Antioxidant effect of astaxanthin on phospholipid peroxidation in human erythrocytes

Abstract

Phospholipid hydroperoxides (PLOOH) accumulate abnormally in the erythrocytes of dementia patients, and dietary xanthophylls (polar carotenoids such as astaxanthin) are hypothesised to prevent the accumulation. In the present study, we conducted a randomised, double-blind, placebo-controlled human trial to assess the efficacy of 12-week astaxanthin supplementation (6 or 12 mg/d) on both astaxanthin and PLOOH levels in the erythrocytes of thirty middle-aged and senior subjects. After 12 weeks of treatment, erythrocyte astaxanthin concentrations were higher in both the 6 and 12 mg astaxanthin groups than in the placebo group. In contrast, erythrocyte PLOOH concentrations were lower in the astaxanthin groups than in the placebo group. In the plasma, somewhat lower PLOOH levels were found after astaxanthin treatment. These results suggest that astaxanthin supplementation results in improved erythrocyte antioxidant status and decreased PLOOH levels, which may contribute to the prevention of dementia.

Full text

Antioxidant effect of astaxanthin on phospholipid peroxidation
in human erythrocytes
Kiyotaka Nakagawa
1
*, Takehiro Kiko
1
, Taiki Miyazawa
1
, Gregor Carpentero Burdeos
1
,
Fumiko Kimura
1
, Akira Satoh
2
and Teruo Miyazawa
1
1
Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University,
Sendai 981-8555, Japan
2
Life Science Institute, Yamaha Motor Company Limited, Shizuoka 437-0061, Japan
(Received 19 July 2010 – Revised 25 November 2010 – Accepted 26 November 2010 – First published online 31 January 2011)
Abstract
Phospholipid hydroperoxides (PLOOH) accumulate abnormally in the erythrocytes of dementia patients, and dietary xanthophylls
(polar carotenoids such as astaxanthin) are hypothesised to prevent the accumulation. In the present study, we conducted a randomised,
double-blind, placebo-controlled human trial to assess the efficacy of 12-week astaxanthin supplementation (6 or 12 mg/d) on both astax-
anthin and PLOOH levels in the erythrocytes of thirty middle-aged and senior subjects. After 12 weeks of treatment, erythrocyte astaxanthin
concentrations were higher in both the 6 and 12 mg astaxanthin groups than in the placebo group. In contrast, erythrocyte PLOOH
concentrations were lower in the astaxanthin groups than in the placebo group. In the plasma, somewhat lower PLOOH levels were
found after astaxanthin treatment. These results suggest that astaxanthin supplementation results in improved erythrocyte antioxidant
status and decreased PLOOH levels, which may contribute to the prevention of dementia.
Key words: Astaxanthin: Phospholipid hydroperoxides: Erythrocytes: Dementia
We have previously confirmed that higher levels of
phospholipid hydroperoxides (PLOOH), the primary
oxidation products of phospholipids (PL)
(1,2)
, accumulate
abnormally in the erythrocytes of dementia patients
(3)
. Such
erythrocytes with high levels of lipid hydroperoxides have
been postulated to have a decreased ability to transport
oxygen to the brain, which may impair blood rheology,
thus facilitating dementia
(4 – 8)
. Recently, we have developed
an HPLC method to determine erythrocyte carotenoid
content
(9)
. Using this method, we gathered evidence that
accumulation of polar oxygenated carotenoids (xanthophylls)
occurs predominantly in human erythrocytes
(9)
, and that a
decrease in xanthophylls and an increase in PLOOH levels in
erythrocytes correlate with the severity of dementia
(10)
. These
findings led to the hypothesis that xanthophyll supplementation
may minimise the accumulation of erythrocyte PLOOH, and that
xanthophylls could be used therapeutically as drugs or func-
tional foods to prevent the disease. Although there is still
scarce information on whether orally administered xantho-
phylls are distributed to human erythrocytes and actually inhibit
erythrocyte PLOOH formation, our recent human study has
revealed antioxidant properties of the xanthophyll lutein
towards erythrocyte PLOOH formation
(11)
. Animal studies
have also supported this hypothesis
(12,13)
.
Among xanthophylls, astaxanthin has recently received
attention for its potent antioxidant activity
(14,15)
. Astaxanthin
is naturally synthesised by plants and algae, and is now com-
mercially available as a food supplement from Haematococcus
alga
(16)
. The recommended daily intake is estimated to be
1–12 mg/d; however, there is not much information regarding
the bioavailability of astaxanthin in humans. To the best of
our knowledge, the occurrence and antioxidant roles of astax-
anthin in human erythrocytes have not been reported.
In this investigation of whether administered astaxanthin is
distributed to erythrocytes and inhibits erythrocyte PLOOH
formation, we conducted a randomised, double-blind, pla-
cebo-controlled human trial. The efficacy of 12-week astax-
anthin supplementation (6 or 12 mg) on both astaxanthin
and PLOOH levels in the erythrocytes of thirty middle-aged
and senior subjects was investigated. For erythrocyte astax-
anthin analysis, a newly developed HPLC coupled with
tandem MS (MS/MS) method was applied. Our findings (the
inhibitory effect of astaxanthin on erythrocyte PLOOH)
* Corresponding author: K. Nakagawa, fax þ 81 22 717 8905, email nkgw@biochem.tohoku.ac.jp
Abbreviations: CL, chemiluminescence; MRM, multiple reaction monitoring; PCOOH, phosphatidylcholine hydroperoxide; PEOOH,
phosphatidylethanolamine hydroperoxide; PL, phospholipid; PLOOH, phospholipid hydroperoxide.
British Journal of Nutrition (2011), 105, 1563–1571 doi:10.1017/S0007114510005398
q The Authors 2011
British Journal of Nutrition

would provide new insights into the possible application of
astaxanthin as an anti-dementia agent.
Subjects and methods
Subjects and materials
The present study was conducted according to the
guidelines laid down in the Declaration of Helsinki, and all
procedures involving human subjects were approved by the
ethics committee of Anti-Aging Science (Tokyo, Japan; ethics
no. I030807). All subjects were recruited from the Anti-Aging
Science volunteer database, and written informed consent
was obtained from all subjects. Exclusion criteria included
pregnancy, lactation and severe medical illness.
Two doses of the test materials were prepared by filling soft
capsules with astaxanthin-rich Haematococcus pluvialis oil
(Puresta
w
; Yamaha Motor Company, Limited, Shizuoka,
Japan)
(17)
. Compositions of the test materials were 75 mg Pure-
sta oil 80 and 145 mg olive oil/capsule for the low dose (equiv-
alent to 6 mg astaxanthin dialcohol); and 150 mg Puresta oil 80
and 70 mg olive oil/capsule for the high dose (equivalent to
12 mg astaxanthin dialcohol). To prepare placebo capsules,
150 mg maize oil was used instead of 150 mg Puresta oil 80.
Placebo capsules were coloured to appear identical to test
capsules.
Supplementation trial
A 12-week randomised, double-blind, placebo-controlled trial
was conducted. A total of thirty healthy subjects (fifteen men
and fifteen women), between 50 and 69 years of age (mean
56·3 (
SD 5·3) years), with a BMI of 27·5 (SD 2·1) kg/m
2
were
recruited, and randomly received 0 (placebo), 6 or 12 mg
astaxanthin. During the 12-week trial, subjects ingested one
of the three astaxanthin-dosed (0, 6 or 12 mg) capsules with
an appropriate amount of water once daily after breakfast.
Before and after the supplementation period (weeks 0 and
12, respectively), anthropometric data (e.g. height, body
weight and blood pressure) and blood samples were collected
from the subjects after they had fasted overnight, adverse
effects were assessed by interviews and self-reports, and com-
pliance was checked by self-reports and returned capsule
counts. Throughout the study period, subjects were instructed
to maintain their usual lifestyle (avoid excessive eating and
drinking, intense exercise and lack of sleep). Dietary intake,
alcohol consumption and physical activity (pedometer
count) during the 3 d before each blood collection (weeks 0
and 12) were also assessed by self-reports.
Measurement of erythrocyte astaxanthin and other
carotenoids
Blood samples were subjected to centrifugation at 1000 g
for 10 min at 48C. After the plasma and buffy coat were removed,
erythrocytes were washed three times with PBS (pH 7·4) to pre-
pare packed cells. For the determination of erythrocyte caroten-
oids (including astaxanthin), packed cells (2·5 ml) were diluted
with 2·5 ml of water and were mixed with 5 ml of 80 m
M-
ethanolic pyrogallol, 1·0 ml of 1·8
M-aqueous KOH and 40 ml
of 1 m
M-ethanolic echinenone (internal standard)
(9)
. After the
addition of 1·25 ml of 0·1
M-aqueous SDS, the sample was
mixed with 15 ml of hexane–dichloromethane (5:1) to extract
erythrocyte carotenoids. The extract was purified by a Sep-
Pak silica cartridge (Waters, Milford, MA, USA), and then was
subjected to HPLC-MS/MS for the determination of astaxanthin.
The HPLC-MS/MS apparatus consisted of a liquid chromato-
graph (Shimadzu, Kyoto, Japan) and a 4000 QTRAP MS/MS
instrument (Applied Biosystems, Foster City, CA, USA). The
MS/MS parameters (e.g. collision energy) were optimised with
an astaxanthin standard under positive atmospheric pressure
chemical ionisation. The standard and erythrocyte extracts
were separated with a C30 carotenoid column (4·6 £ 250 mm,
5 mm; YMC, Kyoto, Japan). The column was eluted using a
binary gradient consisting of the following HPLC solvents: A,
methanol–methyl tert-butyl ether– water (83:15:2; containing
3·9
M-ammonium acetate); B, methanol–methyl tert-butyl
ether–water (8:90:2; containing 2·6
M-ammonium acetate).
The gradient profile was as follows: 0 –12 min, 10–45 % B
linear; 12–24 min, 45–100 % B linear; 24– 38 min, 100 % B. The
flow rate was adjusted to 1 ml/min, and the column temperature
was maintained at 258C. Astaxanthin was detected in the post-
column by MS/MS with multiple reaction monitoring (MRM)
for the transition of the parent ion to the product ion. The
concentration of erythrocyte astaxanthin was calculated
using the standard curve of astaxanthin and was adjusted by
the percentage recovery of the added echinenone (internal
standard). For the determination of other carotenoids,
erythrocyte extracts were analysed by HPLC coupled with
UV diode array detection and atmospheric pressure chemical
ionisation MS
(9)
.
Measurement of erythrocyte phospholipid hydroperoxides
For the determination of erythrocyte PLOOH
(1 –3)
, total lipids
were extracted from packed cells with a mixture of 2-propanol
and chloroform containing butylated hydroxytoluene. PLOOH
(i.e. phosphatidylcholine hydroperoxide (PCOOH) and phos-
phatidylethanolamine hydroperoxide (PEOOH)) in the total
lipids were measured by HPLC with chemiluminescence
(CL) detection. The column was a 4·6 £ 250 mm, 5 mm Fine-
pak SIL NH2-5 (Japan Spectroscopic Company, Tokyo,
Japan), the eluent was 2-propanol–methanol–water
(135:45:20), and the flow rate was 1 ml/min. Post-column CL
detection was carried out using a CLD-100 detector (Tohoku
Electronic Industries Company, Sendai, Japan). A mixture of
luminol and cytochrome c in 50 m
M-borate buffer (pH 10·0)
was used as a hydroperoxide-specific post-column CL reagent.
Calibration was carried out using PCOOH or PEOOH
standards.
Other biochemical measurements
For plasma samples, astaxanthin, other carotenoids and
PLOOH were determined by HPLC-MS/MS, HPLC-UV
(9)
and
HPLC-CL
(1 – 3)
, respectively. Tocopherols in erythrocytes
K. Nakagawa et al.1564
British Journal of Nutrition

and plasma were measured by HPLC with fluorescence
detection
(18)
. Also, blood chemistries such as haematological
and blood biochemical parameters were analysed using
standardised methods.
Statistical analysis
Data are expressed as means and standard deviations.
One-way ANOVA was used to compare means among
the three groups. If a statistically significant difference
(P, 0·05) was detected, Dunnett’s test was performed for com-
parison between the control group and one of the two astax-
anthin groups. For comparison of baseline (week 0) and
post-dosing (week 12) values in each treatment group, a
paired t test was used. These statistical analyses were done
using SPSS for Windows (SPSS Inc., Chicago, IL, USA).
Results
As mentioned in the introduction, the distribution of astax-
anthin in erythrocytes has not been reported, mainly due
to the lack of an analytical method. We, therefore, developed
an HPLC-MS/MS method to analyse for erythrocyte astax-
anthin before we conducted the 12-week astaxanthin sup-
plementation study. In brief, an astaxanthin standard was
analysed by MS/MS with flow injection, and astaxanthin
showed an intense molecular ion at m/z 597 (M þ H)
þ
.
Product ion scanning was conducted for the ion and astax-
anthin-specific fragment ions (e.g. m/z 147) were identified.
These ions (m/z 597 and 147) allowed us to quantitatively
determine erythrocyte astaxanthin concentrations using
HPLC-MS/MS with MRM (Fig. 1(a)).
In the human trial, before administration (baseline),
there were no significant differences in anthropometric para-
meters among the three groups (Table 1). The trial was
completed without any of the subjects withdrawing. The
mean compliance to the prescribed dose for the 6 mg astax-
anthin, 12 mg astaxanthin and placebo groups was 99·5
(
SD 3·7), 98·1 (SD 1·9) and 98·7 (SD 2·4) %, respectively. The
average energy intake during the 12-week trial, as calculated
from the self-reports, did not statistically differ among the
groups. Furthermore, no significant differences were observed
among the groups in the intake of each type of nutrient
(carbohydrate, protein, fat, cholesterol and fibre), alcohol
consumption and pedometer counts. Data from all thirty
subjects were, therefore, included in the statistical analysis.
The results of the physical, haematological and blood bio-
chemical measurements before and after 12 weeks of dosing
are shown in Tables 2 and 3. Some parameters (i.e. Hb, hae-
matocrit, mean corpuscular volume, mean corpuscular Hb,
uric acid, total cholesterol, LDL, fasting glucose and Hb
A1c
)
showed changes from baseline in the astaxanthin groups
(a)
(b)
100
40
Time (min)
0
0
10
Relative intensity (%)
3020
1
10 20 30
Time (min)
0
2
3
100
Relative intensity (%)
0
Fig. 1. Typical multiple reaction monitoring (MRM) and chemiluminescence (CL) chromatograms of (a) astaxanthin and (b) phospholipid hydroperoxides (PLOOH)
in erythrocytes taken (
) before and ( ) after 12-week supplementation of astaxanthin (12 mg/d). Erythrocyte astaxanthin and PLOOH were determined
by HPLC-MS/MS with MRM and HPLC-CL, respectively. Peak identifications are as follows: 1, astaxanthin; 2, phosphatidylcholine hydroperoxide; 3, phosphatidy-
lethanolamine hydroperoxide.
Table 1. Baseline characteristics of the study subjects in the 0 (placebo), 6 and 12 mg astaxanthin groups
(Mean values and standard deviations)
0 mg 6 mg 12 mg
Background factors Mean
SD Mean SD Mean SD P *
Age (years) 56·6 4·4 56·3 6·6 56·1 5·1 0·979
Total number of subjects 10 10 10
Men 5 5 5
Women 5 5 5
Height (cm) 159 11 160 8 164 7 0·407
Weight (kg) 70·3 9·3 70·5 8·1 74·4 5·3 0·429
BMI (kg/m
2
) 27·7 2·1 27·4 2·2 27·6 2·1 0·946
Systolic blood pressure (mmHg) 133 92 124 16 134 18 0·371
Diastolic blood pressure (mmHg) 83·1 10·9 82·3 9·8 90·7 15·0 0·250
* One-way ANOVA test among groups.
Astaxanthin inhibits lipid peroxidation 1565
British Journal of Nutrition

and/or the placebo group. However, these changes were
small and were observed to be within the normal range irre-
spective of the test materials administered. Moreover, no
differences were noted among the three groups in any par-
ameters at baseline or post-dosing. Therefore, the physical
and metabolic states of the subjects were considered to be
randomised homogeneously throughout the trial.
In the typical MRM chromatogram of the erythrocyte extract
taken before and 12 weeks after supplementation, astaxanthin
was clearly detected (Fig. 1(a)). After supplementation, the
erythrocyte astaxanthin concentration significantly increased
and was higher than that of the placebo group (Table 4).
On the other hand, in a typical CL chromatogram of erythro-
cyte total lipids, PCOOH and PEOOH were identified as the
predominant PLOOH forms (Fig. 1(b)). After supplementation,
erythrocyte PLOOH concentration decreased and was lower
than that of the placebo group (Table 4). In plasma, the
only detectable PLOOH was PCOOH, and a somewhat
lower PCOOH level was found after astaxanthin supplemen-
tation (Table 5). In both erythrocytes and plasma, astaxanthin
supplementation did not affect the levels of other carotenoids
(except for small changes in lycopene and lutein) and
tocopherols. These results suggest that upon ingestion of
astaxanthin, it is absorbed, distributed and accumulated in
erythrocytes, where it acts as an antioxidant molecule, thereby
reducing PLOOH, an index of oxidative stress.
Discussion
In recent years, medical and nutritional experts have seriously
considered the antioxidant properties of food constituents,
since the reactive oxygen species-mediated peroxidation of
Table 2. Changes in physical and haematological parameters before and after the
12-week administration of 0, 6 or 12 mg astaxanthin
(Mean values and standard deviations)
0 mg 6 mg 12 mg
Parameters Mean
SD Mean SD Mean SD P †
Weight (kg)
Before 70·3 9·3 70·5 8·1 74·4 5·3 0·429
After 69·0 9·2 69·6 7·5 74·3 5·9 0·248
BMI (kg/m
2
)
Before 27·7 2·1 27·4 2·2 27·6 2·1 0·946
After 27·1 2·2 27·1 2·2 27·6 2·5 0·872
Systolic blood pressure (mmHg)
Before 133 18 124 16 134 18 0·371
After 134 16 127 17 134 16 0·555
Diastolic blood pressure (mmHg)
Before 83·1 10·9 82·3 9·8 90·7 15·0 0·250
After 81·4 9·7 84·2 9·2 86·1 9·1 0·532
Heart rate (bpm)
Before 70·5 12·5 71·8 11·2 70·9 4·0 0·956
After 68·7 11·6 71·1 7·1 75·6 6·2 0·213
Leucocytes (£ 10
9
/l)
Before 4·6 0·6 5·4 1·0 5·7 1·7 0·096
After 4·9 0·8 5·1 0·8 6·1 1·9 0·112
Erythrocytes (£ 10
12
/l)
Before 4·5 0·4 4·7 0·3 4·7 0·4 0·587
After 4·5 0·4 4·7 0·3 4·7 0·5 0·375
Hb (g/l)
Before 144 11 145 11 142 27 0·916
After 137** 11 142 13 141 23 0·799
Haematocrit (%)
Before 44·9 3·1 45·4 3·8 44·1 6·9 0·839
After 42·8** 2·9 44·1 4·1 43·9 5·2 0·750
Corpuscular volume (fl)
Before 99·1 6·3 96·0 3·3 94·8 12·9 0·522
After 96·3** 4·7 94·7** 3·3 94·0 12·1 0·783
Corpuscular Hb (pg/cell)
Before 31·7 2·1 30·8 1·1 30·5 5·3 0·735
After 30·9** 1·7 30·4** 1·2 30·3* 5·1 0·193
Corpuscular Hb concentration (%)
Before 32·0 0·8 32·1 0·8 32·0 2·1 0·983
After 32·1 0·6 32·1 0·6 32·0 2·1 0·988
Platelets (£ l0
9
/l)
Before 237 47 225 41 261 67 0·320
After 212 55 228 28 264 82 0·151
bpm, Beats/min.
Mean values were significantly different in the paired t test between before and after astaxanthin
administration: *P , 0·05, **P, 0·01.
† One-way ANOVA test among groups.
K. Nakagawa et al.1566
British Journal of Nutrition

biological molecules (e.g. lipids) has been postulated to
induce a variety of pathological events such as atherogenesis,
ageing and dementia. Although many in vitro studies on the
antioxidant properties of food constituents have been
reported, little is known about the biological functions of diet-
ary antioxidants in vivo (especially in humans), except for a
few major antioxidants (e.g. tocopherols and ascorbic acid).
Since the bioavailability of food constituents is limited by
their digestibility and metabolic fate, oral administration
trials are favoured in evaluating their biological functions.
The present randomised, double-blind, placebo-controlled
human trial shows that when human subjects ingest asta-
xanthin, it is absorbed, distributed and accumulated in
erythrocytes, where it exhibits antioxidative effects (inhibition
of erythrocyte PLOOH). It is interesting to note that the
antioxidative effect observed in the present study was
Table 3. Changes in blood biochemical parameters before and after the 12-week administration
of 0, 6 or 12 mg astaxanthin
(Mean values and standard deviations)
0 mg 6 mg 12 mg
Parameters Mean
SD Mean SD Mean SD P †
Total protein (g/l)
Before 73·0 2 ·3 74·6 2·1 71·9 3·5 0·950
After 72·6 2·1 73·9 1·8 71·5 3·8 0·162
Albumin (g/l)
Before 45·0 1 ·6 45·7 1·1 44·2 2·2 0·161
After 44·6 1·0 45·4 1·1 43·8 2·2 0·081
Total bilirubin (mg/l)
Before 8·0 2·9 7·0 2·1 7·0 2·7 0·615
After 7·4 1·9 7·2 2·9 7·3 2·9 0·986
GOT (U/l)
Before 22·6 6 ·9 25·0 9·2 23·0 8·4 0·786
After 21·0 5·6 21·3 3·9 23·6 11·0 0·700
GPT (U/l)
Before 25·7 17·6 30·6 15·2 24·1 12·9 0·620
After 21·2 13·0 24 ·2 8·0 25·0 15·6 0·779
ALP (U/l)
Before 225 67 238 55 203 72 0·489
After 225 58 236 58 197 60 0·336
g-GTP (U/l)
Before 40·8 23·6 35·1 15·2 41·3 31·5 0·820
After 37·3 19·4 30·7 11·4 41·6 38·5 0·638
Urea (mg/l)
Before 129 25 144 31 146 24 0·340
After 139 29 128 25 135 29 0·671
Creatinine (mg/l)
Before 7·0 1·5 7·3 1·4 7·8 2·1 0·543
After 7·0 1·5 7·2 1·5 8·0 1·9 0·404
Uric acid (mg/l)
Before 52·0 11·8 62·6 19·2 57·8 16·8 0·357
After 53·1 10·4 57 ·9* 16·0 56·3 13·7 0·721
Total cholesterol (mg/l)
Before 2180 440 2260 390 2030 230 0·372
After 2040* 430 2150 320 1960 150 0·401
HDL (mg/l)
Before 588 147 688 190 588 121 0·268
After 581 130 655 147 562 105 0·250
LDL (mg/l)
Before 1350 350 1320 370 1170 200 0·396
After 1220* 370 1240 290 1120 190 0·629
TAG (mg/l)
Before 1250 520 1110 780 1250 730 0·869
After 1140 410 1160 670 1360 1140 0 ·790
Fasting glucose (mg/l)
Before 1010 60 1020 110 1010 60 0·948
After 1040** 50 1040 110 1050 110 0·985
Hb
Alc
(%)
Before 5·1 0·3 5·1 0·3 5·1 0·3 0·982
After 5·2 0·3 5·2 0·2 5·2** 0·3 1·000
GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; ALP, alkaline phosphatase;
g-GTP, g-glutamic transpeptidase.
Mean values were significantly different in the paired t test between before and after astaxanthin administration:
*P, 0·05, **P, 0·01.
† One-way ANOVA test among groups.
Astaxanthin inhibits lipid peroxidation 1567
British Journal of Nutrition

produced by a relatively short-term supplementation with
astaxanthin (12 weeks).
In the present study, since the distribution of astaxanthin
in erythrocytes had not previously been reported, we
developed an HPLC-MS/MS method to analyse the erythrocyte
astaxanthin content before we conducted the astaxanthin sup-
plementation study. Using MS/MS, we found that protonated
astaxanthin tended to generate product ions (e.g. m/z 147).
Table 4. Changes in carotenoids, phospholipid hydroperoxides (PLOOH) and tocopherol contents in
erythrocytes taken before and after the 12-week administration of 0, 6 or 12 mg astaxanthin
(Mean values and standard deviations)
0 mg 6 mg 12 mg
Parameters Mean
SD Mean SD Mean SD P ‡
Carteonoids (pmol/ml packed cells)
Astaxanthin
Before 4·0 1·7 2·9 1·3 2·8 1·0 0·112
After 3·6 1·2 35·7**†† 11·1 44·9**†† 24·0 0·000
Lutein
Before 43·3 6·8 39·8 7·5 39·5 6·1 0·391
After 43·2 8·5 42·1* 8·0 40·6 7·4 0·765
Zeaxanthin
Before 7·8 2·1 7·1 1·4 7·6 2·1 0·662
After 7·8 2·6 6·9 1·2 7·4 1·8 0·603
b-Cryptoxanthin
Before 10·5 2·6 9·9 3·0 9 ·6 2·1 0·735
After 10·1 2·4 9·5 2·7 9·4 2·6 0·802
a-Carotene
Before 1·2 0·4 0·9 0·4 1·1 0·3 0·234
After 1·1 0·4 0·9 0·3 1·1 0·3 0·151
b-Carotene
Before 3·9 0·3 3·8 0·4 3·7 0·4 0·678
After 3·9 0·4 3·8 0·5 3·8 0·6 0·902
Lycopene
Before 0·7 0·1 0·6 0·1 0·7 0·2 0·231
After 0·7 0·1 0·6 0·1 0·6* 0·2 0·273
Xanthophylls§
Before 65·5 10·3 59·5 11·1 59·5 9·1 0·407
After 64·7 11·4 94·2**†† 20·9 102·4**†† 20·6 0·000
Non-polar carotenoidsk
Before 5·7 0·6 5·3 0·7 5·5 0·7 0·350
After 5·7 0·7 5·3 0·6 5·5 0·9 0·421
Total carotenoids
Before 71·3 10·6 64·8 11·6 65·1 9·8 0·397
After 70·4 11·7 99·4**†† 21·5 107·9**†† 20·5 0·000
PLOOH (pmol/ml packed cells)
PCOOH
Before 8·8 6·6 8·7 4·6 12·3 7·0 0·340
After 9·1 6·3 5·2** 2·7 6·6** 2·3 0·122
PEOOH
Before 5·1 2·4 4·6 3·6 6·3 6·5 0·706
After 5·8 2·6 2·8*† 1·8 3·0† 2·5 0·011
Total PLOOH{
Before 13·9 7·6 13·3 6·1 18·6 11·6 0·353
After 14·9 8·3 8·0**† 3·8 9·7**† 4·0 0·031
Tocopherols (nmol/ml packed cells)
a-Tocopherol
Before 7·8 2·4 7·2 2·6 6·9 1·0 0·663
After 8·4 1·9 7·8 0·9 7·6 0·8 0·365
g-Tocopherol
Before 0·9 0·4 0·8 0·5 0·9 0·3 0·872
After 0·8 0·3 0·8 0·4 0·8 0·3 0·998
Total tocopherols
Before 8·7 2·3 8·0 2·6 7·8 1·1 0·652
After 9·2 2·0 8·6 1·0 8·4 0·9 0·372
PCOOH, phosphatidylcholine hydroperoxide; PEOOH, phosphatidylethanolamine hydroperoxide.
Mean values were significantly different in the paired t test between before and after astaxanthin administration:
*P, 0·05, **P, 0·01.
Mean values were significantly different in Dunnett’s test between the control group and one of the two astaxanthin
groups: †P , 0·05, ††P, 0·01.
‡ One-way ANOVA test among groups.
§ Xanthophylls are the sum of astaxanthin, lutein, zeaxanthin and b-cryptoxanthin.
k Non-polar carotenoids are sum of a-carotene, b-carotene and lycopene.
{ PLOOH are the sum of PCOOH and PEOOH.
K. Nakagawa et al.1568
British Journal of Nutrition

The product ion indicated that MRM could be adapted to the
HPLC-MS/MS analysis of astaxanthin. Under optimised con-
ditions, the detection limit of the HPLC-MS/MS with the
MRM method was very sensitive at 0·02 pmol astaxanthin/
injection. The characteristics and advantages of our HPLC-
MS/MS method are as follows. The method was selective
and sensitive enough to measure astaxanthin in erythrocytes
(Fig. 1(a)) as well as in the plasma. Also, the method was
sufficiently simple and convenient to be applicable to a
large number of samples. The method, therefore, would be
a powerful tool for studying the metabolic fate of astaxanthin
as well as its bioavailability.
Until now, there are few reports concerning human
erythrocyte carotenoids. Some studies have successfully
detected erythrocyte carotenoids (mainly b-carotene)
(19)
,
while other studies have been unable to detect these
Table 5. Changes in carotenoids, phospholipid hydroperoxides (PLOOH) and tocopherols contents
in plasma before and after the 12-week administration of 0, 6 or 12 mg astaxanthin
(Mean values and standard deviations)
0 mg 6 mg 12 mg
Parameters Mean
SD Mean SD Mean SD P ‡
Carteonoids (pmol/ml)
Astaxanthin
Before 9 4 6 3 8 6 0·383
After 8 4 86**†† 30 109**†† 49 0·000
Lutein
Before 520 82 480 88 473 75 0·398
After 529 104 489 107 484 108 0·584
Zeaxanthin
Before 140 37 127 25 137 37 0·663
After 139 46 124 22 134 33 0·607
b-Cryptoxanthin
Before 283 71 266 80 259 56 0·740
After 273 66 256 73 253 71 0·802
a-Carotene
Before 131 43 100 45 130 37 0·182
After 130 44 97 39 127 37 0·147
b-Carotene
Before 673 50 662 70 651 68 0·742
After 679 63 667 80 660 103 0·883
Lycopene
Before 153 20 131 26 136 35 0·210
After 143* 19 122* 25 128* 38 0·275
Xanthophylls§
Before 952 174 880 183 877 156 0·549
After 950 178 956** 188 980** 202 0·931
Non-polar carotenoidsk
Before 957 89 892 106 917 111 0·375
After 951 102 887 97 916 144 0·475
Total carotenoids
Before 1909 238 1772 261 1794 253 0·434
After 1901 237 1843* 264 1896* 320 0·872
PLOOH (pmol/ml)
PCOOH
Before 26·2 8·0 18·7† 5·6 18·2* 6·4 0·021
After 27·4 7·2 15·6*†† 3·4 13·9†† 4·4 0·000
Tocopherols (nmol/ml)
a-Tocopherol
Before 44·7 9·3 42·8 17·6 36·9 5·6 0·328
After 45·1 10·5 39·5 8·5 38·9 10 ·7 0·326
g-Tocopherol
Before 4·1 2·0 3·2 0·9 3·8 1·9 0·483
After 3·8 0·8 3·1 0·8 3·8 1·4 0·302
Total tocopherols
Before 48·8 8·7 46·0 17·4 40·6 6·6 0·314
After 48·8 10·4 42·6 8·8 42·7 11 ·0 0·304
PCOOH, phosphatidylcholine hydroperoxide; PEOOH, phosphatidylethanolamine hydroperoxide.
Mean values were significantly different in the paired t test between before and after astaxanthin administration:
*P, 0·05, **P, 0·01.
Mean values were significantly different in Dunnett’s test between the control group and one of the two astaxanthin
groups: †P,0·05, ††P, 0·01.
‡ One-way ANOVA test among groups.
§ Xanthophylls are the sum of astaxanthin, lutein, zeaxanthin and b-cryptoxanthin.
k Non-polar carotenoids are sum of a-carotene, b-carotene and lycopene.
Astaxanthin inhibits lipid peroxidation 1569
British Journal of Nutrition

species
(20)
. Incorporation of a carotenoid (b-carotene) into
erythrocytes after oral supplementation has been described
in some reports
(21)
. However, there has been no study evalu-
ating whether administered carotenoids other than b-carotene
are distributed to erythrocytes, except for our recent human
study of the xanthophyll lutein
(11)
. In the present study,
using the newly developed HPLC-MS/MS analysis method,
incorporation of astaxanthin into erythrocytes after oral sup-
plementation was established (Fig. 1(a)). Because both the
erythrocyte and plasma astaxanthin concentrations increased
(Tables 4 and 5), it seems likely that astaxanthin in plasma
lipoprotein particles is partly transferred into erythrocytes.
By this hypothesis, astaxanthin would be located on the
outer region of plasma lipoproteins, which would facilitate
its transfer to erythrocytes. On the other hand, the concen-
trations of endogenous antioxidants (i.e. carotenoids and
tocopherols) showed virtually no change before and after
astaxanthin supplementation (Tables 4 and 5). This is advan-
tageous for elucidating the antioxidant contribution of astax-
anthin. By the way, it was known that blood carotenoid
concentration in females is somewhat higher than that in
males. In the present study, we compared sex difference in
blood carotenoids, but no statistical differences were observed
between males and females in each carotenoid at baseline or
post-dosing.
In the present study, to evaluate peroxidisability, we
measured the PLOOH content. Because PLOOH are the
primary oxidation products of PL, an increase in PLOOH
directly reflects in vivo oxidative stress
(1 – 3,22,23)
. As has been
observed, astaxanthin supplementation clearly reduced the
erythrocyte PLOOH concentration (Fig. 1(b)), indicating that
astaxanthin incorporation into erythrocytes attenuated PL per-
oxidation of erythrocyte membranes. On the other hand, the
antioxidant effect of astaxanthin seemed to be more apparent
on the erythrocyte membrane, as compared with the plasma
(Tables 4 and 5). Erythrocytes are rich in PUFA in their PL
bilayer, and contain high concentrations of molecular
oxygen and ferrous ions as constituents of oxyhaemoglobin.
The oxidation of Hb is accompanied by the formation of
superoxides, a source of reactive oxygen species. Therefore,
erythrocyte membrane PL would be more susceptible to per-
oxidation than other organelle membranes, even though
they are protected by several antioxidative systems such as
superoxide dismutase, catalase and glutathione peroxidase.
For plasma PCOOH, unexpectedly, at baseline (week 0),
groups taken 6 or 12 mg astaxanthin showed significantly
less PCOOH than the group taken 0 mg astaxanthin. Because
no differences were observed among the three groups in other
parameters (e.g. haematological and blood biochemical
values), it might be other factor(s) affecting plasma PCOOH
before the start of the study. This possibility needs further
investigation.
In the present study, when comparing erythrocyte PCOOH
between the placebo and astaxanthin groups, PCOOH levels
after astaxanthin supplementation (5·2 and 6·6 pmol/ml
packed cells for 6 and 12 mg astaxanthin groups, respectively)
were somewhat lower but not statistically significant as com-
pared with those of the placebo group (9·1 pmol/ml packed
cells; P¼0·122; Table 4). In contrast, PEOOH levels after astax-
anthin supplementation (2·8 and 3·0 pmol/ml packed cells for
6 and 12 mg astaxanthin groups, respectively) were signifi-
cantly lower (P¼0·011) than those of the placebo group
(5·8 pmol/ml packed cells). PLOOH (sum of PCOOH and
PEOOH) is, therefore, considered to show significant changes
(P¼0·031) between the placebo (14·9 pmol/ml packed cells)
and astaxanthin groups (8·0 and 9·7 pmol/ml packed cells
for 6 and 12 mg astaxanthin groups, respectively). Considering
these, the antioxidant effect of astaxanthin appears likely to be
through the reduction of erythrocyte PEOOH rather than
PCOOH. This possibility needs to be clarified in future studies.
On the other hand, for the efficacy of astaxanthin, inhibitory
effects of the 6 mg astaxanthin group on PCOOH and
PEOOH were as good as or even better than those of the
12 mg astaxanthin group (Tables 4 and 5). Concentrations of
erythrocytes and plasma astaxanthin were not different
between the 6 and 12 mg astaxanthin groups, suggesting that
6 mg astaxanthin is effective enough to show antioxidative
benefit in vivo. Thus, to estimate the optimal dose of astax-
anthin, we are now conducting a human study by administer-
ing 3–6 mg astaxanthin to volunteers.
Among the carotenoids (xanthophylls), astaxanthin has
recently received increased scientific interest due to its
potent antioxidant activity and hence possible anti-metabolic
syndrome, anti-brain ageing and anti-atopic dermatitis
effects
(24 –26)
. We have previously found that there was a
higher accumulation of PLOOH in the erythrocytes of dementia
patients
(3)
. Erythrocytes high in lipid hydroperoxides have been
suggested to have a decreased ability to transport oxygen to the
brain and may impair blood rheology, thus facilitating demen-
tia
(4 –8)
. In the present study, orally administered astaxanthin
was incorporated into erythrocytes, and erythrocyte PLOOH
levels decreased. On the basis of these points, it seems that simi-
lar to lutein
(11)
, astaxanthin has the potential to act as an import-
ant antioxidant in erythrocytes, and thereby may contribute to
the prevention of dementia. This possibility warrants the testing
of astaxanthin in other models of dementia with a realistic pro-
spect of its use as a human therapy.
Acknowledgements
The present study was supported in part by Grants-in-Aid for
Scientific Research (KAKENHI; 20228002) of the Japanese
Society for the Promotion of Science ( JSPS; Tokyo, Japan).
K. N. was involved in data collection, data analysis, data
interpretation, literature search and manuscript preparation.
T. K., T. M., G. C. B., F. K. and A. S. were involved in data col-
lection, data analysis and data interpretation. A. S. and T. M.
were involved in the study design, data interpretation and
review of the manuscript. None of the authors has conflicts
of interest with respect to the present study.
References
1. Miyazawa T, Yasuda K, Fujimoto K, et al. (1988) Presence
of phosphatidylcholine hydroperoxide in human plasma.
J Biochem 103, 744 –746.
K. Nakagawa et al.1570
British Journal of Nutrition

2. Miyazawa T, Suzuki T, Fujimoto K, et al. (1992) Chemilumi-
nescent simultaneous determination of phosphatidylcholine
hydroperoxide and phosphatidylethanolamine hydroper-
oxide in the liver and brain of the rat. J Lipid Res 33, 1051–1059.
3. Miyazawa T, Suzuki T, Yasuda K, et al. (1992) Accumu-
lation of phospholipid hydroperoxides in red blood cell
membranes in Alzheimer disease. In Oxygen Radicals,
pp. 327–330 [K Yagi, M Kondo, E Niki and T Yoshikawa,
editors]. Amsterdam: Elsevier Science Publishing.
4. Bosman GJ, Bartholomeus IG, de Man AJ, et al. (1991)
Erythrocyte membrane characteristics indicate abnormal
cellular aging in patients with Alzheimer’s disease. Neurobiol
Aging 12, 13 –18.
5. Goodall HB, Reid AH, Findlay DJ, et al. (1994) Irregular
distortion of the erythrocytes (acanthocytes, spur cells) in
senile dementia. Dis Markers 12, 23–41.
6. Solerte SB, Ceresini G, Ferrari E, et al. (2000) Hemorheologi-
cal changes and overproduction of cytokines from immune
cells in mild to moderate dementia of the Alzheimer’s type:
adverse effects on cerebromicrovascular system. Neurobiol
Aging 21, 271 – 281.
7. Ajmani RS, Metter EJ, Jaykumar R, et al. (2000) Hemo-
dynamic changes during aging associated with cerebral
blood flow and impaired cognitive function. Neurobiol
Aging 21, 257 – 269.
8. Mohanty JG, Eckley DM, Williamson JD, et al. (2008) Do red
blood cell-b-amyloid interactions alter oxygen delivery in
Alzheimer’s disease? Adv Exp Med Biol 614, 29 –35.
9. Nakagawa K, Kiko T, Hatade K, et al. (2008) Development of
an HPLC-based assay for carotenoids in human red blood
cells: application to clinical studies. Anal Biochem 381,
129–134.
10. Nakagawa K, Miyazawa T, Kiko T, et al. (2010) Effect of
carotenoids towards lipid peroxidation in vivo. J JSMUFF 6,
123–130 [in Japanese].
11. Nakagawa K, Kiko T, Hatade K, et al. (2009) Antioxidant
effect of lutein towards phospholipid hydroperoxidation in
human erythrocytes. Br J Nutr 102, 1280 –1284.
12. Nakagawa K, Fujimoto K & Miyazawa T (1996) b-Carotene
as a high-potency antioxidant to prevent the formation of
phospholipid hydroperoxides in red blood cells of mice.
Biochim Biophys Acta 1299, 110 –116.
13. Asai A, Nakagawa K & Miyazawa T (1999) Antioxidative
effects of turmeric, rosemary and capsicum extracts on
membrane phospholipid peroxidation and liver lipid metab-
olism in mice. Biosci Biotechnol Biochem 63, 2118– 2122.
14. Higuera-Ciapara I, Fe
´
lix-Valenzuela L & Goycoolea FM
(2006) Astaxanthin: a review of its chemistry and appli-
cations. Crit Rev Food Sci Nutr 46, 185 – 196.
15. Hussein G, Sankawa U, Goto H, et al. (2006) Astaxanthin,
a carotenoid with potential in human health and nutrition.
J Nat Prod 69, 443–449.
16. Guerin M, Huntley ME & Olaizola M (2003) Haematococcus
astaxanthin: applications for human health and nutrition.
Trends Biotechnol 21, 210– 216.
17. Satoh A, Ishikura M, Murakami N, et al. (2010) The
innovation of technology for microalgae cultivation and its
application in functional foods and the nutraceutical
industry. In Biotechnology in Functional Foods and Nutra-
ceuticals, pp. 313– 329 [D Bagchi, FC Lau and DK Ghosh,
editors]. London: Taylor & Francis Group, CRC Press.
18. Ikeda S, Tohyama T, Yoshimura H, et al. (2003) Dietary
a-tocopherol decreases a-tocotrienol but not g-tocotrienol
concentration in rats. J Nutr 133, 428–434.
19. Fotouhi N, Meydani M, Santos MS, et al. (1996) Carotenoid and
tocopherol concentrations in plasma, peripheral blood mono-
nuclear cells, and red blood cells after long-term b-carotene
supplementation in men. Am J Clin Nutr 63, 553–558.
20. Norkus EP, Bhagavan HN & Nair PP (1990) Relationship
between individual carotenoids in plasma, platelets and
red blood cells (RBC) of adult subjects. FASEB J 4, A1774.
21. Murata T, Tamai H, Morinobu T, et al. (1992) Determination
of b-carotene in plasma, blood cells and buccal mucosa by
electrochemical detection. Lipids 27, 840–843.
22. Miyazawa T (1989) Determination of phospholipid hydro-
peroxides in human blood plasma by a chemilumines-
cence-HPLC assay. Free Radic Biol Med 7, 209–217.
23. Moriya K, Nakagawa K, Santa T, et al. (2001) Oxidative stress
in the absence of inflammation in a mouse model for
hepatitis C virus-associated hepatocarcinogenesis. Cancer
Res 61, 4365 –4370.
24. Uchiyama A & Okada Y (2008) Clinical efficacy of astax-
anthin-containing Haematococcus pluvialis extract for the
volunteers at risk of metabolic syndrome. J Clin Biochem
Nutr 43, 38 –43.
25. Satoh A, Tsuji S, Okada Y, et al. (2009) Preliminary clinical
evaluation of toxicity and efficacy of a new astaxanthin-
rich Haematococcus pluvialis extract. J Clin Biochem Nutr
44, 280 –284.
26. Satoh A, Kawamura T, Horibe T, et al. (2009) Effect of the
intake of astaxanthin-containing Haematococcus pluvialis
extract on the severity, immunofunction and physiological
function in patients with atopic dermatitis. J Environ Derma-
tol Cutan Allergol 3 429–438 [in Japanese].
Astaxanthin inhibits lipid peroxidation 1571
British Journal of Nutrition