Cholesterol photosensitized oxidation in food and biological systems

Biochimie 95 (2013) 473e481

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Biochimie journal homepage: www.elsevier.com/locate/biochi

Review

Cholesterol photosensitized oxidation in food and biological systems Vladimiro Cardenia a, Maria Teresa Rodriguez-Estrada b, *, Emanuele Boselli c, Giovanni Lercker b a

Inter-Departmental Centre for Agri-Food Industrial Research, Alma Mater Studiorum-Università di Bologna, Piazza Goidanich 60, 47521 Cesena, Italy Department of Food Science, Alma Mater Studiorum-Università di Bologna, Viale G. Fanin 40, 40127 Bologna, Italy c Department of Agricultural, Food and Environmental Sciences, Università Politecnica delle Marche, Ancona, Italy b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 April 2012 Accepted 10 July 2012 Available online 22 July 2012

Lipid oxidation is one of the main chemical degradations occurring in biological systems and leads to the formation of compounds that are related to aging and various chronic and degenerative diseases. The extent of oxidation will depend on the presence of antioxidants/pro-oxidants, the unsaturation degree of fatty acids, and environmental conditions. Lipid oxidation can also affect other molecules that have double bonds in their chemical structures, such as cholesterol. Cholesterol oxidation products (COPs) have been studied in depth, because of their negative and controversial biological effects. The formation of COPs can be particularly favored in the presence of light and photosensitizers, since they generate excited singlet oxygen that rapidly reacts with the double bond by a non radical mechanism and without any induction period. The present review intends to provide an overall and critical picture of cholesterol photosensitized oxidation in food and biological systems, and its possible impact on human health and well-being. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Cholesterol photooxidation Food Skin Lens Packaging

1. Introduction Cholesterol is a monounsaturated molecule located in the cell membrane and it is involved in membrane permeability and fluidity. The A ring of the molecule is exposed to the outer side of the double phospholipid layer, with the hydroxyl group interacting with the polar head groups of the membrane phospholipids, while the side chain is situated among the alkyl chain of phospholipids [1,2]. Due to the presence of a double bond in position 5,6 of the B ring, cholesterol can oxidize, following similar oxidative pathways as monounsaturated fatty acids [3e5]. Cholesterol tends to oxidize first in an undamaged cell membrane and its oxidative instability could be further favored by the presence of a relatively large amount of polyunsaturated fatty acids (PUFA) in the cell membrane [6]. Cholesterol oxidation products (COPs) can be generated by autoxidation, photosensitized oxidation and enzymatic oxidation [2e7]; the types and concentrations of the single COPs produced

Abbreviations: 5a-HPC, 5a-hydroperoxycholesterol; 6a-HPC, 6a-hydroperoxycholesterol; 6b-HPC, 6b-hydroperoxycholesterol; 7a-HC, 7a-hydroxycholesterol; 7a-HPC, 7a-hydroperoxycholesterol; 7b-HC, 7b-hydroxycholesterol; 7b-HPC, 7b-hydroperoxycholesterol; 7-KC, 7-ketocholesterol; a-EC, 5a,6a-epoxycholesterol; b-EC, 5b,6b-epoxycholesterol; COPs, cholesterol oxidation products; d, days; FA, fatty acids; h, hours; PUFA, polyunsaturated fatty acids; triol, cholestanetriol; UFA, unsaturated fatty acids. * Corresponding author. Tel.: þ39 051 2096011; fax: þ39 051 2096017. E-mail address: [email protected] (M.T. Rodriguez-Estrada). 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2012.07.012

will depend on the oxidative mechanism they originate from, as well as on environmental conditions (temperature, light, water activity) and the presence of trace elements and biomolecules with prooxidant and/or antioxidant activities. COPs have been studied in depth in food [5,7e11], as they are likely to be involved in several chronic and degenerative diseases, disturbance of cell functionality and lipid metabolism [7,10,12,13]. While the role of photosensitized oxidation on the production of dietary COPs has been well documented [8e11,14e18], there is little information dealing with photosensitized oxidation of biological tissues that are daily exposed to light, such as skin, retina and lens. This review intends to provide a picture on cholesterol photosensitized oxidation on food and biological tissues, in order to better understand its impact on food quality, biological tissue integrity and consumer health. 1.1. Cholesterol photosensitized oxidation Cholesterol photooxidation reactions are classified as either type I (free radical mechanism) or type II (singlet oxygen mediated) [19,20]. Cholesterol photosensitized oxidation requires singlet oxygen (1O2) for the initiation phase to occur, being mainly generated when photosensitizers absorb light, become electronically excited and interact with triplet oxygen to convert it into reactive singlet oxygen. The latter is more reactive (32,000 times for monounsaturated structures and 1600 times for diunsaturated ones) than triplet oxygen [21], as it has higher redox potential and a lower energy of

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activation. When cholesterol photosensitized oxidation takes place, 1 O2 attacks the steroid molecule by an ene addition mechanism on either side of the double bond [22]. In reactions mediated by 1O2, 5ahydroperoxycholesterol (5a-HPC), 6a-hydroperoxycholesterol (6aHPC) and 6b-hydroperoxycholesterol (6b-HPC) are the only three hydroperoxides that are generated, 5a-HPC being the most abundant one [3,23] (Fig. 1). The latter can rearrange and give rise to both 7ahydroperoxycholesterol (7a-HPC) and 7b-hydroperoxycholesterol (7b-HPC), which are usually present at different concentration levels as the a epimer is less favored from a thermodynamics standpoint [2,3,5]. Hydroperoxides are then rapidly converted into hydroxyl and keto derivatives by a dismutation reaction, giving rise to 7ahydroxycholesterol (7a-HC) and 7b-hydroxycholesterol (7b-HC), together with 7-ketocholesterol (7-KC) (Fig. 1). As observed for hydroperoxides, 7a-HC is commonly found at lower concentrations levels than its corresponding epimer. Depending on the lighting conditions, it might be possible that 7-HCs and 7-KC are interconvertible [24]; this behavior could be also influenced by an oxidant environment. While the formation of such oxidation compounds occurs, hydroperoxides can also follow a bimolecular reaction pathway, which involves the interaction with a cholesterol molecule that generates 5a,6a-epoxycholesterol (a-EC) and 5b,6b-epoxycholesterol (b-EC) (Fig. 1). In presence of water and acidic conditions, the epoxy derivatives can undergo ring opening and, thus, produce cholestanetriol (CT, triol). The formation of side-chain COPs is also possible due the presence of tertiary atoms at C-20 and C-25 in the side chain of the cholesterol molecule [4]; although the oxidation mechanisms are similar to those of the ring structure, a lower extent of side-chain oxidation is usually observed [3]. It is important to consider that, in any case, the trend of photosensitized oxidation can be affected by the surrounding environment, which will impact the type and amount of the oxidation products that are generated. 2. Photosensitized cholesterol oxidation in food Over the past 35 years, COPs have been studied in food systems, as they exhibit different negative biological effects at different

concentrations [7,10,11,25], including atherogenesis, cytotoxicity, mutagenesis, apoptosis, carcinogenesis, selective estrogen receptor modulation and inhibition of cholesterol biosynthesis and membrane functions [7,10e13,26e30]. Among COPs, triol is considered as one of the most toxic oxidation products, even at low concentration levels [7,10,25]. Although dietary COPs might represent a risk for human health, no toxicity limit for such compounds has been specified yet, so the threshold of toxicological concern (TTC) for unclassified compounds (0.15 mg per person per day) [31] can be utilized as reference. Table 1 reports the COPs content found in food subjected to photosensitized oxidation. 2.1. Egg and egg products Eggs have a very high content of cholesterol (about 400 mg/ 100 g of edible portion) [32], so the formation of COPS has been widely studied in egg powders and egg products. The large consumption of industrialized egg-containing foods, such as bakery products and pasta, has favored the utilization of egg as powders [33], which are characterized by a better microbiological safety, a smaller volume as compared to unshelled or liquid eggs [34] and a low water content. The most common systems to produce egg powders are freeze-drying and spray-drying; the first provides the best ingredient overall quality but, due to its relatively high cost, spray-drying is most widely used at commercial level. The quality of the egg powder lipid fraction can be greatly influenced by processing and storage conditions [35,36]. One of the most critical chemical modifications that can occur is lipid oxidation, including cholesterol oxidation, due to the egg powders’ large surface area. In fact, because of the high surface activity of sterols, they tend to migrate to the oilewater interface, where oxidative stress is high [1]; this behavior would suggest that cholesterol locates at the interface before water removal, remaining at the surface level of the egg powder and being thus directly exposed to air and consequently to oxidation. The large surface area will be further decisive when egg powder and egg-containing food are subjected to light

Fig. 1. Oxidation cholesterol pathways.

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Table 1 Amount of COPs generated by photosensitized oxidation. The experimental and analytical conditions are described. Food product/biological sample/model system Egg and egg products Egg Commercial spray-dried whole egg

Dried egg yolk powder Egg pasta With pasteurized eggs obtained from hens bred with organic methods (POE) With pasteurized eggs from conventional breeding (PCE) With pasteurized spray-dried eggs (SPCE) obtained from conventional breeding Meat and meat products Beef (sliced)

Horse (sliced) Pork (sliced) Turkey (sliced breast)

Dairy products Butter Butter made with sour cream (frozen 6 months); sweet cream; sour cream butter

Brined white (Nabulsi) cheese 9 months Sliced yellow cheeseb Grated yellow cheeseb Feta type cheeseb Not irradiated, 4  C at day 0

Seafood and seafood products Shrimp Sardine (whole) Biological tissues Retinac

Experimental conditions

Total COPs (mg/kg sample)

Not irradiated, stored 8 months at 20  C Not irradiated, stored 8 months at room temperature Exposed to light, stored 8 months at room temperature Stored for 1 year and then UV-irradiated for 3 weeks

9.8 39.6

Fluorescent lamp (6000 K and 32 W), room temperature for 12 months Dark storage, room temperature for 12 months Fluorescent lamp (6000 K and 32 W), room temperature for 12 months Dark storage, room temperature for 12 months Fluorescent lamp (6000 K and 32 W), room temperature for 12 months Dark storage, room temperature for 12 months

52e201.2

[42]

4151

[41]

7a-HC, 7b-HC, a-EC, b-EC, 7-KC

52e121.4 43.8e209.6 43.8e117.3 50.6e159.3 50.6e106.0

0.1e0.33

7a-HC, 7b-HC, b-EC, a-EC, 20-HC, 7-KC

[15]

3.8e7.9

7a-HC, 7b-HC, b-EC, a-EC, triol, 7-KC 7a-HC, 7b-HC, b-EC, a-EC, 7-KC

[16]

0.03e10

7a-HC, 7b-HC, b-EC, a-EC, triol, 7-KC

[14]

Fluorescent lamp (1500 Lux), 4  C for 20 days Daylight lamp (1700 lux), 4  C for 42 days Daylight lamp (1700 lux), 20  C for 42 days Lamp for food (1050 lux), 4  C for 42 days Lamp for food (1050 lux), 20  C for 42 days UV lamp at 20  C for 4 days Fluorescent light and daylight Room temperature Light protected Fluorescent light (520 lux), 4  C for 55 days Not irradiated, 4  C at day 0 Fluorescent light (520 lux), 4  C for 72 days Not irradiated, 4  C at day 0 Fluorescent light (520 lux), 4  C for 30 days in open can Not irradiated, 4  C at day 0

Qualitative determination 0.87; 0.44; 0.41 35.66; 68.96; 79.47 25.59; 12.98; 26.13 39.56; 49.89; 65.51 8.27; 8.29; 8.47 52

5a-HC, 7a-HC, 7b-HC 7a-HC, 7b-HC, 7-KC

[78] [79]

7-KC

[85]

7a-HC, 7b-HC, a-EC, b-EC, 7KC

[80]

Sun-dried (4e8 h) Daylight fluorescent lamp; normal atmosphere; 4 h at 4  C

6.74e54.87 0.6e3.7

7a-HC, 7b-HC, 25-HC, 7-KC 7a-HC, 7b-HC, b-EC, a-EC, Triol, 7-KC

[87] [88]

Fresh monkey eyes (5e7 years of age)

1e1.5 5e8

7-KC (unesterified)

[107]

13.1 0.4 0.99 1.38

7b-HC, a-EC, 20-HC, 25-HC, 7-KC

[114]

5-HPC, 7a-HPC, 7b-HPC

[122]

0.5e1.0

Neural retina Pigment epithelium and choriocapillaris Cataract Normal lens

HR-1 hairless mice skina

UVA protected UVA projection lamp, 47 J/cm2 (8 h)

c

7a-HC, 7b-HC, a-EC, b-EC, 7-KC

51.9

Warm tone fluorescent lamp; normal atmosphere (8h) and modified atmosphere (32%O2) (8d); transparent film Daylight fluorescent lamp; normal atmosphere; 8h; transparent/red film Daylight fluorescent lamp; normal atmosphere; 3 days at 8  C; Warm tone fluorescent lamp/daylight lamp; 0e11 days; transparent film; normal atmosphere

Cataract

a

Ref.

[18]

Human lensc

b

Identified COPs

23 8.3 7.6 35.3 5 245.7

[17]

7.5

nmol/g of skin. ppm lipid. pmol/nmol of free cholesterol.

exposure, as it will favor cholesterol photooxidation. However, eggs contain carotenoids, which act as quenchers of the triplet state of chlorophyll, a very efficient photosensitizer, and of active oxygen species (singlet oxygen and free radicals). Sujak et al. [37] reported that lutein and zeaxanthin are able to protect egg yolk lecithin liposomal membranes against free radical attack with almost the same efficacy [37]; in fact, zeaxanthin appeared to be a better

photoprotector during prolonged UV exposure, while the shorter protective efficacy of lutein was attributed to the photooxidation of the carotenoid itself [37]. According to Wrona et al. [38], the presence of zeaxanthin can have a noticeable inhibitory impact on photosensitized cholesterol oxidation, exhibiting a dosedependent inhibitory effect on the accumulation of 5a-HPC, 7aHPC and 7b-HPC; furthermore, the zeaxanthin concentrations

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needed to significantly inhibit singlet oxygen-derived cholesterol hydroperoxides were lower than those required to inhibit accumulation of free radical-derived cholesterol hydroperoxides. As supported by these studies, the type and concentration of carotenoids in egg and egg-containing products seem to play a determinant role on the cholesterol oxidative behavior, together with the storage conditions. Chicoye et al. [39] observed that the levels of COPs (7-KC, 7a-HC, 7b-HC, 5,6b-EC, and triol) in egg yolk powder increased when subjected to 5-h summer sunlight irradiation or to 280-h fluorescent light (40 W). Herian and Lee [40] studied the formation of the 7-hydroxy derivatives in dry egg nog mix exposed to fluorescent light (40 W) for 105 days at 23  C, where they observed an increase of such COPs during the first 80 days of storage. Van de Bovenkamp et al. [41] found a very high level of COPs in dried egg yolk powder (4151 mg/kg of sample), after 1 year of storage followed by irradiation with UV light for 3 weeks. Fontana et al. [42] also reported that 8-month storage under daylight led to a higher COP content in commercial spray-dried egg, as compared to that of the unexposed sample; moreover, the ratio of C-7 COPs to epoxy derivatives was lower in egg subjected to light exposure, which suggests that cholesterol follows different oxidation pathways according to the lighting conditions. Recently, Verardo et al. [18] studied the effect of two packaging conditions on the accumulation of COPs in egg pasta prepared with different egg products during 12 months of storage at room temperature. Three different egg coproducts, were used: pasteurized eggs obtained from hens bred with organic methods (POE), pasteurized eggs from conventional breeding (PCE) and pasteurized spray-dried eggs (SPCE) obtained from conventional breeding. At time zero, the pasta had about 50 mg of COPs/kg of fat, whereas COP levels significantly increased during storage. PCE, SPCE and POE spaghetti stored in the dark showed a content of total COPs that was 2.0, 2.0, and 1.5 times lower than those of samples stored with typical pasta packaging. To avoid light-induced oxidation in egg powders and eggcontaining food, suitable lighting conditions and packaging with high light barriers are suggested. In addition, to partially contrast the effects of processing technology and storage, dietary supplementation with antioxidants (such as carotenoids and tocopherols) should be performed to modify egg lipid stability [35,43,44]. 2.2. Meat and meat products Raw meat usually contains low amounts of COPs, but they tend to greatly increase after exposure to light, heat, metals, oxygen, and/or extensive processing and storage [8e11,45,46]; 7-KC is usually the most abundant COP present in meat exposed to light [8e11,47]. Meat characteristics (fatty acid unsaturation level, pigment type and content, endogenous antioxidant category and concentration) will influence cholesterol photosensitized oxidation. However, the oxidative stability of meat will also depend on the holding time and storage conditions, such as irradiation and packaging [15e17,48,49] and on the anti/prooxidant balance [50], so dietary supplementation with antioxidants, such as a-tocopheryl acetate, might improve its stability [17,51e53]. The lamp attributes (radiation energy expressed as color temperature, K) will contribute to the overall oxidative behavior [54]. In general, meat pigments tend to absorb more in the blue and green parts of the light spectrum (6000 K), rather than in the red one (3000 K) [14]; such absorption characteristics will be influenced by the content of total iron, its oxidation status, the occurrence of covalent and ionic complexes with molecular oxygen and water, respectively [55]. To limit light absorption, appropriate packaging material (that transmits wavelengths between 490 and 589 nm and/

or with aluminum foil as light and gas barrier) [56] and conditions (modified atmosphere and vacuum) [57] are helpful strategies. Cholesterol photosensitized oxidation of turkey, beef and horse meat was studied [14e16] considering the atmosphere composition (normal or modified with high percentage of oxygen), the lighting equipment (fluorescent light with at 6000 or 3000 K) and the wrapping film properties (transparent or red plastic layers). The results obtained with these meat matrices were quite consistent, even though they had a different initial oxidative status that could be ascribed to the different PUFA profile, pigment level (horse > beef > poultry) [58e60], cholesterol content and holding time after slaughtering. Fresh sliced turkey breast was exposed in a bench fridge (4  C) in a vessel wrapped with plastic film and the effects of two irradiation sources were compared [14]. COPs displayed a bell-shaped trend during light exposure; they reached their maximum value after just one day of daylight fluorescence lamp exposure (6000 K), while it took up to 7 days when meat was kept under dark storage or subjected to a warm tone light (3000 K). For these reasons, it was strongly suggested to use a warm tone lamp as a display light in the retail bench fridges. The most abundant COP, in almost all cases, was 7-KC, which corresponded to about one third of total COPs; 7aHC, 7b-HC, b-EC, a-EC and triol were also detected. In a subsequent study, raw beef slices packed in a 32% oxygen atmosphere and standard atmosphere, were exposed to a warm tone lamp [15]; in the first case, the photooxidation process affected the whole thickness of the beef slice, while it was a superficial process in meat packed in standard atmosphere. The main cholesterol oxide was 7-KC (about 30% of total COPs), followed by 7b-HC, 7a-HC and b-EC; the cholesterol oxidation ratio (calculated as % COPs/cholesterol) ranged between 0.02% and 0.3%. The average COPs content in modified atmosphere (1.5e5.2 mg COPs/kg meat) was twice as much as in normal air conditions (0.4e2.7 mg COPs/kg meat), thus evincing that the prooxidant effect of the oxygen-enriched atmosphere was not compensated by the use of a warm tone lamp. Similar conclusions were attained by Ahn et al. [49] on cooked meat and by Nam et al. [48] on raw turkey leg, beef, and pork loin meat oxidation. Due to the high susceptibility of horse meat slices to oxidation, the use of a light-protecting red film for wrapping the vessels packed with sliced horse meat was evaluated and compared with a transparent film with a similar oxygen transmission rate [16]. The red film reduced the formation of COPs with respect to a transparent film. Major COPs found were 7-KC, 7a-HC and 7b-HC, which accounted for 76.5e83.2% of total COPs; the other COPs detected were b-EC, a-EC and triol. After 8 h of light exposure, cholesterol oxidation ratio was about 1.3% and 0.9% in samples wrapped with transparent and red film, respectively; these values, however, were much higher than those found in photooxidized beef meat (0.02e0.3%) [15], and could be ascribed to the greater PUFA level and pigment content of horse meat. Cardenia et al. [17] evaluated the effect of both dietary supplementation (with high-oleic sunflower oil and/or a-tocopheryl acetate) and light exposure on the extent of cholesterol oxidation in pork meat slices during storage in a commercial retail bench refrigerator. Meat slices were packed in vessels with transparent shrink film, stored at dark and under white fluorescent light (color temperature ¼ 3800 K) for 3 days at 8  C. Total COP amount ranged from 0.5 to 1.1 mg/kg meat, while the cholesterol oxidation rate varied from 0.1 to 0.3%. The most abundant COPs were 7-KC and b-EC, followed by a-EC, 7b-HC and 7a-HC; no triol was detected. 7-KC content was lower in samples supplemented with vitamin E compared with unsupplemented samples, thus confirming its antioxidant efficacy. A similar trend was also reported by Monahan et al. [61]. However, vitamin E exhibited a higher antioxidant

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activity during dark storage, which could be ascribed to its different scavenging capacity concerning radicals and singlet oxygen [62]; in fact, vitamin E has been found to display a lower scavenging affinity for the latter, being thus a more suitable antioxidant for autoxidized and/or thermoxidized samples rather than for photooxidized ones. In all these studies carried out on photooxidized meat [14e17], it was noticed that COPs, which are secondary oxidation products, also decomposed and/or reacted with other molecules (such as proteins) [63,64], generating compounds that could not be detected under the analytical conditions used. Sliced meat products have become very popular among consumers over the past few years, because they are practical and ready-to-eat. Photosensitized oxidation will be particularly critical in sliced meat products, because of the very large surface to volume ratio. Zanardi et al. [65] evaluated lipid oxidation in Milano-type fermented sausages as related to packing conditions and extended storage under fluorescent light. Matured sausages were sliced and packed under vacuum or in protective atmosphere (100% N2) and exposed in a display cabinet to mimic commercial conditions of light (12-h illumination cycle per day under fluorescent lamps (6500 K) and temperature (4  C)) for a total period of 2 months. During storage, COPs increased significantly, parallel to the increasing brown scores; 7-KC was the most abundant oxide, followed by a-EC and 7b-HC. Protective atmosphere was more efficient than vacuum in controlling fatty acid oxidation and, to a lesser extent, cholesterol and pigment degradation; in fact, cholesterol oxides gradually increased (from 0.52 to 1.37 mg/kg, equivalent to 0.06e0.15% oxidized cholesterol) but remained lower than those of vacuum-packed sausages (from 0.50 to 1.90 mg/kg, equivalent to 0.06e0.21% oxidized cholesterol). 2.2.1. Passive and active packaging for meat oxidation control One of the main strategies to prevent and control photosensitized lipid and cholesterol oxidation is packaging, as the technologist can operate at diverse levels (type of inner atmosphere, properties of the wrapping film (color, thickness, permeability to oxygen, light and moisture), simple vs. combined films). Multilayer gas barrier bags for prime cuts of meat and/or modified atmosphere packaging are the late 20th century innovations developed to protect food products from oxidation [66,67]. However, literature data show that even very small amounts of oxygen, i.e. 1e200 ppm (or mg of oxygen per kg of food), may cause a substantial quality loss. The levels of residual oxygen in most packaging systems are much higher, e.g. between 0.1% in vacuum packs and 2% in gas flushed packaging processes [68]. Among the latest innovations, active packaging is a challenging approach in order to prolong the food shelf-life by reducing nutrient losses and/ or protecting the initial quality of the food product [69]. Recently, active packaging systems that reduce the contact of food products with oxygen, have been developed. As the utilization of active packaging implies an additional cost, this type of packaging is mainly used for high-quality and/or longterm storage food products, such as meat and meat products. In meat and meat products, active packaging systems are asked to delay the oxidation process and reduce discoloration, so they often include oxygen scavengers. Kerry et al. [70] extensively described the use of both active and intelligent packaging systems for meat and muscle-based products. Oxygen scavengers are the alternative to vacuum and gas flushing technologies in order to prolong the shelf-life of fresh meat, because these techniques do not always assure the complete removal of oxygen; in the presence of oxygen scavengers, instead, the gas is trapped as soon as it permeates through the packaging film. Oxygen removers are usually based on oxidation of iron powder, but ascorbic acid, photosensitive dyes, unsaturated fatty acids (e.g. oleic and linoleic

477

acids) and enzymatic systems (e.g. glucose oxidase/catalase and alcohol oxidase) can also be employed. These reducing compounds are placed into small sachets, labels or pads and they can be combined with carbon dioxide emitters in sachet or tablets to help preventing partial packing collapse due to the decrease of the oxygen content. Different studies report that meat discoloration can be efficiently prevented by using such devices; for instance, Gill and McGinnis [71] demonstrated that a quite large number of oxygen scavengers can reduce the oxygen content of the meat pack to less than 10 ppm after a 2 h-storage at 1.5  C. However, oxygen absorbers can also be impregnated into packaging films; this seems to be the most preferable solution, since there is no risk of incidental ruptures of the container. In addition, the absence of foreign objects into the meat packing is usually preferred and would not lead to accidental ingestion by the consumer. 2.3. Milk and dairy products Milk contains approximately 120 mg of cholesterol per kg of milk fat [72], which is associated with the milk fat globule membrane [73]. It is well-known that the risk of COPs formation in fresh milk or fresh dairy products is low, due to milk fat organization into globules, to the reducing environment and to the low content of polyunsaturated fatty acids (PUFA) and prooxidant trace elements (such as iron and copper) [74]. Sander et al. [75] (detected 9 mg of 5,6a-EC and 1 mg of 5,6b-EC per kg of milk) used for cheese manufacture. However, the natural occurrence of photosensitizers (such as riboflavin, protoporphyrin), favors cholesterol photooxidation. This was confirmed by Chen et al. [76], where the combined effect of riboflavin and fatty acid methyl esters on cholesterol photooxidation, exposed to light at 25  C for 28 days, was tested in a model system. The authors reported that both riboflavin and fatty acid methyl esters could promote COPs formation; moreover, riboflavin displayed a more pronounced effect on cholesterol oxidation than fatty acid methyl esters [76]. 7KC was the most abundant COP, followed by 5,6b-EC, 5,6a-EC, 7aHC and 7b-HC; 3,5-cholestadien-7-one was also found, probably due to 7-KC dehydration [76]. Photooxidation in dairy products can be minimized by the utilization of suitable packaging and storage conditions, as they can block light, from ultraviolet radiation (UV) through the entire visible region [77]. However, when dairy products are directly subjected to light exposure, they can undergo light-induced oxidation and generate COPs. In any case, the natural acidity ascribable to the presence of lactic acid, can also contribute to the demolition of hydroperoxides, thus releasing radical species. Luby et al. [78] exposed butter to fluorescent light (1500 lux) and, after 20 days of treatment, 7a-HC and 7b-HC were identified; moreover, COPs were concentrated at the butter block surface, which highlights the photo-sensitization effect. Hiesberger et al. [79] stored three types of butter (fresh sweet cream butter, fresh sour cream butter and sour cream butter previously frozen for 6 months) under different light (daylight lamp L18w/11, light for food L18w/76 and UV lamp 40w/79 K) and temperature conditions (refrigerator (4  C) and room temperature (20  C)) for 6 weeks. After 23-days exposure, COPs level ranged from 0.12 to 1.66 mg/kg matrix, whereas COPs dramatically increased (36.2 mg/kg matrix) in sour cream butter stored under daylight lamp exposure for 42 days at room temperature. On the other hand, the light for food at refrigerator temperature generated more COPs than the daylight lamp, while UV lamp exposure for 4 days also led to detectable COPs amounts [79]. Nielsen et al. [80] reported that the exposure of sliced yellow cheese to fluorescent light for 55 days did not affect COPs level. On the contrary, when grated cheese was exposed to light for a longer

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period (72 days), 1.12 mg/kg of 7-KC were detected, followed by 7bHC (0.47 mg/kg) and 7a-HC (0.36 mg/kg); low levels (<0.10 mg/kg) of a-EC and b-EC were also found. In addition, in feta cheese stored in an open can, the highest value of COPs (246 mg/kg lipid) was detected after 30 days of storage and the 7-KC displayed a very marked accumulation (220 mg/kg lipids) [80]. Al-Ismail et al. [81] investigated the effect of light (daylight) on 7-KC formation in brined Nabulsi cheese stored for 9 months. The authors reported a quite low concentration of 7-KC (1.2 mg/kg) in freshly prepared cheese, which indicated a high oxidative stability of cholesterol during cheese processing; however, 7-KC concentration was 1.6 and 2.3 times higher in cheese exposed to light for 6 and 9 months, respectively, as compared to 7-KC levels found in cheese samples stored in light-protected conditions (jars covered with aluminum foil) [81]. In fact, the brined Nabulsi cheese reached 2.9 and 1.8 mg of 7-KC/kg after 6 months of storage under light exposure and light protected conditions, respectively, while 9month storage led to the corresponding formation of 5.2 and 2.3 mg of 7-KC/kg [81]. Finally, these findings lead to the conclusion that the type of light and packaging represent useful strategies to prevent and control the formation of cholesterol oxidation products in stored milk and dairy products. 2.4. Seafood and seafood products Fish is characterized by a large content of highly unsaturated fatty acids, which makes it particularly prone to lipid oxidation; however, in some species, the lipid fraction also presents elevated levels of cholesterol, thus favoring the formation of COPs [46]. These factors, if associated to light exposure during processing and/ or storage conditions, will further favor cholesterol oxidation [82]. Chen et al. [83] detected COPs in small sun-dried fish Spratelloides gracilis and Decapterus maruadsi, stored under normal atmosphere at room temperature for 3 months. 7a-HC, 7b-HC, 7-KC and 5,6a-EC were identified and the total level of COPs ranged from 4.82 to 65.7 mg/kg. To our knowledge, however, there are no studies published on photosensitized cholesterol oxidation of fish, under controlled light-exposure conditions. Baldacci et al. [84] evaluated photosensitized oxidation of cholesterol in sardines (Sardina pilchardus), during storage at commercial retail conditions. COPs detected in sardines stored under a daylight lamp (3800 K) for 4 h at 4  C were compared with those found on samples stored at dark. The authors reported a general increase of lipid oxidation after light exposure; cholesterol oxidation ratio varied from 0.1 to 0.9% and the most common COPs (7a-HC, 7b-HC, 5,6a-EC, 5,6b-EC, triol and 7-KC) were found. Relevant amounts of epoxy derivates were detected, which might be partly due to the interaction of sterols with hydrogen peroxide released by microbial enzymes naturally present in muscle tissues [9]. The impact of photosensitized oxidation on the COPs content was more pronounced as storage time increased and 4 h of light exposure led to the highest content of each COP, confirming that photosensitized oxidation affected COPs formation. Shrimps are crustaceans characterized by a high content of cholesterol [85], which is not affected by seasonability [86]. Luzia et al. [86] detected about 1650 mg/kg of cholesterol on wet weight basis in seabob shrimp, while Soto-Rodriguez et al. [87] found slightly lower cholesterol levels (1100e1492 mg/kg) in sun-dried shrimp. The presence of pigments and carotenoids, such as astaxanthin, can affect oxidation [88]. Although Meléndez-Martínez et al. [89] suggested that the antioxidant mechanism of astaxanthin is similar to that of b-carotene (singlet oxygen quencher), Bragadóttir et al. [90] highlighted that its oxidation products can act as prooxidants.

Sampaio et al. [91] evaluated the COPs content in salted dried shrimp. The shrimps were cooked in brine, followed by drainage for 4e8 h, with direct sunlight incidence. The main COPs were 7b-HC (34.6e72.6 mg/kg), 7a-HC (5.0e12.1 mg/kg), 7-KC (7.4e32.7 mg/kg) and 25-HC (2.4e22.9 mg/kg). Soto-Rodríguez et al. [87] quantified the eight major COPs (7a-HC, 7b-HC, a-EC, b-EC, 20-HC, 7-KC, triol, 25-HC) in sun-dried shrimps (traditional Mexican foods), and the total level of oxysterol ranged from 131 to 254 mg/kg. In both studies [87,91], the authors concluded that the processing and storage conditions led a high extent of cholesterol oxidation and that some modifications should be implemented in the production process to lower cholesterol degradation. 3. Photosensitized cholesterol oxidation in biological tissues 3.1. Retina and lens There are many studies that have investigated the multiple biological effects of oxysterols as related to the development of different diseases [10,27,64,92e95]. The retina is a light-capturing tissue that faces a photooxidative environment, involving challenges that are not encountered by other neurological tissues or organs [96]. The retina is able to form cholesterol, in order to maintain its dynamic steady-state lipid composition [97]; moreover, the retina is able to accumulate lipids and LDL in the choriocapillaris and Bruch’s membrane [98,99], thus oxysterols are originated by LDL oxidation. Oxysterols are involved in the regulation of cholesterol homeostasis and are also produced as intermediates in bile acid synthesis in the liver [100]. Javitt [101] reported that oxysterols are potential modulators of gene expression with specific oxysterol structures functioning as ligands for nuclear receptors. However, the excess formation and accumulation of oxysterols lead to pathological effects in cells and tissues. Several studies have suggested that there is a correlation between oxysterols accumulation and the increased IL-8 cytokine production and secretion by retinal pigment epithelium cells, as well as by other non-related retinal cell types [102e104]. Dugas et al. [105] proved that 7b-HC, 7-KC, and 25-HC have cytotoxic, oxidative, inflammatory, and/or angiogenic activities on ARPE-19 cells, and that COPS present in retina not only have the ability to induce IL8, but they also enhance the secretion of the vascular endothelium growth factor (VEGF). Catarino et al. [100] have recently suggested the use of 25-HC as a new molecular target for treatment of age-related macular degeneration; in fact, the authors reported that 25-HC is an inducer of IL-8 expression and secretion in ARPE-19 cells, in opposition to 7-KC, 7b-HC and cholesterol. Bretillon et al. [106] determined the presence of 24-HC in retina, whereas Moreira et al. [107] found traces of side-chain hydroxylated oxysterols and quantifiable levels of 7-KC in neural retina (1e1.5 pmol/nmol of free cholesterol) and pigment epithelium and choriocapillaris (5e8 pmol/nmol of free cholesterol). According to these results, it was hypothesized that cholesterol photooxidation, mediated by singlet oxygen, was the main oxidation mechanism involved. However, the author was able to find only 7-KC and no intermediates were detected. Moreover, the presence of photosensitizer molecules (such as A2E and all-trans retinoic acid) that are abundant in the retina, showed low photosensitizer activity [108,109]. It is possible that cholesterol oxidation may be mainly catalyzed by the presence of iron; in fact, it is well known that, in the presence of light, ferritin releases iron [110]. In addition, Yurkova et al. [111] found that oxidation by hydrogen peroxide can cause cytochrome c to release iron and oxidize cardiolipin via a free radical-mediated mechanism. Free metals are known to break down hydroperoxides, thus leading to the formation of radicals, as

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it happens when exposed to light. To our knowledge no study has reported direct interaction of light with photosensitizer, even though it is possible that the light is involved in cholesterol oxidation but not by singlet oxygen mediated mechanism. Cholesterol represents approximately 40% of total lipids of human lens fibers; photooxidation can therefore affect the level and/or partition of cholesterol and may alter optical lens properties, leading to the formation of cataract [112]. 7-KC may disrupt the highly regulated differentiation program of the lens epithelial cells, thus compromising lens growth and transparency [113]. Furthermore, 7-KC was found in human cataracts, but it was not detected in clear lenses [114]. 3.2. Skin The skin is a biological barrier, which separates the body from the environment and thus is exposed to solar light (ultraviolet irradiation). In addition, the chronic exposure of human skin to solar ultraviolet is known to induce photoaging by enhancing the oxidative stress. In particular, Bruls et al. [115] reported different interactions of UVA and UVB with skin; in fact, the latter (280e320 nm) attacks the epidermis but is not able to react with the dermis, while the UVA (320e400 nm) can penetrate the dermis leading to an oxidative damage. The oxidative process in the skin is enhanced by the presence of chromophores, which act as photosensitizer leading to photosensitized oxidation mediated by singlet oxygen [116]. Yamazaki et al. [117] determined free and ester forms of cholesterol 7a- and 7b-hydroperoxides (7-HPCs) in human skin lipids obtained from Japanese volunteers before and after sunlight exposure. The authors reported that the level of 7-HPCs before sunlight exposure were 3.76e32.6 pmol/cm2 skin, while after 3 h of sunlight exposure, the concentrations were 10.1e166 pmol/cm2 skin (1.4e11.7 folds higher than those found in unexposed samples). Minami et al. [118] found 7a-HPC, 7b-HPC and 5a-HPC in irradiated human keratinocyte cell line by UVA (Black-light blue fluorescent lamp, 2.5 J/cm2) in the presence of hematoporphyrin; this implies that hematoporphyrin acted as a type II photosensitizer, generating singlet oxygen by energy transfer. In addition, these authors investigated the effect of UVA irradiation in skin of hairless mice and the level of 5a-HPC was significantly higher than that found in non-irradiated samples, which indicates that singlet oxygen participated in the peroxidation of skin cholesterol. In order to prevent the oxidation, the skin is equipped with enzymatic and non-enzymatic antioxidant systems [118]. In addition, Thiele et al. [119,120] identified vitamin E as the predominant antioxidant present in the uppermost human skin layers, the stratum corneum and skin surface lipids. Mudiyanselage et al. [121] demonstrated that human sebum contains high levels of atocopherol, and suggested that the biologic role of the high concentrations of vitamin E present in skin surface lipids may serve as protection against oxidation, acting as radical scavengers. However, a helpful strategy to reduce the oxidation by singlet oxygen-mediated reactions may be the use of a singlet oxygen quencher, such as carotenoids. Terao et al. [122] demonstrated that dietary carotenoids seem to participate in the prevention of photooxidative stress by accumulating as antioxidants in the skin. In addition, the authors reported that the exposure of hairless mice to UVA led to the accumulation of 5a-HPC in skin lipids and the presence of 7a-HPC, 7b-HPC and 5a-HPC enhanced the activity of metalloproteinase-9, responsible of collagenase activity on collagen type IV (basement membrane of the skin) [122]. These results suggest that dietary b-carotene may impact metalloproteinase-9, thus reducing the formation of singlet oxygen and cholesterol oxidation products.

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4. Conclusions Cholesterol photosensitized oxidation is one of the main chemical degradations that occurs in food and biological tissues, leading to the formation of compounds that are related to aging, various chronic and degenerative diseases. The extent of such degradation will depend on the presence of antioxidants/prooxidants, the unsaturation degree of fatty acids, and environmental conditions. During photosensitized oxidation, not only the primary oxidation products (e.g. peroxides) are converted into secondary products, but also COPs are transformed into other products (i.e. protein-COPs binding) that cannot be detected by the standard chromatographic methods. COPs levels in food subjected to light exposure will greatly vary according to the matrix composition, surface/volume ratio, processing, packaging and storage conditions; in these cases, COPs formation can be minimized by adding antioxidants through feeding (such as vitamin E) or surface spraying before packaging (i.e. fat-soluble or watersoluble antioxidants), using low processing temperature, proper packaging (with low oxygen permeability, suitable light filters, colored/reflecting films, and protective atmosphere/vacuum) and appropriate lighting conditions (source, color, energy, distance). Furthermore, the combined use of some of these strategies during commercial retail storage, can efficiently prevent cholesterol photooxidation without modifying the food product composition and sensory properties. Although the cholesterol oxidation rate does not usually exceed 0.9% of cholesterol in food, further research is required on the actual toxicity levels of single and mixed COPs, to better ascertain if the COPs content in photooxidized food represents a risk for human health. On the other hand, the intake of antioxidant with a noticeable activity of singlet oxygen quenchers and radical scavengers (such as carotenoids, tocopherols and biophenols, respectively), could represent a useful strategy to suppress the photo-mediated damage in skin, as well as photoaging. Finally, further research is required about the COPs binding capacity and specificity with respect to proteins, in particular 7-KC as it is involved in the inflammatory pathways of retina and lens and this would allow to better understand how 7-KC initiates such inflammation processes. Acknowledgments This research was supported by the Inter-Departmental Centre for Agri-Food Industrial Research and the RFO-2010 funding. References [1] L. Cercaci, M.T. Rodriguez-Estrada, G. Lercker, E.A. Decker, Phytosterol oxidation in oil-in-water emulsions and bulk oil, Food Chem. 102 (2007) 161e167. [2] L.L. Smith, Cholesterol Autoxidation, Plenum Press, New York (USA), 1981. [3] L.L. Smith, The oxidation of cholesterol, in: S.K. Peng, R.J. Morin (Eds.), Biological Effects of Cholesterol Oxides, CRC Press, London (UK), 1981, pp. 7e31. [4] L.L. Smith, Review of progress in sterol oxidation: 1987e1995, Lipids 31 (1996) 453e487. [5] G. Lercker, M.T. Rodriguez-Estrada, Cholesterol oxidation: presence of 7ketocholesterol in different food products, J. Food Comp. Anal. 13 (2000) 625e631. [6] J.D. Wood, R.I. Richardson, G.R. Nute, A.V. Fisher, M.M. Campo, E. Kasapidou, P.R. Sheard, M. Enser, Effects of fatty acids on meat quality: a review, Meat Sci. 66 (2004) 21e32. [7] G.J.Jr. Schroepfer, Oxysterol: modulation of cholesterol metabolism and other processes, Phys. Rev. 80 (2000) 361e554. [8] P. Paniangvait, A.J. King, A.D. Jones, B.G. German, Cholesterol oxides in foods of animal origin, J. Food Sci. 52 (1995) 57e62. [9] S.J. Hur, G.B. Park, S.T. Joo, Formation of cholesterol oxidation products (COPs) in animal products, Food Control 18 (2007) 939e947. [10] A. Otaegui-Arrazola, M. Menendez-Carreno, D. Ansorena, I. Astiasaran, Oxysterols: a world to explore, Food Chem. Toxicol. 48 (2010) 3289e3303.

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