Biochimie 95 (2013) 473e481

Contents lists available at SciVerse ScienceDirect

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-Universit di Bologna, Piazza Goidanich 60, 47521 Cesena, Italy
Department of Food Science, Alma Mater Studiorum-Universit di Bologna, Viale G. Fanin 40, 40127 Bologna, Italy
c
Department of Agricultural, Food and Environmental Sciences, Universit 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.
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
uidity. 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
rst 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.: 39 051 2096011; fax: 39 051 2096017.
E-mail address: maria.rodriguez@unibo.it (M.T. Rodriguez-Estrada).
0300-9084/$ e see front matter 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 classied 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

474

V. Cardenia et al. / Biochimie 95 (2013) 473e481

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 inuenced 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 specied yet, so the threshold of toxicological concern (TTC) for
unclassied 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 rst 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 inuenced by processing and storage conditions [35,36]. One of the most critical
chemical modications 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.

V. Cardenia et al. / Biochimie 95 (2013) 473e481

475

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 uorescent 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 (unesteried)

[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)

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

51.9

Warm tone uorescent lamp; normal

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

Cataract

Ref.

[18]

Human lensc

Identied 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 efcient 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 efcacy [37]; in fact, zeaxanthin appeared to be a better

photoprotector during prolonged UV exposure, while the shorter

protective efcacy 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

476

V. Cardenia et al. / Biochimie 95 (2013) 473e481

needed to signicantly 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 uorescent light (40 W). Herian and Lee [40] studied the formation of the
7-hydroxy derivatives in dry egg nog mix exposed to uorescent
light (40 W) for 105 days at 23  C, where they observed an increase
of such COPs during the rst 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 signicantly 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 inuence 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 inuenced 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
(modied 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 modied with high percentage of oxygen), the
lighting equipment (uorescent light with at 6000 or 3000 K) and
the wrapping lm 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 prole, 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 lm 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 uorescence 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 rst case, the photooxidation process
affected the whole thickness of the beef slice, while it was
a supercial 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 modied 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 lm for wrapping the vessels
packed with sliced horse meat was evaluated and compared with
a transparent lm with a similar oxygen transmission rate [16]. The
red lm reduced the formation of COPs with respect to a transparent lm. 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 lm, 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 sunower 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 lm, stored at dark and under white uorescent light (color
temperature 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 conrming its
antioxidant efcacy. A similar trend was also reported by Monahan
et al. [61]. However, vitamin E exhibited a higher antioxidant

V. Cardenia et al. / Biochimie 95 (2013) 473e481

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 afnity
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 uorescent 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 uorescent
lamps (6500 K) and temperature (4  C)) for a total period of 2
months. During storage, COPs increased signicantly, parallel to the
increasing brown scores; 7-KC was the most abundant oxide, followed by a-EC and 7b-HC. Protective atmosphere was more efcient 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 lm (color, thickness, permeability to oxygen, light and moisture), simple vs. combined lms).
Multilayer gas barrier bags for prime cuts of meat and/or modied
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 ushed 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 ushing 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 lm. 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 efciently 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 lms; 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 riboavin, protoporphyrin), favors cholesterol photooxidation. This was conrmed by Chen et al. [76], where the
combined effect of riboavin 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
riboavin and fatty acid methyl esters could promote COPs
formation; moreover, riboavin 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 uorescent light (1500 lux)
and, after 20 days of treatment, 7a-HC and 7b-HC were identied;
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 uorescent light for 55 days did not affect COPs level. On
the contrary, when grated cheese was exposed to light for a longer

478

V. Cardenia et al. / Biochimie 95 (2013) 473e481

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 ndings 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 sh 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 identied 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 sh, 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, conrming 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 Melndez-Martnez
et al. [89] suggested that the antioxidant mechanism of astaxanthin
is similar to that of b-carotene (singlet oxygen quencher), Bragadttir 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-Rodrguez et al. [87] quantied
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 modications 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 Bruchs 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 specic 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, inammatory, 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 quantiable 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 nd 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

V. Cardenia et al. / Biochimie 95 (2013) 473e481

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 bers; 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
uorescent 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 signicantly
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] identied 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.

479

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 lters,
colored/reecting lms, 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 efciently 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 specicity with respect to
proteins, in particular 7-KC as it is involved in the inammatory
pathways of retina and lens and this would allow to better
understand how 7-KC initiates such inammation 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.

480

V. Cardenia et al. / Biochimie 95 (2013) 473e481

[11] F. Guardiola, P.C. Dutta, R. Codony, G.P. Savage, Cholesterol and Phytosterol
Oxidation Products: Analysis, Occurrence and Biological Effects, AOCS Press,
Champaign IL (USA), 2002.
[12] S. Garcia-Cruset, K.L.H. Carpenter, R. Codony, F. Guardiola, Cholesterol
oxidation products and atherosclerosis, in: F. Guardiola, P.C. Dutta,
R. Codony, G.P. Savage (Eds.), Cholesterol and Phytosterols Oxidation Products: Analysis, Occurrence, and Biological Effects, AOCS Press, Champaign IL
(USA), 2002, pp. 241e277.
[13] K. Osada, Cholesterol oxidation products: other biological effects, in:
F. Guardiola, P.C. Dutta, R. Codony, G.P. Savage (Eds.), Cholesterol and
Phytosterols Oxidation Products: Analysis, Occurrence, and Biological Effects,
AOCS Press, Champaign IL (USA), 2002, pp. 278e318.
[14] E. Boselli, M.F. Caboni, M.T. Rodriguez-Estrada, T.G. Toschi, M. Daniel,
G. Lercker, Photooxidation of cholesterol and lipids of turkey meat during
storage under commercial retail condition, Food Chem. 91 (2005) 705e713.
[15] E. Boselli, M.T. Rodriguez-Estrada, G. Fedrizzi, M.F. Caboni, Cholesterol
photosensitised oxidation of beef meat under standard and modied
atmosphere at retail conditions, Meat Sci. 81 (2009) 224e229.
[16] E. Boselli, M.T. Rodriguez-Estrada, F. Ferioli, M.F. Caboni, G. Lercker,
Cholesterol photosensitised oxidation of horse meat slices stored under
different packaging lms, Meat Sci. 85 (2010) 500e505.
[17] V. Cardenia, M.T. Rodriguez-Estrada, F. Cumella, L. Sardi, G. Della Casa,
G. Lercker, Oxidative stability of pork meat lipids as related to high-oleic
sunower oil and vitamin E diet supplementation and storage conditions,
Meat Sci. 88 (2011) 271e279.
[18] V. Verardo, F. Pasini, G. Iafelice, M.C. Messia, E. Marconi, M.F. Caboni, Inuence of storage conditions on cholesterol oxidation in dried egg pasta,
J. Agric. Food Chem. 58 (2010) 3586e3590.
[19] A.W. Girotti, New trends in photobiology: photosensitized oxidation of
cholesterol in biological systems: reaction pathways, cytotoxic effects and
defense mechanisms, J. Photochem. Photobiol. B 13 (1992) 105e118.
[20] C.S. Foote, Mechanisms of photosensitized oxidation, Science 162 (1968)
963e970.
[21] E.N. Frankel, Lipid Oxidation, The Oily Press Ltd, Dundee (UK), 1998.
[22] M.J. Kulig, L.L. Smith, Sterol metabolism. XXV. Cholesterol oxidation by
singlet molecular oxygen, J. Org. Chem. 38 (1973) 3639e3642.
[23] W. Korytowski, A.W. Girotti, Singlet oxygen adducts of cholesterol: photogeneration and reductive turnover in membrane systems, Photochem. Photobiol. 70 (1999) 484e489.
[24] J.H. Nielson, C.E. Olsen, L.H. Skibsted, Cholesterol oxidation in a heterogeneous system initiated by water-soluble radicals, Food Chem. 56 (1996)
33e37.
[25] B. Sottero, P. Gamba, S. Gargiulo, G. Leonarduzzi, G. Poli, Cholesterol oxidation products and disease: an emerging topic of interest in medicinal
chemistry, Curr. Med. Chem. 16 (2009) 685e705.
[26] S. Bsinger, W. Luf, E. Brandl, Oxysterols: their occurrence and biological
effects, Int. Dairy J. 3 (1993) 1e33.
[27] A. Vejux, G. Lizard, Cytotoxic effects of oxysterols associated with human
diseases: induction of cell death (apoptosis and/or oncosis), oxidative and
inammatory activities, and phospholipidosis, Mol. Aspects Med. 30 (2009)
153e170.
[28] C. Mascia, M. Maina, E. Chiarpotto, G. Leonarduzzi, G. Poli, F. Biasi, Proinammatory effect of cholesterol and its oxidation products on CaCo-2
human enterocyte-like cells: effective protection by epigallocatechin-3gallate, Free Radic. Biol. Med. 49 (2010) 2049e2057.
[29] N. Shibata, C.K. Glass, Macrophages, oxysterols and atherosclerosis, Circ. J. 74
(2010) 2045e2051.
[30] M. Umetani, P.W. Shaul, 27-Hydroxycholesterol: the rst identied endogenous SERM, Trends Endocrin. Metab. 22 (2011) 130e135.
[31] R. Kroes, A.G. Renwick, M. Cheeseman, J. Kleiner, I. Mangelsdorf, A. Piersma,
B. Schilter, J. Schlatter, F. Van Schothorst, J.G. Vos, G. Wurtzen, Structurebased thresholds of toxicological concern (TTC): guidance for application to
substances present at low levels in the diet, Food Chem. Toxicol. 42 (2004)
65e83.
[32] J. Galobart, F. Guardiola, Formation of content of cholesterol oxidation
products in egg and egg products, in: F. Guardiola, P.C. Dutta, R. Codony,
G.P. Savage (Eds.), Cholesterol and Phytosterols Oxidation Products: Analysis,
Occurrence, and Biological Effects, AOCS Press, Champaign IL (USA), 2002, pp.
124e146.
[33] M.R. Mazalli, N. Bragagnolo, Effect of storage on cholesterol oxide formation
and fatty acid alterations in eggs powder, J. Agric. Food Chem. 55 (2007)
2743e2748.
[34] D.H. Berquist, Egg dehydration, in: W.J. Stadelman, O.J. Cotterill (Eds.), Egg Science
and Technology, fourth ed., Haworth, Binghamton N.Y., 1995, pp. 335e376.
[35] J. Galobart, F. Guardiola, A.C. Barroeta, S. Lpez-Ferrer, M.D. Baucells, Inuence of dietary supplementation with a-tocopheryl acetate and canthaxanthin on cholesterol oxidation in u-3 and u-6 fatty acid-enriched spray-dried
eggs, J. Food Sci. 67 (2002) 2460e2466.
[36] M.F. Caboni, E. Boselli, M.C. Messia, V. Velazco, A. Fratianni, G. Panli,
E. Marconi, Effect of processing and storage on the chemical quality markers
of spray-dried whole egg, Food Chem. 92 (2005) 293e303.
[37] A. Sujak, J. Gabrielska, W. Grudziski, R. Borc, P. Mazurek, W.I. Gruszecki,
Lutein and zeaxanthin as protectors of lipid membranes against oxidative
damage: the structural aspects, Arch. Biochem. Biophys. 371 (1999)
301e307.

[38] M. Wrona, W. Korytowski, M. Ranowska, T. Sarna, T.G. Truscott, Cooperation of antioxidants in protection against photosensitized oxidation, Free
Radic. Biol. Med. 35 (2003) 1319e1329.
[39] E. Chicoye, W.D. Powrie, O. Fennema, Photooxidation of cholesterol in spraydried egg yolk upon irradiation, J. Food Sci. 33 (1968) 581e587.
[40] A.M. Herian, K. Lee, 7a- and 7b-hydroxycholesterols formed in a dry egg nog
mix exposed to uorescent light, J. Food Sci. 50 (1985) 276e277.
[41] P. Van de Bovenkamp, T.G. Kosmeijer-Schuil, M.B. Katan, Quantication of
oxysterols in dutch foods: egg products and mixed diets, Lipids 23 (1988)
1079e1085.
[42] A. Fontana, F. Antoniazzi, M.L. Ciavata, E. Trivellone, G. Cimino, 1H-NRM
study of cholesterol autoxidation in egg powder and cookies exposed to
adverse storage, J. Food Sci. 58 (1993) 1286e1290.
[43] A. Obara, M. Obiedzinski, T. Kolczak, The effect of water activity on cholesterol oxidation in spray- and freeze-dried egg powders, Food Chem. 95
(2005) 173e179.
[44] L. Rizzi, M. Simioli, G. Martelli, R. Paganelli, L. Sardi, Effects of organic farming
on egg quality and welfare of laying hens, Worlds Poult. Sci. J. 62 (2006) 165.
[45] J. Kerry, D.A. Gilroy, N.M. OBrien, Formation and content of cholesterol
oxidation products in meat and meat products, in: F. Guardiola, P.C. Dutta,
R. Codony, G.P. Savage (Eds.), Cholesterol and Phytosterols Oxidation Products: Analysis, Occurrence, and Biological Effects, AOCS Press, Champaign IL
(USA), 2002, pp. 162e185.
[46] T. Ohshima, Formation and content of cholesterol oxidation products in
seafood and seafood products, in: F. Guardiola, P.C. Dutta, R. Codony,
G.P. Savage (Eds.), Cholesterol and Phytosterols Oxidation Products: Analysis,
Occurrence, and Biological Effects, AOCS Press, Champaign IL (USA), 2002, pp.
187e203.
[47] G. Maerker, Cholesterol autoxidation e current status, J. Am. Oil Chem. Soc.
64 (1987) 388e392.
[48] K.C. Nam, M.C. Du, C. Jo, D.U. Ahn, Cholesterol oxidation products in irradiated raw meat with different packaging and storage time, Meat Sci. 58
(2001) 431e435.
[49] D. Ahn, K.C. Nam, M. Du, C. Jo, Effect of irradiation and packaging conditions
after cooking on the formation of cholesterol and lipid oxidation products in
meats during storage, Meat Sci. 57 (2001) 413e418.
[50] E.A. Decker, Z. Xu, Minimizing rancidity in muscle foods, J. Food Technol. 52
(1998) 54e59.
[51] E. Boselli, D. Pacetti, P. Lucci, P. Di Lecce, N. Frega, Supplementation with
high-oleic sunower oil and a-tocopheryl acetate: effects on meat pork
lipids, Eur. J. Lipid Sci. Technol. 110 (2008) 381e391.
[52] K. Eder, G. Mller, H. Kluge, F. Hirche, C. Brandsch, Concentrations of oxysterols in meat and meat products from pigs fed diets differing in the type of
fat (palm oil or soybean oil) and vitamin E concentrations, Meat Sci. 70
(2005) 15e23.
[53] Q. Guo, B.T. Richert, J.R. Burgess, D.M. Webel, D.E. Orr, M. Blair, G.E. Fitzner,
D.D. Hall, A.L. Grant, D.E. Gerrard, Effects of dietary vitamin E and fat
supplementation on pork quality, J. Anim. Sci. 84 (2006) 3089e3099.
[54] F.J. Francis, F.M. Clydesdale, Food Colorimetry: Theory and Applications, The
Avi Publishing Company Inc., Westport CT (USA), 1975, pp. 292e318.
[55] F.J. Francis, Pigments and other colorants, in: O. Fennema, Marcel Dekker
(Eds.), Food Chemistry, second ed., New York (USA), (1985), pp. 545e584.
[56] M. Bekblet, Light effects on food, J. Food Prot. 53 (1990) 430e440.
[57] M. Du, K.C. Nam, D.U. Ahn, Cholesterol and lipid oxidation products in
cooked meat as affected by raw-meat packaging and irradiation and by
cooked-meat packaging and storage time, J. Food Sci. 66 (2001) 1396e1401.
[58] J.D. Wood, M. Enser, A.V. Fisher, G.R. Nute, P.R. Sheard, R.I. Richardson,
S.I. Hughes, F.M. Whittington, Fat deposition, fatty acid composition and
meat quality: a review, Meat Sci. 78 (2008) 343e358.
[59] D. Pacetti, F. Alberti, E. Boselli, N.G. Frega, Characterization of furan fatty
acids in Adriatic sh, Food Chem. 122 (2010) 209e215.
[60] J.C. De Almeida, M.S. Perassolo, J.L. Camargo, N. Bragagnolo, J.L. Gross, Fatty
acid composition and cholesterol content of beef and chicken meat in
Southern Brazil, Braz. J. Pharm. Sci. 42 (2006) 109e117.
[61] F.J. Monahan, J.I. Gray, A.M. Booren, E.R. Miller, D.J. Buckley, P.A. Morrissey,
E.A. Gomaa, Inuence of dietary treatment on lipid and cholesterol oxidation
in pork, J. Agric. Food Chem. 40 (1992) 1310e1315.
[62] H.J. Kim, D.B. Min, Chemistry of lipid oxidation, in: C.O. Akoh, D.B. Min (Eds.),
Food Lipids: Chemistry, Nutrition, and Biotechnology, third ed., CRC Press,
Boca Raton (FL, USA), 2008, pp. 299e364.
[63] M.T. Rodriguez-Estrada, G. Penazzi, M.F. Caboni, G. Bertacco, G. Lercker,
Effect of different cooking methods on some lipid and protein components of
beef hamburger, Meat Sci. 45 (1997) 365e375.
[64] V.M. Olkkonen, R. Hynynen, Interaction of oxysterols with membranes and
proteins, Mol. Aspects Med. 30 (2009) 123e133.
[65] E. Zanardi, V. Dorigoni, A. Badiani, R. Chizzolini, Lipid and colour stability of
Milano-type sausages: effect of packing conditions, Meat Sci. 61 (2002) 7e14.
[66] A.L. Brody, B. Bugusu, J.H. Han, C. Koelsch Sand, T.H. Mc Hugh, Innovative
food packaging solutions, J. Food Sci. 73 (2008) R107eR116.
[67] P.N. Skandamis, G.J.E. Nyachas, Preservation of fresh meat with active and
modied atmosphere packaging conditions, Int. J. Food Microbiol. 79 (2002)
35e45.
[68] S. Amberg-Schwab, U. Weber, Development of oxygen scavenger and indicator systems for the enhancement and indication of food quality,
Fraunhofer ISC Annual Report (2006) 32e35.

V. Cardenia et al. / Biochimie 95 (2013) 473e481

[69] T.P. Labuza, An introduction to active packaging for foods, Food Technol. 50
(1996) 68e71.
[70] J.P. Kerry, M.N. OGrady, S.A. Hogan, Past, current and potential utilization of
active and intelligent packaging systems for meat and muscle-based products: a review, Meat Sci. 74 (2006) 113e130.
[71] C.O. Gill, J.C. McGinnis, The use of oxygen scavengers to prevent the transient
discoloration of ground beef packaged under controlled, oxygen-depleted
atmospheres, Meat Sci. 41 (1995) 19e27.
[72] H.G. Walte, Die Natrliche Variation des Cholesteringehaltes in der Rohmilch. Dissertation. Universitt Kiel (1994) 1e109.
[73] N.O.V. Sonntag, Baileys Industrial Oil and Fat Products, fourth ed., vol. 1,
Wiley-Interscience, New York, 1979.
[74] R. Sieber, Oxidised cholesterol on milk and dairy products, Inter. Dairy J. 15
(2005) 191e206.
[75] B.D. Sander, D.E. Smith, P.B. Addis, Effects of processing stage and storage
conditions on cholesterol oxidation products in butter and Cheddar cheese,
J. Diary Sci. 71 (1998) 3173e3178.
[76] J.T. Chen, Y.F. Lu, P.C. Hu, B.H. Chen, Cholesterol photooxidation as affected
by combination of riboavin and fatty acid methyl esters, Food Chem. 81
(2003) 421e431.
[77] J.B. Webster, S.E. Duncan, J.E. Marcy, S.F. OKeefe, Controlling light oxidation
avour in milk by blocking riboavin excitation wavelengths by interference,
J. Food Sci. 74 (2009) S390eS398.
[78] J.M. Luby, J.I. Gray, B.R. Harte, Effects of packaging and light source on the
oxidation stability of cholesterol in butter, J. Food Sci. 51 (1986) 908e911.
[79] J. Hiesberger, W. Luf, Oxidation of cholesterol in butter during storage-effects
of light and temperature, Eur. Food Res. Technol. 211 (2000) 161e164.
[80] J.H. Nielsen, C.E. Olsen, C. Duedahl, L.H. Skibsted, Isolation and quantication
of cholesterol oxides in dairy products by selected ion monitoring mass
spectrometry, J. Dairy Res. 62 (1995) 101e113.
[81] K.M. Al-Ismail, M.A. Humied, Effect of processing and storage of brined white
(Nabulsi) cheese on fat and cholesterol oxidation, J. Sci. Food Agric. 83 (2003)
39e43.
[82] F.R. De Oliveira, G.M. Lira, Oxidation of cholesterol in sh (review), B. CEPPA
27 (2009) 143e152.
[83] J.S. Chen, G.C. Yen, Cholesterol oxidation products in small sun-dried sh,
Food Chem. 50 (1994) 167e170.
[84] E. Baldacci, Study about cholesterol oxidation in fresh sardines (Sardina pilchardus) subjected to photosensitized oxidation, Master thesis on Health
Biology, Alma Mater Studiorum-Universit di Bologna 2011.
[85] I. Astiasarn, D. Ansorena, M. Echarte, A. Conchillo, M. Menndez-Carreo,
Cholesterol oxidation products in common cooked foods, An. R. Acad. Nac.
Farm 73 (2007) 1159e1174.
[86] L.A. Luzia, G.R. Sampaio, C.M.N. Castellucci, E.A.F.S. Torres, The inuence of
season on the lipid proles of ve commercially important species of Brazilian sh, Food Chem. 83 (2003) 93e97.
Soto-Rodrguez,
P.J.
Campillo-Velzquez,
J.
Ortega-Martnez,
[87] I.
M.T. Rodriguez-Estrada, G. Lercker, H.S. Garcia, Cholesterol oxidation in
traditional Mexican dried and deep-fried food products, J. Food Compos.
Anal. 21 (2008) 489e495.
[88] E.J. Vernon-Carter, J.T. Ponce-Palafox, R. Pedroza-Islas, Pigmentation of
Pacic white shrimp (Penaeus vannamei) using Aztec marigold (Tagetes
erecta) extracts as the carotenoid source, Arch. Latinoam. Nutr. 46 (1996)
243e246.
[89] A. Melndez-Martnez, I. Vicario, F. Heredia, Importancia nutricional de los
pigmentos carotenoids, Arch. Latinoam. Nutr. 54 (2004) 149e154.
[90] M. Bragadttir, Endogenous antioxidants in sh, Masters thesis (2001)
Department of Food Science, University of Iceland 2001.
[91] G.R. Sampaio, D.H.M. Bastos, R.A.M. Soares, Y.S. Queiroz, E.A.F.S. Torres, Fatty
acids and cholesterol oxidation in salted and dried shrimp, Food Chem. 95
(2006) 344e351.
[92] S. Lordan, J.J. Mackrill, N.M. OBrien, Oxysterols and mechanisms of apoptotic
signaling: implications in the pathology of degenerative diseases, J. Nutr.
Biochem. 20 (2009) 321e336.
[93] G. Poli, B. Sottero, S. Gargiulo, G. Leonarduzzi, Cholesterol oxidation products
in the vascular remodeling due to atherosclerosis, Mol. Aspects Med. 30
(2009) 180e189.
[94] I. Bjrkhem, A. Cedazo-Minguez, V. Leoni, S. Meaney, Oxysterols and
neurodegenerative diseases, Mol. Aspects Med. 30 (2009) 171e179.
[95] A.J. Brown, W. Jessup, Oxysterols: sources, cellular storage and metabolism,
and new insights into their roles in cholesterol homeostasis, Mol. Aspects
Med. 30 (2009) 111e122.
[96] I.R. Rodriguez, I.M. Larrayoz, Cholesterol oxidation in the retina: implications
of 7KCh formation in chronic inammation and age-related macular
degeneration, J. Lipid Res. 51 (2010) 2847e2862.
[97] S.J. Fliesler, R. Florman, L.M. Rapp, S.J. Pittler, R.K. Keller, In vivo biosynthesis
of cholesterol in the rat retina, FEBS Lett. 335 (1993) 234e238.
[98] N. Tserentsoodol, J. Sztein, M. Campos, N.V. Gordiyenko, R.N. Fariss, J.W. Lee,
S.J. Fliesler, I.R. Rodriguez, Uptake of cholesterol by the retina occurs

[99]

[100]

[101]
[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]
[110]

[111]
[112]

[113]
[114]
[115]

[116]

[117]

[118]

[119]

[120]

[121]

[122]

481

primarily via a low density lipoprotein receptor-mediated process, Mol. Vis.

12 (2006) 1306e1318.
J.W. Ruberti, C.A. Curcio, C.L. Millican, B.P. Menco, J.D. Huang, M. Johnson,
Quick-freeze/deep-etch visualization of age-related lipid accumulation in
Bruchs membrane, Invest. Ophthalmol. Vis. Sci. 44 (2003) 1753e1759.
S. Catarino, C.F. Bento, A. Brito, E. Murteira, A.F. Fernandes, P. Pereira,
Regulation of the expression of interleukin-8 induced by 25hydroxycholesterol in retinal pigment epithelium cells, Acta Ophthalmol.
(2012). http://dx.doi.org/10.1111/j.1755e3768.2011.02350.x.
N.B. Javitt, Oxysterols: novel biologic roles for the 21st century, Steroids 73
(2008) 149e157.
B. Bai, K. Yamamoto, H. Sato, H. Sugiura, T. Tanaka, Combined effect of
25-hydroxycholesterol and IL-1beta on IL-8 production in human colon
carcinoma cell line (Caco-2), Inammation 29 (2005) 141e146.
C. Erridge, D.J. Webb, C.M. Spickett, 25-Hydroxycholesterol, 7beta-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression
independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages, Free Radic. Res. 41 (2007) 260e266.
I.M. Larrayoz, J.D. Huang, J.W. Lee, I. Pascual, I.R. Rodriguez,
7-Ketocholesterol-induced inammation: involvement of multiple
kinase signaling pathways via NFkappaB but indepen- dently of reactive
oxygen species formation, Invest. Ophthalmol. Vis. Sci. 51 (2010)
4942e4955.
B. Dugas, S. Charbonnier, M. Baarine, K. Ragot, D. Delmas, F. Mntrier,
J. Lherminier, L. Malvitte, T. Khalfaoui, A. Bron, C. Creuzot-Garcher,
N. Latruffe, G. Lizard, Effects of oxysterols on cell viability, inammatory
cytokines, VEGF, and reactive oxygen species production on human retinal
cells: cytoprotective effects and prevention of VEGF secretion by resveratrol,
Eur. J. Nutr. 49 (2010) 435e446.
L. Bretillon, U. Diczfalusy, I. Bjorkhem, M.A. Maire, L. Martine, C. Joffre,
N. Acar, A. Bron, C. Creuzot-Garcher, Cholesterol- 24S-hydroxylase
(CYP46A1) is specically expressed in neurons of the neural retina, Curr. Eye
Res. 32 (2007) 361e366.
E.F. Moreira, I.M. Larrayoz, J.W. Lee, I.R. Rodriguez, 7-Ketocholesterol is
present in lipid deposits in the primate retina: potential implication in the
induction of VEGF and CNV formation, Invest. Ophthalmol. Vis. Sci. 50 (2009)
523e532.
A. Pawlak, M. Wrona, M. Rozanowska, M. Zareba, L.E. Lamb, J.E. Roberts,
J.D. Simon, T. Sarna, Comparison of the aerobic photoreactivity of A2E with
its precursor retinal, Photochem. Photobiol. 77 (2003) 253e258.
H.S. Harper, E.R. Gaillard, Studies of all-trans-retinal as a photooxidizing
agent, Photochem. Photobiol. 73 (2001) 71e76.
K. Ohishi, X.M. Zhang, S. Moriwaki, T. Hiramitsu, S. Matsugo, Iron release
analyses from ferritin by visible light irradiation, Free Radic. Res. 39 (2005)
875e882.
I. Yurkova, D. Huster, J. Arnhold, Free radical fragmentation of cardiolipin by
cytochrome c, Chem. Phys. Lipids 158 (2009) 16e21.
A. Vejux, M. Samadi, G. Lizard, Contribution of cholesterol and oxysterols in
the physiopathology of cataract: implication for the development of pharmacological treatments, Article ID 471947, J. Ophthalmol. (2011) 6.
H. Girao, F. Shang, P. Pereira, 7-Ketocholesterol stimulates differentiation of
lens epithelial cells, Mol. Vis. 9 (2003) 497e501.
H. Girao, M.C. Mota, J. Ramalho, P. Pereira, Cholesterol oxides accumulate in
human cataracts, Exp. Eye Res. 66 (1998) 645e652.
W.A. Bruls, H. Van Weedlden, J.C. Van der Leun, Transmission of UV-radiation
through human epidermal layers as a factor inuencing the minimal
erythema dose, Photochem. Photobiol. 39 (1984) 63e67.
J. Krutmann, Ultraviolet A radiation-induced biological effects in human
skin: relevance for photoaging and photodermatosis, J. Dermatol. Sci. 23
(2000) S22eS26.
S. Yamazaki, N. Ozawa, A. Hiratsuka, T. Watabe, Increases in cholesterol 7hydroperoxides in lipids of human skin by sunlight exposure, Free Radic.
Biol. Med. 26 (1999) 1126e1133.
Y. Minami, S. Yokoi, M. Setoyama, N. Bando, S. Takeda, Y. Kawai, J. Terao,
Combination of TLC blotting and gas chromatography-mass spectrometry for
analysis of peroxidized cholesterol, Lipids 42 (2007) 1055e1063.
J.J. Thiele, F. Dreher, L. Packer, Antioxidant defense systems in skin, in:
P. Elsner, H. Maibach (Eds.), Drugs Vs. Cosmetics: Cosmeceuticals, Marcel
Dekker, New York, 2000, pp. 145e188.
J.J. Thiele, C. Schroeter, S.N. Hsieh, M. Podda, L. Packer, The
antioxidant network of the stratum corneum, Curr. Probl. Dermatol. 29
(2001) 26e42.
S.E. Mudiyanselage, M. Hamburger, P. Elsner, J.J. Thiele, Ultraviolet A induces
generation of squalene monohydroperoxide isomers in human sebum and
skin surface lipids in vitro and in vivo, J. Invest. Dermatol. 120 (2003)
915e922.
J. Terao, Y. Minami, N. Bando, Singlet molecular oxygen-quenching activity of
carotenoids: relevance to protection of the skin from photoaging, J. Clin.
Biochem. Nutr. 48 (2011) 57e62.