Astaxanthin Protecting Membrane Integrity against Photosensitized

Article pubs.acs.org/JAFC

Astaxanthin Protecting Membrane Integrity against Photosensitized Oxidation through Synergism with Other Carotenoids Hui-Hui Du,† Ran Liang,† Rui-Min Han,† Jian-Ping Zhang,*,† and Leif H. Skibsted*,‡ †

Department of Chemistry, Renmin University of China, Beijing 100872, People’s Republic of China Food Chemistry, Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

‡

ABSTRACT: Incorporation of astaxanthin or zeaxanthin in giant unilamellar vesicles (GUVs) of phosphatidylcholine resulted in a longer lag phase than incorporation of β-carotene or lycopene for the onset of budding induced by chlorophyll a photosensitization and quantified by a dimensionless entropy parameter using optical microscopy and digital image heterogeneity analysis. The lowest initial rate of GUV budding after the lag phase was seen for GUVs with astaxanthin as the least reducing carotenoid, while the lowest final level of entropy appeared for those with lycopene or β-carotene as a more reducing carotenoid. The combination of astaxanthin and lycopene gave optimal protection against budding with respect to both a longer lag phase and lower final level of entropy by combining good electron acceptance and good electron donation. Quenching of singlet oxygen by carotenoids close to chlorophyll a in the membrane interior in parallel with scavenging of superoxide radicals by astaxanthin anchored in the surface may explain the synergism between carotenoids involving both type I and type II photosensitization by chlorophyll a. KEYWORDS: giant unilamellar vesicle, budding, astaxanthin, carotenoid synergism, bilayer integrity

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INTRODUCTION Carotenoids emerged early in evolution in archaebacteria. Astaxanthin (Ast), the more hydrophilic among common tetraterpene carotenoids, is an important protector of the membrane structures in some of these primitive microorganisms and in some yeasts.1,2 Ast (see Scheme 1) may be identified in such microorganisms by a characteristic pink color and is a protector against exposure to intense light and photooxidation. It is, like other tetraterpene carotenoids, an efficient quencher of singlet oxygen, 1O2, in both homogeneous solution and structured media.3−5 Ast is the least reducing among common caroteniods and seems to scavenge the superoxide radical anion, O2• −, through oxidation rather than scavenging other radicals like the hydroxyl radical, HO•, through reduction.5,6 Ast is transferred through food chains in cold oceans from phytoplankton to krill and further to shellfish and salmon. Ast seems important for the oxidative stability of many vulnerable seafoods, especially when they are exposed to light, and it may also explain some of the positive health effects of such seafoods.7−11 Ast has accordingly like most other carotenoids a dual function in foods because they provide characteristic colors and protect foods and beverages against light-induced oxidation. Ast has been found important as a protector of phospholipids in liposomes against oxidation as a model for cell membranes.12 Notably, it was found to be anchored in the membrane/ water interface and to transfer electrons from antioxidants like β-carotene (β-Car) embedded in the membrane interior to radicals in the lipid/water interface. The interaction between Ast and other antioxidants like lycopene (Lyc) and β-Car resulted in clear synergetic antioxidative effects protecting the phospholipids of the membrane. The physical integrity of lipid bilayers in membranes of the food system is an important aspect of food stability. Membranes may become instable as the © 2015 American Chemical Society

result of the formation of hydroperoxides that increase the dipole moment of the phospholipids in the membrane and further result in the collapse of membrane structures.12 To investigate the importance of Ast in stabilizing membranes under oxidative stress, either alone or through interaction with other carotenoids, we have used the giant unilamellar vesicle (GUV) model recently developed.13 This model for cell membranes has been found versatile because it allows use of optical microscopy to follow the changes in the membrane structure.14 One of the first indications of membrane instability and decrease of functionality is budding of the GUVs, which may be readily quantified using digital image heterogeneity analysis. Ast was accordingly incorporated in GUVs together with chlorophyll a (Chl a) as a photosensitizer, and its protective effects on membranes were quantified and compared to effects of other carotenoids incorporated alone or in combinations with Ast.

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MATERIALS AND METHODS

Materials. Soybean L-α-phosphatidylcholine (PC; 23%; product number P5638) and all-trans-β-carotene (hereafter, β-Car; >95%) were purchased from Sigma-Aldrich (St. Louis, MO). Ast (>90%) and zeaxanthin (Zea; >90%) sealed in ampules under argon were supplied by Roche A/S (Hvidovre, Denmark) and used as received. All-translycopene (Lyc; >85%) was extracted from tomato sauce, purified, and recrystallized according to the procedure previously described.15 Methods for extraction and purification of Chl a (>90%) from fresh spinach leaves are described elsewhere.16 Sucrose (≥99%) was purchased from Amresco, Inc. (Solon, OH). Chloroform and methanol [analytical reagent(AR) grade] was purchased from Beijing Chemical Works Received: Revised: Accepted: Published: 9124

July 27, 2015 October 1, 2015 October 1, 2015 October 1, 2015 DOI: 10.1021/acs.jafc.5b03658 J. Agric. Food Chem. 2015, 63, 9124−9130

Article

Journal of Agricultural and Food Chemistry

to the cell to generate a turbid GUV suspension with a volume of approximately 2.5 mL. Optical Microscopy and Digital Image Processing. The experimental setup for fluorescence microscopy was described elsewhere.13 Briefly, 200 μL of liposome suspension was added to a Costar 24-well cell culturing cluster (Corning Incorporated, Corning, NY), which was fixed on the translational stage. Light radiation in 400−440 nm from an ultrahigh-pressure mercury lamp was used for excitation with a power of 0.81 mW and was focused on an area with a diameter of 80 μm. A band-pass filter (500−570 nm) was used to minimize the fluorescence emission from Chl a. Liposome images were collected every 1 s, and each GUV preparation was measured independently for at least 30 times. GUVs unsensitized with Chl a were used as a blank, whereas sensitized GUVs without antioxidant added were used as a control. Evaluation of the liposome physical integrity was performed using digital image processing on the basis of heterogeneity.18,19 Briefly, raw images were digitized into 16-bit grayscale format with an area of 100 × 100 pixels selected as region of interest (ROI). The 16-bit grayscale intensity interval was divided into 256 subintervals by default. Histogram counts (p) over these subintervals were used to calculate the entropy, defined as E = −∑p log2 p, which was used as a statistical scalar for measuring the heterogeneity.19 The budding process of GUVs was characterized by entropy change (ΔE) with reference to the average entropy at minus delay time, i.e., before light irradiation.13

Scheme 1. Molecular Structures of (a) Predominant Species of Soybean PC, (b) Chl a, (c) Lyc, (d) β-Car, (e) Ast, and (f) Zea

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RESULTS AND DISCUSSION The blank soybean PC GUVs used as a membrane model were found resistant to light exposure (400−440 nm) because no change in GUV morphology was observed. Incorporating Chl a (Scheme 1) as a photosensitizer made them sensitive to light exposure as evidenced by the fast increase in entropy and budding (Figures 1−3), which could be quantified using the entropy change ΔE defined in the Materials and Methods.13 It is seen in Figures 1 and 2 that ΔE increased immediately upon light exposure for control and Chl a-sensitized GUVs with β-Car. In contrast, Lyc, Zea, and Ast, each added separately, induced a significant lag phase for the increase of ΔE. The spatial arrangement of the GUVs as a model for membranes is depicted in Scheme 2. In this scheme, Chl a as a hydrophobic molecule lies in the interior of the lipid bilayer together with hydrophobic Lyc (or β-Car), while Ast (or Zea) with polar substituents to ending rings anchors in the water/lipid interfaces. Three effects on the budding process resulting from photosensitization may be identified from the temporal evolution of ΔE during light exposure, as results from the addition of each individual carotenoid investigated (Figures 2 and 3): (i) Lyc, Zea, and Ast each, in contrast to β-Car, resulted in a significant lag phase, during which ΔE was essentially unchanged. (ii) The ΔE increasing rate after the lag phase decreased upon incorporation of each individual carotenoid, especially for the case of Ast. (iii) The final level of heterogeneity was found similar for Ast and Zea, both similar to the control, but for β-Car and Lyc, the levels were found to be comparable and significantly lower. The length of the lag phase increased in the order of control < β-Car < Lyc < Zea < Ast as most clearly seen from Figure 3. Notably, Zea and Ast are both xanthophylls with hydrophilic groups (Scheme 1). Xanthophylls are known to be anchored in the water/lipid interfaces of membranes resulting in the decrease of the membrane fluidity,12,20 and such effects may be important in the initial protection against photosensitized oxidation and result in a lag phase (vide inf ra). The rate of ΔE increase following the initial lag phase obeys another ordering than the length of the lag phase: Ast < Lyc < β-Car < Zea < control (Table 1). Ast accordingly

(Beijing, China). Purified water was prepared using a Milli-Q Academic Water Purification System (Millipore Corp., Billerica, MA). Electroformation of GUVs. GUVs were prepared using an electroformation method with certain modifications.17 Briefly, the cell used for GUV preparation consisted of two indium tin oxide (ITO) glass substrates separated by a Teflon spacer with a thickness of ∼3 mm. The PC reagent was dissolved in chloroform at a concentration of 4 mg/mL. Chl a was predissolved in methanol and used as the photosensitizer. β-Car and Lyc predissolved in chloroform and Zea and Ast predissolved in methanol were used as antioxidants. The final molar ratio of PC reagent and Chl a was 100:1, and the final molar ratio of PC reagent and each individual carotenoid was 200:1. The solutions were deposited on one of the conductive surfaces of ITO glass substrates and then dried in vacuum for 2 h. About 2.5 mL of 100 mM sucrose solution was injected into the cell, which was afterward sealed using silicone grease. Alternative electrical fields from a Rigol DG1022U function generator (Rigol Technologies, Beijing, China) were applied 9125

DOI: 10.1021/acs.jafc.5b03658 J. Agric. Food Chem. 2015, 63, 9124−9130

Article


Figure 1. Images of Chl a-sensitized GUV samples exposed to light (400−440 nm, 160 mW/mm2) for an indicated time at room temperature: (a) control, (b) β-Car, (c) Lyc, (d) Zea, and (e) Ast added.

Figure 2. ΔE as a metric of image heterogeneity versus time for budding of Chl a-sensitized GUVs with the addition of individual carotenoid exposed to light (400−440 nm, 160 mW/mm2). For control, see Figure 3. Statistics: mean ± standard deviation (SD) (n = 6).

subsequent development of ΔE (Table 1). Notably, the two carotenes, Lyc and β-Car, have comparable ΔE increasing rates.

both induces the longest lag phase and results in the slowest ΔE increasing rate, while Zea, which also induces a long lag phase as a xanthophyll, yields little protection during the 9126


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The GUVs with Lyc and β-Car are seen to yield the best protection as evaluated by the final level of heterogeneity. GUVs with Ast and Zea both reach approximately the same final levels of heterogeneity as the control. Two factors seem to control the pattern of protection of GUVs against budding. One could be related to the decreased fluidity of membranes of GUVs as resulting from incorporation of Ast or Zea. Chl a, as a photosensitizer, may operate through two mechanisms as outlined in Figure 4.21 Type II

Figure 3. Comparison of temporal evolution profiles of ΔE for budding of Chl a-sensitized GUVs with individual carotenoid added. The time evolutions are from Figure 2. For clarity, error bars are not shown.

Scheme 2. Ast and Lyc in a Liposomal Membrane

Figure 4. Type I and type II photosensitization by Chl a.16

photosensitization will yield singlet oxygen 1O2, which will diffuse from Chl a with a rate depending upon the fluidity of the membrane in competition with quenching. The 1O2 quenching rates are comparable for the four carotenoids in homogeneous solutions (Table 2), and hence, the other factor

a

Table 2. Second-Order Rate Constant, k2, for Quenching of Singlet Oxygen by Carotenoids in Benzene and in Unilamellar Liposomes, Standard Reduction Potential for Carotenoid Radical Cation versus NHE, E°, in Dichloromethane, Electron Acceptor Capacities, Ra, and Electron Donor Capabilities, Rd, All at Room Temperature k2 (L mol−1 s−1) carotenoid β-Car Lyc Zea Ast a a

Membrane thickness is approximately 4 nm.

lag phase (s)

control β-Car Lyc Zea Ast Ast + β-Car Ast + Zea Ast + Lyc

0.15 1.8 7.3 7.8 8.9 14 17 26

initial rate (s−1) (165 ± 13) (75 ± 5) (65 ± 6) (121 ± 9) (40.4 ± 1.3) (18.3 ± 0.9) (10.3 ± 1.7) (52 ± 4)

× × × × × × × ×

10−3 10−3 10−3 10−3 10−3 10−3 10−3 10−3

ΔE∞

AUC (au)

0.90 0.46 0.37 0.82 0.80 0.32 0.24 0.30

29 14 8.9 17 12 6.7 3.2 2.2

1.3 1.7 1.6 1.1

× × × ×

1010 1010 1010 1010

liposomesa

E° (V)b

Ra c

Rdc

× × × ×

0.84 0.81 0.85 0.97

0.46

1.40

0.49 0.94

1.44 2.10

2.3 2.4 2.3 5.9

1010 1010 1010 109

From ref 22. bFrom refs 25 and 26. cFrom ref 24.

may be important in controlling the oxidation of the phospholipids by 1O2.22 Tentatively, the longer lag phases seen for Ast and Zea relative to Lyc and β-Car may be assigned to a decrease in the rate of diffusion as a result of the decreased fluidity of the membrane by anchoring of Ast and Zea in the membrane surface. A lower diffusion rate will delay the oxidation of the phospholipids at the lipid/water interface but will not prevent the formation of the hydroperoxides that result in a final increase in ΔE and budding. The second factor could be related to the electron-accepting capability of Ast. Ast is the least reducing among the four carotenoids (Table 2). It has a high electron-accepting capability, as provided by quantum chemical calculations, and has been suggested to oxidize the superoxide radical anion

Table 1. Lag Phases, Initial Rates, Final Levels (ΔE∞), and Area under Curve (AUC) of ΔE/t Evolutions for Each GUV Preparationa sample (n = 6)

benzenea

a

Ast + O2• − → Ast• − + O2

AUCs were calculated from the end of lag phases until 31 s for GUV preparations with incorporating single carotenoids or until 40 s for those with combined carotenoids added.

in contrary to most other carotenoids.6,23 Chl a may operate partly through a type I mechanism for photosensitization, resulting in the formation of radicals (Figure 4). O2• −, when produced in the membrane, many react with Ast but not Zea or the carotenes like β-Car and Lyc. This could explain

The final level of heterogeneity was quantified as both the final value for ΔE and the area under the ΔE−t curve in arbitrary units after the lag phase, which are compared in Figure 3 and Table 1. 9127


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Figure 5. Images of Chl a-sensitized GUV samples exposed to light (400−440 nm, 160 mW/mm2) at the indicated time at room temperature: (f) β-Car and Ast, (g) Zea and Ast, (h) Lyc and Ast, and (i) unsensitized blank GUVs.

Figure 6. ΔE as a metric of image heterogeneity versus time for budding of Chl a-sensitized GUVs with the addition of combined carotenoids and blank GUVs exposed to light (400−440 nm, 160 mW/mm2). Statistics: mean ± SD (n = 6).

β-Car but not by Ast and Zea to give the lowest values for final ΔE

the decreased rate in the development of ΔE following the lag phase as seen for Ast but not for the other three carotenoids. What still remains to be explained is, however, the higher final level of ΔE for Zea and Ast compared to β-Car and Lyc. It may be the result of another type of process not directly related to the type I and type II photosensitized oxidation but involving reduction of an initially formed radical precursor to peroxides by the more reducing Lyc and

Lyc + R• + H+ → Lyc• + + RH

The combination of Ast with each of the three other carotenoids clearly showed synergetic effects, which may help to explain in more details the complex pattern of reactions Figures 5 and 6. Ast combined with Lyc resulted in the longest 9128



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lag phase and also the lowest level for the final heterogeneity. The combination of the most reducing and the most oxidizing carotenoid seems optimal for protection of the membrane against photosensitized budding and disintegration. Apparently, the longer lag phase is the result of a decrease in the rate of 1O2 diffusion from type II photosensitization by Chl a via anchoring of Ast, while the lower level for final heterogeneity is the result of reduction of hydroperoxides by the strongly reducing Lyc. The combination of Ast and β-Car shows a similar protective effect, confirming that a carotenoid with a high electron-accepting capability together with another carotenoid with a strong electron-donating capability results in prominent synergistic effects (Figure 7). Carotenoids have

ACKNOWLEDGMENTS The authors are indebted to Professor Peng Yang at the School of Chemistry and Chemical Engineering, Shaanxi Normal University, for his help in GUV preparation.

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ABBREVIATIONS USED Ast, astaxanthin; Lyc, all-trans-lycopene; Zea, zeaxanthin; β-Car, all-trans-β-carotene; Chl a, chlorophyll a; PC, L-α-phosphatidylcholine; GUV, giant unilamellar vesicle; 1O2, singlet oxygen; O2• −, superoxide radical anion; HO•, hydroxyl radical; ROI, region of interest; AUC, area under curve

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REFERENCES

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Figure 7. Comparison of time evolution of ΔE as a metric of heterogeneity for budding of Chl a-sensitized GUVs with combined carotenoids and blank GUVs. The time evolutions are from Figure 6. For clarity, error bars are not shown.

both the capability for electron donation and for electron acceptance depending upon their structures.24−26 Most carotenoids are good electron donors, but the conclusion from the present study seems to be that combination of carotenoids being good electron donors with those being good electron acceptors like Ast may yield the optimal protection against photosensitized degradation of membranes. The use of GUVs for the study of oxidative processes in membranes is promising because spatial aspects of oxidation may be understood on the basis of the distribution of Chl a as a photosensitizer located in the lipophilic interior and potential antioxidants with distribution depending upon their hydrophilic/lipophilic balance (Scheme 2).

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Article

AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-10-62516604. E-mail: [email protected]. edu.cn. *Telephone: +45-3533-3221. E-mail: [email protected]. Funding

This work has been supported by the Natural Science Foundation of China (Grant 21173265), the Fundamental Research Funds for the Central Universities, and the Research Funds of Renmin University of China (RUC; 10XNI007, 14XNLQ04, and 14XNH058). Notes

The authors declare no competing financial interest. 9129


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Astaxanthin Protecting Membrane Integrity against Photosensitized

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