Antioxidant Effect of Astaxanthin
on Phospholipid Peroxidation
in Human Erythrocytes

This section is compiled by Frank M. Painter, D.C.
Send all comments or additions to:    Frankp@chiro.org

FROM:   Brit J Nutrition 2011 (Jun);   105 (11):   1563-1571 ~ FULL TEXT

Nakagawa K, Kiko T, Miyazawa T, Carpentero Burdeos G, Kimura F, Satoh A, Miyazawa T.

Food and Biodynamic Chemistry Laboratory,
Graduate School of Agricultural Science,
Tohoku University,
Sendai 981-8555, Japan.

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Dementias are disorders with symptoms of memory loss and a decrease in cognition and the ability to reason. Dementia, sometimes called "senility," is not a part of the normal aging process, and shows that some other disease may be present. Dementia affects a person's ability to be successful in carrying out their activities of daily living. Accurate diagnosis of the disease that is present is necessary for treating the dementia properly. Astaxanthin belongs to a group of compounds called carotenoids. While b-carotene is a vitamin A precursor, astaxanthin cannot be converted to vitamin A. In laboratory studies, astaxanthin has been reported to be typically at least 10 times more potent as an antioxidant than the other standard carotenoids such as canthaxanthin, b-carotene, lutein, lycopene, tunaxanthin and zeaxanthin. Astaxanthin provides the rich pink color observed in various aquatic species including salmon, crabs, lobster, shrimp and even some nonaquatic species such as the flamingo (whose diet includes some astaxanthin-producing organisms). A study published in the British Journal of Nutrition investigated the effect of astaxanthin on phospholipid hydroperoxides, which are known to accumulate abnormally in the red blood cells of people with dementia. The study included 30 healthy volunteers between the ages of 50 and 69 years who were randomly assigned to receive either 6 mg or 12 mg of astaxanthin or 0 mg placebo per day for 12 weeks. The results revealed that after 12 weeks of treatment, levels of phospholipid hydroperoxides were significantly lower in erythrocytes following astaxanthin supplementation with reductions in the order of about 40 and 50 percent in the 6 and 12 mg groups, respectively, compared with no significant change in the placebo group. These findings suggest that supplementation with astaxanthin improved erythrocyte antioxidant status and decreased phospholipid hydroperoxides levels, which may help prevent dementia. From:   Astaxanthin May Prevent Dementia

The 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.

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From the FULL TEXT Article:

Introduction

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 functional foods to prevent the disease. Although there is still scarce information on whether orally administered xanthophylls 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 commercially 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 astaxanthin 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, placebo-controlled human trial. The efficacy of 12-week astaxanthin 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 astaxanthin analysis, a newly developed HPLC coupled with tandem MS (MS/MS) method was applied. Our findings (the inhibitory effect of astaxanthin on erythrocyte PLOOH) would provide new insights into the possible application of astaxanthin as an anti-dementia agent.

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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 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 dietary 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 astaxanthin, 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 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 supplementation study. Using MS/MS, we found that protonated astaxanthin tended to generate product ions (e.g. m/z 147). The product ion indicated that MRM could be adapted to the HPLC-MS/MS analysis of astaxanthin. Under optimised conditions, 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 β-carotene) [19], while other studies have been unable to detect these species [20]. Incorporation of a carotenoid (β-carotene) into erythrocytes after oral supplementation has been described in some reports [21]. However, there has been no study evaluating whether administered carotenoids other than β-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 supplementation 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 concentrations of endogenous antioxidants (i.e. carotenoids and tocopherols) showed virtually no change before and after astaxanthin supplementation (Tables 4 and 5). This is advantageous for elucidating the antioxidant contribution of astaxanthin. 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 peroxidation 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 peroxidation 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 compared with those of the placebo group (9·1 pmol/ml packed cells; P = 0·122; Table 4). In contrast, PEOOH levels after astaxanthin supplementation (2·8 and 3·0 pmol/ml packed cells for 6 and 12 mg astaxanthin groups, respectively) were significantly lower (P = 0·011) 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 (P = 0·031) 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 astaxanthin, we are now conducting a human study by administering 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 dementia [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 similar to lutein [11], astaxanthin has the potential to act as an important 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 prospect of its use as a human therapy.

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