Biochimica et Biophysica Acta 1426 (1999) 119^125
E¡ect of astaxanthin rich red yeast (Pha¤a rhodozyma) on oxidative
stress in rainbow trout
Toshiki Nakano *, Tomoaki Kanmuri 1 , Minoru Sato, Masaaki Takeuchi
Marine Biochemistry Laboratory, Faculty of Agriculture, Tohoku University, Aoba-ku, Sendai 981-8555, Japan
Received 9 July 1998; received in revised form 22 October 1998; accepted 22 October 1998
Abstract
The antioxidative biological effect of dietary red yeast, Phaffia rhodozyma, which is rich in astaxanthin, on rainbow trout,
Oncorhynchus mykiss, was examined. The levels of serum transaminase (glutamic-pyruvic transaminase and glutamicoxaloacetic transaminase) activities and of lipid peroxides (LPO) of fish fed oxidized oil were significantly higher than those
of the control fish fed non-oxidized oil. However, the supply of red yeast considerably decreased both enzyme activities and
LPO level. Furthermore, the serum lipid (triglycerides, total cholesterol and phospholipids) concentrations were also
significantly decreased. Especially, the serum triglyceride level of fish fed the red yeast was as low as that of the control. It
was also observed that there were no significant differences in muscle LPO levels between the fish fed red yeast and the
control. The present results suggest for the first time that dietary red yeast may effectively suppress the LPO generation of
tissue and normalize liver function as well as improving muscle pigmentation of trout. Thus, red yeast should have a reducing
effect on oxidized oil-induced oxidative stress in fish. ß 1999 Elsevier Science B.V. All rights reserved.
Keywords: Oxidative stress; Astaxanthin; Lipid peroxide; Liver function; (Rainbow trout); (Pha¤a rhodozyma)
1. Introduction
It is thought that oxidative stress leads to an increased risk of developing several diseases in mammals, e.g. mutations, cancer, in£ammation, cardiovascular disease [1^6]. In particular, some of the
strong oxidants that occur in the bodies of both
mammals and ¢sh are reactive oxygen species such
as superoxide radical, singlet oxygen and lipid per-
* Corresponding author. Fax: +81 (22) 717-8739;
E-mail: [email protected]
1
Present address: Fisheries Agency, Chiyoda-ku, Tokyo 1000013, Japan.
oxides (LPO). Aerobic organisms have both enzymatic and non-enzymatic defensive systems against
oxidative stress [3^6]. In both mammals and ¢sh,
insu¤cient ingestion of nutritional antioxidants has
been linked to a decrease in the ability to defend
against and increased susceptibility to oxidative
stress and some diseases [1^5].
It is known that aquaculture feeds contain a large
amount of highly unsaturated fatty acids (HUFA)
which are easily oxidized. The oxidized HUFA might
lead to oxidative stress in ¢sh. Some ¢sh diseases,
such as muscular dystrophy, hemolysis and jaundice,
are thought to be catalyzed by oxidative damage to
tissue [3^5]. Furthermore, certain pollutants, such as
poly chlorinated biphenyl, paraquat (methyl viologen) and heavy metals, are known to induce lipid
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 1 4 5 - 7
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120
T. Nakano et al. / Biochimica et Biophysica Acta 1426 (1999) 119^125
peroxidation in ¢sh tissue [3^5,7]. In the course of
studies on the antioxidant defensive abilities of ¢sh
[8^13], the defensive abilities of cultured ¢sh have
been found insu¤cient and increasing defensive abilities of ¢sh is very important.
The antioxidative activity of astaxanthin (ASX)
against certain kinds of reactive oxygen under several
experimental conditions has been observed to be
stronger than that of K-tocopherol [14]. It is reported
that carotenoids and K-tocopherol show a synergistic
antioxidative action [15]. In vivo, both ASX and Ktocopherol are thought to be located in membranes
which contain a large amount of HUFA [16,17].
Carotenoids are thought to increase the membrane's
mechanical strength [16]. Moreover, at low oxygen
pressure in most tissues under physiological conditions, some investigators report that certain kinds
of carotenoids exhibit potent radical trapping activity [18]. Some kinds of carotenoids show positive
biological activities in mammals without exhibiting
any cytotoxicity [14,19^21]. Therefore, the carotenoids appear to have special properties that no
other antioxidants show [22]. In ¢sh, several experiments have been done to improve their defensive to
oxidants by administering an antioxidant such as
K-tocopherol [3^5,23,24]. Recently, it was observed
that the dietary red yeast, Pha¤a rhodozyma, which
is rich in ASX, and synthetic ASX decrease the LPO
level and transaminase activities in the serum of
healthy rainbow trout Oncorhynchus mykiss [25,26].
The ¢sh liver is rich in glutamic-pyrubic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT). Because the activities of serum
non-speci¢c enzymes, such as GPT and GOT, should
be related to cell damage in speci¢c tissue, the activities of transaminase in ¢sh serum are very useful as
an index for the diagnosis of liver function [27].
Therefore, it was suggested that the red yeast and
synthetic ASX seemed to protect the liver from the
cell damage. Unfortunately, most of the research on
dietary ASX (including the red yeast) has been done
in terms of muscle pigmentation [28,29], and only a
few studies have reported the biological e¡ects of
ASX in ¢sh [30,31].
In the present study, we examined the e¡ect of red
yeast administration on oxidative stress in trout. We
also discussed the relationship between ASX, oxidative stress and the health of ¢sh.
2. Materials and methods
2.1. Experimental design, animals, rearing system and
diets
Unpigmented rainbow trout O. mykiss (0-yr old,
approximate body weights: 15 g) were obtained from
a local hatchery, the Tanii Trout Farm in Zao town,
Miyagi, Japan. During 4 months of acclimatization
prior to commencement of the feeding trials, the ¢sh
were kept in 103 l £ow-through ¢berglass tanks and
fed commercial trout pellets free from ASX. The
commercial trout pellets, Seseragi #3, the main ingredient of which is sardine meal, were purchased
from Nippon Formula Feed MFG, Yokohama, Japan. After the acclimatization, the ¢sh were individually weighed and divided into three groups of 10
healthy ¢sh (approximate body weight: 60 g) each.
They were then reared in 60 l £ow-through glass
tanks over a 2-month period at 16³C. The ¢sh were
fed to satiation two times daily. The composition of
the experimental diet is presented in Table 1. The
oxidized oil was prepared by vigorous bubbling air
through pollack liver oil at 40³C for 120 h. The peroxide value (POV) of the oxidized oil was titrimetrically measured according to the o¤cial method of
the Japan Oil Chemical Society [32] and was found
to be 500 mEq/kg. The concentration of ASX in the
diet was adjusted to 50 mg/kg [28]. The red yeast, P.
rhodozyma, and synthetic ASX were kindly provided
by KI Chemical, Shizuoka, Japan. The red yeast was
harvested and thoroughly washed with distilled
water. The red yeast cells were then treated with
NaOH solution, followed by neutralizing, freeze-drying and milling. As a result, the yeast cell walls were
found to be signi¢cantly destroyed. The ASX content
of the freeze-dried yeast was 4.9 mg/g of yeast. The
freeze-dried yeast preparation was thoroughly mixed
with the dry ingredients before pelleting. All the diets
were newly prepared every 3 weeks and stored in a
dark vacuum at 330³C in small aliquots to avoid
deterioration before use.
2.2. Collection of blood and muscle
At the end of the feeding trial, the ¢sh were fasted
for 1 day, then weighed and blood was individually
collected from the caudal vessels under MS222
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121
Table 1
Composition of experimental diets
Ingredient
Diet
White ¢sh meal (g/kg)
K-Starch (g/kg)
Pollack liver oil (non-oxidized) (g/kg)
Oxidized pollack liver oila (g/kg)
Mineral mixtureb (g/kg)
Vitamin mixturec (g/kg)
Red yeastd (g/kg)
Cellulose (g/kg)
1
2
3
600
200
100
0
40
10.05
0
49.95
600
200
0
100
40
10.05
0
49.95
600
200
0
100
40
10.05
10.87
39.08
a
Oxidized pollack liver oil was prepared by bubbling air through the oil at 40³C for 120 h.
Minerals added at the following levels (g/kg of diet): Ca lactate, 13.08; KH2 PO4 , 9.59; MgSO4 W7aq, 5.48; Ca(H2 PO4 )aq, 5.43;
NaH2 PO4 W2aq, 3.49; NaCl, 1.47; Fe citrate, 1.19; ZnSO4 , 0.12; MnSO4 , 0.03; CoCl2 , 0.03; AlCl3 , 0.006; KI, 0.006; CuCl2 , 0.004.
c
Vitamins added at the following levels (g/kg of diet): vitamin B1 , 0.055; vitamin B2 , 0.2; vitamin B6 , 0.05; vitamin B12 , 0.0001; nicotinic acid, 0.75; d-Ca pantothenate, 0.5; inositol, 2; biotin, 0.005; folic acid, 0.015; ascorbic acid, 1; vitamin K3 , 0.04; K-tocopherol,
0.1; choline chloride, 5; vitamin A, 20 000 IU; vitamin D3 , 4000 IU.
d
Pha¤a rhodozyma, see text for details.
b
(m-aminobenzoic acid ethyl ester methanesulfonate)
anesthesia. Serum from the blood was placed in ice
water and was quickly used for the determination.
The ¢sh were then gutted and skinned. The muscle
was collected and quickly frozen at 380³C for later
analysis.
2.3. Determination of serum enzyme activity, LPO
level and lipid contents
Serum enzyme activities, GPT (EC 2.6.1.2) and
GOT (EC 2.6.1.1), were spectrophotometrically determined using Determiner GPT and GOT kits and
were expressed in Karmen units. The serum LPO
level was enzymatically measured by the Determiner
LPO kit using cumen hydroperoxide as the standard
and was expressed in nmol of lipid peroxide per dl of
serum. Serum lipid contents (triglycerides, TG; total
cholesterol, TC; phospholipids, PL) were enzymatically estimated using Determiner TG, TC and PL
kits and were expressed in mg of lipid per dl of
serum. All assays were carried out on six or seven
samples according to instructions (Kyowa Medecs,
Tokyo, Japan).
2.4. Determination of muscle LPO and ASX
Muscle LPO was measured in 10% homogenates
Table 2
Growth performance of rainbow trout fed diet containing red yeasta
Parameter
Initial weight (g)
Final weight (g)c
Weight gain (%)
DFI (%)d
FERe
Diet
b
1
2
3
59.5 þ 6.8
134.3 þ 44.0
125.8
1.35
0.86
60.2 þ 6.1
133.8 þ 18.0
122.3
1.56
0.82
61.9 þ 6.3
147.8 þ 17.5
138.7
1.56
1.07
a
Values are mean þ S.D. (n = 10).
Statistical signi¢cance was not detected.
d
Daily feed intake = (100Ufeed intake/total days/[(initial weight+¢nal weight)/2]).
e
Feed e¤ciency ratio = (wet weight gain/dry matter intake).
b;c
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T. Nakano et al. / Biochimica et Biophysica Acta 1426 (1999) 119^125
Fig. 1. E¡ect of red yeast on serum glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) activities of rainbow trout fed oxidized oil. The results are expressed
in Karmen units. Data points represent mean þ S.D. (n = 6).
There are signi¢cant di¡erences (P 6 0.05) among di¡erent letters. (1) Control; (2) oxidized oil; (3) oxidized oil+red yeast.
of muscle (1 g wet weight of muscle+9 ml of 1.15%
KCl) using the method of Uchiyama and Mihara [33]
and was expressed as the amount of total thiobarbituric acid reactive substances (TBARS). The ASX
content of muscle was determined according to the
method described by Tsukuda [34]. LPO and ASX
assays were carried out on seven and ¢ve samples,
respectively, and their mean values were expressed as
nmol of malondialdehyde (MDA) per g of tissue and
mg of ASX per kg of tissue.
2.5. Statistical analysis
All data were subjected to a one-way analysis of
variance (ANOVA). Signi¢cant di¡erences between
means were ranked using Fisher's least signi¢cant
di¡erence (lsd) test at the 5% level [35].
Fig. 2. E¡ect of red yeast on serum lipid peroxides level of
rainbow trout fed oxidized oil. The results are expressed in
nmol of lipid peroxide per dl of serum using cumen hydroperoxide as the standard. Data points represent mean þ S.D.
(n = 6). There are signi¢cant di¡erences (P 6 0.05) among di¡erent letters. (1) Control ; (2) oxidized oil; (3) oxidized oil+red
yeast.
3. Results
3.1. Growth performance
There were no mortalities during the feeding trial.
At the end of the feeding trial, there were no signi¢cant di¡erences among the groups (diets 1^3) in the
following items (Table 2): (1) average body weight;
(2) weight gain; (3) daily feed intake (DFI)
(= 100Ufeed intake/total days/[(initial body weight+
¢nal body weight)/2]); and (4) feed e¤ciency ratio
(FER) (= wet weight gain/dry matter intake).
3.1. Enzyme activity, LPO level and lipid contents of
serum
The activities of GPT and GOT in the serum from
Fig. 3. E¡ect of red yeast on serum lipid levels (TG, triglycerides; TC, total cholesterol; PL, phospholipids) of rainbow trout fed oxidized oil. The results are expressed as mg of lipid per dl of serum. Data points represent mean þ S.D. (n = 6). There are signi¢cant differences (P 6 0.05) among di¡erent letters. (1) Control; (2) oxidized oil; (3) oxidized oil+red yeast.
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T. Nakano et al. / Biochimica et Biophysica Acta 1426 (1999) 119^125
123
yeast (diet 3) signi¢cantly decreased the LPO level
and there were no signi¢cant di¡erences in the
LPO levels between the red yeast-fed group and the
control.
4. Discussion
Fig. 4. E¡ect of red yeast on muscle lipid peroxides level of
rainbow trout fed oxidized oil. The results are expressed as the
amount of total thiobarbituric acid reactive substances
(TBARS) per g of tissue. Data points represent mean þ S.D.
(n = 7). There are signi¢cant di¡erences (P 6 0.05) among di¡erent letters. (1) Control; (2) oxidized oil; (3) oxidized oil+red
yeast.
the three groups (diets 1^3) of ¢sh are shown in Fig.
1. Oxidized oil (diet 2)-fed ¢sh showed signi¢cantly
higher activities of both GPT and GOT than those in
the control (diet 1). However, the red yeast (diet 3)
signi¢cantly decreased both activities, and there were
no signi¢cant di¡erences in those enzyme activities
between the red yeast-fed group and the control.
As shown in Fig. 2, while the LPO level of diet 2
was increased 6-fold over that of the control, the red
yeast (diet 3) signi¢cantly decreased the LPO. The
group fed the red yeast exhibited no signi¢cant differences in LPO level compared to the control.
The levels of TG, TC and PL in the serum of ¢sh
fed diets 1^3 were compared (Fig. 3). The group fed
diet 2 had signi¢cantly higher TG, TC and PL levels
than the control. On the other hand, surprisingly, the
red yeast group (diet 3) had signi¢cantly decreased
TG, TC and PL levels. In particular, the level of TG
of the ¢sh fed the red yeast was found to be as low as
that of the control.
3.2. ASX and LPO levels of muscle
The mean amounts of ASX in ¢sh fed diets 1, 2
and 3 were 0.134, 0.097 and 1.102 mg/kg of muscle,
respectively. There were signi¢cant di¡erences in the
mean ASX levels of muscle: diet 3 s diet 1 = diet 2.
The LPO level in muscle for the three dietary
groups is shown in Fig. 4. The muscle from the
group fed diet 2 was observed to have a signi¢cantly
higher level of LPO than that of the control. The red
Oxidized oil contains LPO and secondary products
such as aldehydes, ketones, alcohol and epoxides
[36,37]. LPO is further decomposed into a variety
of oxygen-containing radicals. The resulting radicals
attack almost all cell components, such as proteins,
lipids, nucleic acids and membranes, initiate free radical chain reactions, and induce oxidative stress
[6,37,38]. The serum LPO level is considered to be
one of the sensitive indicators of tissue damage derived from oxidative stress [39]. In the present study,
the serum LPO level of ¢sh fed oxidized oil (diet 2)
was observed to be signi¢cantly higher than that of
the control (diet 1) (Fig. 2). Hence, the present result
suggested that lipid peroxidation in trout tissue was
accelerated by oxidized oil administration. On the
other hand, the red yeast was shown to e¡ectively
inhibit the accumulation of LPO in serum (diet 3)
(Fig. 2). In mammalian serum, most LPO are reported to exist in lipoprotein [39]. Both mammalian
and ¢sh lipoproteins contain triglycerides, cholesterol
and phospholipids which are high susceptibility to
oxidation [40,41]. The oxidative modi¢cation of lipoproteins causes signi¢cant damage to the endothelial
cells of blood vessels, followed by disturbance of
the blood cycle system [42,43]. Thus, serum LPO is
considered to be a trigger, which causes several serious diseases. Dietary carotenoids and K-tocopherol
are found to exist in the lipoproteins of both mammalian and ¢sh serums [44^46]. Recently, K-tocopherol concentration in the serum of trout fed red
yeast is observed to increase [47]. In ¢sh serum, there
are many kinds of circulating small molecule antioxidants (e.g. ascorbic acid and bilirubin) [48,49].
Hence, dietary ASX and K-tocopherol might act synergistically with other circulating antioxidants to decrease the susceptibility of lipoproteins to oxidation
and normalize the blood cycle system in ¢sh. LPO
production in muscle was also observed to be signi¢cantly decreased after the red yeast administration
(Fig. 4). The muscle of ¢sh fed red yeast accumulated
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T. Nakano et al. / Biochimica et Biophysica Acta 1426 (1999) 119^125
a signi¢cant amount of ASX. The level of muscle Ktocopherol is reported to be increased by dietary
synthetic ASX in both Atlantic salmon and trout
[47,50]. Thus, similar to serum, membrane-bound
antioxidants, ASX and K-tocopherol, might protect
the membranal lipids and proteins from oxidation
and, consequently maintaining both membrane dynamics and function. On the other hand, there are
some reports that describe that dietary carotenoids,
e.g. L-carotene or canthaxanthin, are not able to decrease the plasma LPO level in mice and chicks
[51,52]. However, in these cases, the administration
levels of carotenoids seem to be very high compared
to the present study. An excessive dose of antioxidants such as K-tocopherol is observed to act as a
pro-oxidant and to promote lipid peroxidation in
both body and the model system [46,53].
The highly toxic secondary products in oxidized oil
are known to accumulate and to cause damage in the
liver [37,54]. In the present study, both the GPT and
GOT activities in the serum of ¢sh fed oxidized oil
(diet 2) increased. Accordingly, some damage appeared to happen in the liver of ¢sh fed oxidized
oil. On the other hand, these transaminase activity
levels were signi¢cantly decreased by the red yeast
administration (Fig. 1). The dietary ASX shows an
improvement in the histology of the ¢sh liver structure [55]. In addition, ASX is reported to inhibit
reactive oxygen-induced cell death in cultured trout
¢broblasts [56]. As a result, the liver of ¢sh fed oxidized oil might be protected by the red yeast administration against damage and toxicity derived from
oxidized oil. The present observation is similar to
the report of rats administered L-carotene [57].
The concentration of serum lipid is known to be
often in£uenced by both the physiological state and
environmental conditions, such as age, diet, temperature and so on [58^60]. In particular, orally ingested
substance seem to be one of most important in£uential factors. Because in the liver almost all nutrients
and xenobiotics are taken in and metabolized, the
liver can generally be called the main organ for lipid
metabolism [58]. In the present study, the lipid concentration of oxidized oil (diet 2)-fed ¢sh serum was
observed to increase so much that malfunction in the
lipid metabolism of ¢sh was possible. The red yeast
could signi¢cantly decrease the lipid concentration in
serum (Fig. 3). Accordingly, the serum lipid concen-
tration-lowering e¡ect of red yeast is attributed to its
antioxidative action which normalizes the dysfunction of the lipid metabolism in the liver.
In summary, the present results suggest for the
¢rst time that red yeast reduces the oxidative stress
in trout dramatically. Salmonids are known to be
unable to synthesize carotenoids de novo, so they
depend on the accumulation of pigments from their
diet [28]. Many sources of dietary carotenoids have
been used for the coloration of cultured ¢sh
[28,29,61]. Red yeast is one such dietary carotenoid
source and, therefore, our ¢ndings would provide
novel signi¢cance to the use of red yeast in aquaculture.
The main factor for the antioxidative biological
activity of the red yeast may be ASX. The red yeast
appears to contain unknown substances, which a¡ect
trout, other than ASX [25,26]. Further investigation
(which is now in progress) is required to reveal the
properties of such unknown substances.
Acknowledgements
We are grateful to Mr. T. Miwa, KI Chemical,
Japan, for providing the red yeast. We thank Dr.
M. Hata, Dr. T. Yamaguchi and Mr. T. Kawasaki,
Tohoku University, Japan, and Dr. M. Tokuda, National Research Institute of Aquaculture, Japan, for
valuable suggestions and technical assistants. We
also acknowledge Mr. Walter Wyman, School Board
of Naruko town, Miyagi, Japan, for reading the
manuscript. This study was supported in part by a
Grant-in-Aid for Scienti¢c Research from the Ministry of Education, Science, Sports, and Culture of
Japan for T.N.
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