARS. This
indicates that lipid oxidation products, including lipid
peroxides, were transported to the intestines in Atlantic salmon and that aldehydes were absorbed to
a larger extent than the lipid hydroperoxides. It is
possible that the fish discriminate against lipid hydroperoxides in the absorption and that this represents
another protection mechanism.
There were no differences in the concentration of
-tocopherol in the tissues of Atlantic salmon fed
oxidised and control feed. This is in contrast to the
results of Baker and Davis (1997) who found a reduction in liver -tocopherol to 15% of original level in
African catfish (Clarias gariepinus) after feeding oxidised feed for two weeks. Similar results were found
by Hung et al. (1981) but Cowey et al. (1984) failed to
find any reduction in tissue -tocopherol in response
to feeding of moderately oxidised oil. In the present
study, the liver concentration of -tocopherol was already quite low compared to the 200 mg kg1 found in

216

Figure 3. Feed intake (g fish1 day1 ) in cannulated Atlantic salmon fed the oxidised and control diets in Experiment 2. (n = 5 and 3,
respectively. Bars represent standard deviations).

several previous studies with salmon fed similar levels

of vitamin E (Hamre 1995). The fish in the present
study needed a long time to adapt to the experimental
diet and rearing facilities, during which they were not
feeding well. Secondly, the control diet, which was
used for the acclimatisation period, already contained
quite high levels of oxidation products and the feeding period in the present experiment was quite short.
These could be reasons why we did not find an effect
of oxidised oil on tissue vitamin E concentration. The
lack of difference in tissue ascorbic acid concentration
is in line with the results of Hung et al. (1980), who fed
diets increasing in levels of oxidation products with
similar levels of vitamin C to rainbow trout without
finding any effect on tissue vitamin C concentrations.
Oxidised lipids can be detrimental to fish health.
The most toxic substances are probably lipid hydroperoxydes, which if they are taken up into the body,
will initiate in vivo lipid oxidation, damage protein
and nucleic acids and cause severe lesions and subsequently high mortality (Cortesi and Privett 1992).
Atlantic salmon seems to feed quite well on an oxidised diet. The discrimination against oxidised feed
started at a level of oxidation products well above that
found in commercial diets, but the level of TBARS in
plasma fed this level of oxidation products (the control diet) was similar to that usually found in Atlantic
salmon (unpublished results). On the other hand, Atlantic salmon probably have similar mechanisms as

mammals to prevent absorption of lipid hydroperoxides. The fact that the PV was similar in tissues of fish
fed oxidised and control diets, and that the PV in the
contents of the large intestine was 7-fold higher in fish
fed the oxidised compared to the control diet, indicates
that very little lipid peroxides were absorbed.
On the other hand, aldehydes, here represented by
MDA, appeared both in plasma and tissues of fish fed
oxidised feed. Draper and Hadley (1990) argue that
very little free MDA is present in feeds due to rapid reaction with free amino groups of protein, phospholipid
and nucleic acids, and that absorption of these adducts
is less toxic than absorption of MDA itself. Further,
very little free MDA is detected in tissues, plasma and
urine in rats fed diets enriched with MDA, probably
due to a very rapid binding to various molecules and
rapid metabolism and excretion (Draper et al. 1986;
Grune et al. 1997). This could be different in fish,
where the feed may contain more than 40% of fish oil
very rich in n-3 PUFA. Our method of TBARS determination separates protein from MDA extracted in the
water:methanol phase of a Bligh and Dyer extraction.
No step is applied to hydrolyse bound MDA from protein but water-soluble complexes may be hydrolysed
in the process where the MDA-TBA adduct is formed
in the presence of tri-carboxylic acid. Free MDA at
concentrations down to 107 M has been shown to
be mutagenic for bacteria and in cultures of mammalian lymphoma cells and skin fibroblasts (Draper

217

Figure 4. TBARS in plasma and red blood cells (nmol ml1 og nmol g1 ) of Atlantic salmon fed the oxidised and control diets in Experiment 2
(n = 5 and 3, respectively). Significant differences are marked with asterisks. Bars represent standard deviations.

and Hadley 1990). The concentrations found in Atlantic salmon plasma were 105 to 106 M. Administration of MDA in the drinking water of mice lead
to dose dependent appearance of neoplastic lesions in
the liver, and mortality at the highest supplementation
levels, but the doses given were quite high compared
to a normal human diet (Draper and Hadley 1990).

Libondi et al. (1994) showed that -crystallin of the

bovine lens was modified and crosslinked after incubation with 102 M MDA and proposed that increased
lipid oxidation and formation of MDA could lead to
cataracts, a significant problem in the fish farming
industry.

218
The accumulated evidence indicates that animals
are protected against absorption of lipid hydroperoxides, firstly by avoidance due to unpleasant flavour and
odour of oxidised lipids, secondly by the decomposition of lipid hydroperoxides in the stomach, thirdly by
limited absorption of hydroperoxides from the intestine and lastly by the GI-GPX in the mucosa. Based
on our data we conclude that Atlantic salmon has a low
ability to discriminate by taste against diets with low
and intermediate levels of oxidised fat. Further, we
suggest that Atlantic salmon has the same protection
mechanisms, to prevent uptake of lipid hydroperoxides as those found in higher vertebrates. This as long
as the dose is not too high or the antioxidant defence,
here represented by GPX and GSH, is not compromised. We also conclude that an increased dietary
level of aldehydes will result in elevated blood and tissue levels of the same compounds increasing the risk
of pathological changes.
Oxidised lipids may also compromise the antioxidant defence by reducing tissue levels of antioxidant
substances, such as vitamin E (Hung et al. 1981; Liu
and Hung 1995, 1996; Baker and Davis 1997), and
thereby lead to a secondary vitamin E/antioxidant deficiency and subsequently increased in vivo oxidative
stress (Hamre et al. 1994). The possible impact of
oxidation products on fish health and the low discrimination by Atlantic salmon against oxidised feeds,
underline the importance that care should be taken to
minimise oxidation products in fish feeds.

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