Journal of Photochemistry and Photobiology B: Biology 97 (2009) 71–76

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

An ex vivo methodology to assess the lipid peroxidation in stratum corneum

Cristina Alonso a,*, Clara Barba a, Laia Rubio a, Sonya Scott c, Anna Kilimnik d, Luisa Coderch a,Jaime Notario b, José Luis Parra a

a Department of Chemical Technology, Institute of Advanced Chemistry of Catalonia IQAC, Barcelona, Spainb Department of Dermatology, University Hospital of Bellvitge, U.B Barcelona, Spainc AgResearch Limited, Lincoln Research Centre, Christchurch, New Zealandd National Institute of Water and Atmospheric Research Limited (NIWA), Wellington, New Zealand

a r t i c l e i n f o

Article history:Received 11 May 2009Received in revised form 23 July 2009Accepted 10 August 2009Available online 15 August 2009

Keywords:AntioxidantEx vivoLipid peroxidationStratum corneumUV irradiation

1011-1344/$ - see front matter � 2009 Elsevier B.V. Adoi:10.1016/j.jphotobiol.2009.08.003

* Corresponding author. Address: IQAC, Jordi GiroSpain. Tel.: +34 934 006 100; fax: +34 932 045 904.

E-mail address: cristina.alonso@iqac.csic.es (C. Alo

a b s t r a c t

Environmental risks, particularly UV radiation, provide a challenge to the function of the skin barrier. Pro-tective measures such as the use of antioxidant products represent a possible method of providing pro-tection to the skin.

This paper reports the development of a non-invasive ex vivo method using tape strips of the outermostlayers of stratum corneum (SC) from human volunteers in order to determine the effectiveness of an anti-oxidant emulsion topically applied to prevent lipid peroxidation (LPO) in the horny layer after an UV irra-diation exposure. Two different formulations were used: formulation (A), containing Vitamin A, E and C,and formulation (B) containing fish extract. Both formulations were topically applied in vivo on volunteerforearms; then, a tape stripping of the SC of each volunteer was carried out. The lipid peroxidation wasmeasured ex vivo after an UV irradiation of the SC samples. The amount of SC stripped to evaluate differ-ences in lipid peroxidation, the UV irradiation intensity to form lipid peroxides and the accuracy of lipidperoxide analysis were optimized in this methodology using formulation (A). After an exposure applica-tion of seven days, a group of three strips of the outermost layers of SC of volunteers was irradiated withan intensity of 182.7 J/cm2 to quantify the LPO inhibition.

The percentage of LPO inhibition obtained after topical application of both formulations was in therange of 40–58% demonstrating the effectiveness of the formulations topically applied against lipid per-oxidation on human SC. This methodology may be used as a quality control tool to determine ex vivo thepercentage of the LPO inhibition on human SC for a variety of antioxidants topically applied.

� 2009 Elsevier B.V. All rights reserved.

1. Introduction

The role of the skin as a multipurpose defensive system (e.g.biochemical, biological, immunological, physical, mechanical, ther-mal) has long been an active field of research of investigative der-matology [1]. UV radiation poses an environmental threat to theskin by increasing the risk of photooxidative damage due to reac-tive oxygen species (ROS) such as the superoxide anion radical,hydrogen peroxide, hydroxyl radical, and singlet oxygen [2,3].

The epidermis is composed mainly of keratinocytes, rich in ROSdetoxifying enzymes, such as superoxide dismutase, catalase, thio-redoxin reductase, and glutathione peroxidase, and in low-molec-ular-mass antioxidant molecules, such as tocopherol, glutathione,and ascorbic acid; thus, it provides some natural protection againstoxidative stress [4]. Studies have shown that cutaneous photopro-tection involve antioxidant strategies that prevent damage to SC

ll rights reserved.

na 18-26, 08034 Barcelona,

nso).

lipids and proteins [5,6] which are directly exposed to a prooxida-tive environment [7]. Moreover, some systemically applied antiox-idants accumulate in the outermost layer of epidermis, SC, and playan important role against UV-induced photodamage in skin. Thephotoprotective effects of Vitamin E and C have been studiedextensively [8,9]. Significantly reduced acute skin responses suchas sunburn cell formation, lipid peroxidation, DNA adducts andUVA-induced binding of photosensitizers have been demonstratedwhen those vitamins are applied on skin.

A large number of natural extracts have been shown to containcompounds that have antioxidant activity. The majority of theseare plant extracts, for example green tea [10], grapes [11] and blue-berries [12]. Very few fish extracts have been tested for antioxidantactivity and only initial ROS scavenging activity assays have beenpreviously reported [13].

Oxidative damage of lipids and proteins [14,15] is known to bean immediate consequence following UV radiation of human skin[16,17]. Lipid peroxidation in skin appears as a result of photooxi-dative damage. The basic reactions of lipid peroxidation are due tothe high reactivity of methylene groups of the polyunsaturated

72 C. Alonso et al. / Journal of Photochemistry and Photobiology B: Biology 97 (2009) 71–76

fatty acids to oxidizing agents. The lipid peroxidation can be deter-mined by measuring the losses of unsaturated fatty acids, theamounts of primary peroxidation products (hydroperoxides) andthe amounts of secondary products/end products (aldehydes, pen-tane and ethane).

Malondialdehyde (MDA) is one of several low-molecular-weight end products formed via the decomposition of certain pri-mary and secondary lipid peroxidation products [18]. The quanti-fication of MDA (as the free aldehyde or its thiobarbituricderivative) may be used as a measurement to detect and quantifylipid peroxidation in an injured tissue. The thiobarbituric acid(TBA) test measures amounts of MDA formed to quantify lipid per-oxidation. Several authors have been used in vitro methodologiesto determine the amount of LPO species formed. Svobodova et al.[19] used a human keracinocyte as an appropriate experimentalin vitro model to measure the evolution of ROS generation byTBA test. Prasad et al. [20] used human lymphocytes to evaluatethe activity of antioxidant enzymes and TBA reacting substancesafter UV exposure. Another in vitro model to examine the protec-tive effect of antioxidants on UV-induced lipid peroxidation isbased on use of human fibroblasts [21–23]. Other authors com-pared the effect of sunscreen and antioxidants on reconstructedepidermis (with or without melanocytes) [24] or in a simple skinlipid model system and complex skin adapted lipid system [25]after UV irradiation.

The effect of a topical antioxidant formulation using in vivomethodologies has also been studied. Rahman et al. [26] evaluatedthe efficacy of Vitamin E in preventing the oxidative stress re-sponse in a 12-O-tetradecanolyphorbal-13-acetate application intumor promotion mice, with the consequence sacrifice of animalsafter the application. For in vivo methodologies the gold standardis the use of human volunteers. Schneider et al. [27] measuredthe photodamage by LPO in a study surveyed by personal dosime-try to obtain a correlation analysis of acquired dose and amount ofLPO. Other authors released studies to demonstrate the efficacy ofantioxidant compounds upon skin parameters of human subjects,taking a dietary supplement and topical formulation containingantioxidant compounds [28,29]. All these in vivo methodologiesare invasively techniques for volunteers because some erythemasmaybe induced in human skin using UV radiation. A new techniquewas used by Moeskops et al. [30] which biomarkers of lipid perox-idation were monitored using proton-transfer reaction. This tech-nique detect trace of gases emitted by human skin in vivo non-invasively but, to induce lipid peroxidation the volunteer wereirradiated with UV light.

Brown [31] measured antioxidant benefits of skincare productsinducing LPO in SC layers sampled by D-squame from forearms ofvolunteers. In the present study a similar non-invasive ex vivomethodology is proposed. Thus, a method using tape strips of theoutermost layers of SC from human volunteers has been developedin order to determine the effectiveness of two formulations topi-cally applied to prevent lipid peroxidation in the horny layer ofSC after an UV irradiation exposure.

Table 2Age and phototype of volunteers (Emulsion B).

Volunteer 11 12 13 14 15 16 17 18 19 20 Mean ± SD

Age 29 30 53 47 54 53 29 40 29 31 39.5 ± 11.2Phototype III IV IV IV III IV III IV III II

Table 1Age and phototype of volunteers (Emulsion A).

Volunteer 1 2 3 4 5 6 7 8 9 10 Mean ± SD

Age 34 30 28 28 37 49 30 40 30 28 33.4 ± 6.9Phototype III IV III III III III IV IV IV III

2. Materials and methods

2.1. Topical formulations applied

Two different emulsions were investigated in this study.Formulation (A) is an antioxidant formulation containing Vita-

min A acetate (0.1%), Vitamin E (1%) and Vitamin C (5%) (suppliedby Acofarm, Barcelona, Spain) in a conventional base emulsion:Lanette wax 20%w/w (Acofarm, Barcelona, Spain), Tween 801%w/w (Fagron Iberica, Barcelona, Spain), Nipasol 0.03%w/w (Fa-gron Iberica, Barcelona, Spain) and Deionised water (to 100%w/w).

Formulation (B) is a Base Hand Crème (AgResearch Limited,New Zealand) containing a sterile, water-soluble fish extract (2%).The fish extract had been earlier identified as having significantin vitro antioxidant activity (NIWA, New Zealand). The base creamformulation (without antioxidants) has the following ingredients:Crodamol GTCC (10.0%w/w), Polawax NF (5.0%w/w), Stearic acid(3.0%w/w), Crodacol C90 EP (2.0%w/w), Ultrez21 (2%solution)(8.0%w/w), Phenova (0.80%w/w) supplied by Croda (England) andDeionised water (to 100%w/w). This formulation was prepared byAgResearch Limited (New Zealand).

A skin area of 18 � 5 cm2 was marked in the volunteer forearms.270 mg of selected emulsion was applied and distributed homoge-nously with a gloved finger in order to apply about 3 mg/cm2 ofthose preparations, according to OCDE guidelines [32,33]. Formula-tions were applied by each volunteer twice a day over a period of7 days on one forearm. A placebo emulsion without any antioxidantor fish extract was applied on the other forearm as a control.

2.2. Volunteers

The experimental protocol for the antioxidant formulation (A)was conducted with 10 healthy Caucasian volunteers (all women)with phototypes III and IV [34]. The mean age of the volunteerswas 33.4 ± 6.9 years old (range 28–49 years) (Table 1).

The study with the fish extract formulation (B) was done with10 healthy Caucasian volunteers (all females) with skin types II,III, and IV [34] and with a mean age of 39.5 ± 11.2 years old (range29–54) (Table 2).

The volunteers refrained from using cosmetics, body oils, sunsc-reens or moisturizers on their arms 7 days prior to the study andduring the study except the two formulations selected.

The participants were given a detailed description of the study,and the corresponding written consents were obtained. The studydesign was approved by the local Ethics Committee (IQAC-CSIC,Barcelona, Spain).

2.3. Tape stripping and lipid extraction

The tape stripping of the SC of each forearm was carried out onthe seventh day on a conditioned room at 25 ± 1 �C and about 50%of relative humidity, by D-SquameTM tapes (/ = 22 mm, CuDerm,Dallas, USA) previously pressed onto the skin with a roller andstripped in one quick move. A piece of paper was placed betweentape and roller avoiding a transfer of emulsion from the adjacentpart of the skin to the adhesive tape. The weight of the tapes wasdetermined directly (Sartorius BP121S, Göttingen, Germany) be-fore and after stripping, obtaining then the weight of stratum cor-neum removed. Then, half of the strips were fixed in a glass plateand irradiated using a light source simulating UV solar radiation(3.045 J/min cm2, Suntest CPS, Atlas, USA). This radiation is doubleof the maximum radiation of one day of June in Catalonia (1.50–

C. Alonso et al. / Journal of Photochemistry and Photobiology B: Biology 97 (2009) 71–76 73

1.84 J/min cm2 equivalent to 21–26.5 MJ/m2 per day [35]). More-over, the UV irradiation intensity was modified with three differentexposure times: 30 min (91.35 J/cm2), 60 min (182.7 J/cm2), and120 min (365.4 J/cm2). The SC lipid extraction from the stripswas made with methanol (Merck Darmstadt, Germany) and after-wards sonicated in a Labsonic 1510 device (B. Braun, Melsungen,Germany) for 15 min.

2.4. Thiobarbituric acid-reactive species (TBARS) assay

Lipid peroxides (LPO species) were measured by thiobarbituricacid (TBA) assay [36] with some modifications. The thiobarbituricacid-reactive species (TBARS) were quantified by spectrophotome-try at 534 nm (Cary 300 Bio UV–Visible Spectrophotometer, Varian,USA). At low pH and elevated temperature, MDA readily partici-pates in nucleophilic addition reaction with TBA, generating ared, fluorescent 1:2 MDA:TBA adduct according to the reactionshown in Fig. 1.

The results obtained were expressed as malonaldehyde bis(di-methyl acetal) equivalents (lM MDA) using a standard curve forpure MDA–TBA complex. The calibration curve was obtained byusing MDA (Sigma, St Louis, MO, USA) at different concentrations(5–40 lM). Also, negative and positive controls were quantifiedusing 0 and 100 lM of MDA. The calibration curve was preparedon each day of the experimental study.

Briefly, the LPO species containing in the samples (0.5 mL) wasadded to aliquots (1 mL) of a solution made up with 0.4% of TBA(Sigma, St Louis, MO, USA) and 15% of trichloroacetic acid (TCA)(Merck, Darmstadt, Germany) in 100 mL of HCl solution (0.25 M).The mixture was incubated for 1 h in a water boiling bath. FreshTBA/TCA stock solution was prepared on each day of analysis.

2.5. Determination of LPO

The linearity graphs of standard MDA solutions were obtainedwith the mean value of the triplicate analysis by TBARS assay

Fig. 2. Scheme of the experimental procedure used. Th

Fig. 1. Reaction proposed for the detection of M

indicated in Section 2.4. The linearity of standard MDA sampleswas found in the concentration range of 0–40 lM. A general linearregression equation from the experimental analysis was obtainedABS = 0.0576 [MDA] + 0.0271(correlation coefficient, r2 = 0.999).The repeatability and reproducibility of the analytical methodwas confirmed from the absorbance of the MDA positive control(100 lM MDA). The mean value obtained for this MDA control(100 lM) was 2.3632 ± 0.0037. The results for each experimentalanalysis indicate a good repeatability and intra-day precision withan acceptable R.S.D. (<0.50%).

A general scheme of the experimental protocol used is shown inFig. 2.

This methodology permits the evaluation of the lipid peroxidesspecies from the outermost layers of the stratum corneum formedby UV irradiation.

Following the experimental procedure indicated in Fig. 2, thequantity of LPO formed from the unprotected skin (placebo),non-irradiated (Po) and irradiated (Px), and amount of LPO formedfrom protected skin (antioxidant emulsion), non-irradiated (Ao)and irradiated (Ax), respectively, were calculated from absorbancevalues obtained of SC extraction samples by MDA determinations.Therefore, the following four values, expressed as lM MDA fromlinear equation, were obtained for each volunteer:

Po: SC lipid peroxides from strips of forearm without antioxi-dant emulsion and without UV irradiation.Px: SC lipid peroxides from strips of forearm without antioxi-dant emulsion but with UV irradiation.Ao: SC lipid peroxides from strips of forearm with antioxidantemulsion without UV irradiation.Ax: SC lipid peroxides from strips of forearm with antioxidantemulsion with UV irradiation.

The percentage of LPO inhibition (% LPO inhib.) was determinedfrom the difference in values of amount of LPO formed between theSC placebos and SC antioxidant samples, applying the Eq. (1).

e different abbreviations are explained in the text.

DA after a lipid peroxidation process [36].

74 C. Alonso et al. / Journal of Photochemistry and Photobiology B: Biology 97 (2009) 71–76

LPOð% inhibitionÞ ¼ ð1� ½Ax� Ao�=½Px� Po�Þ � 100 ð1Þ

In practical terms, a 0% of LPO inhibition means that the antiox-idant does not protect at all the skin against UV irradiation. On thecontrary, a 100% of LPO inhibition means that the formulation usedhas a complete antioxidant capacity.

2.6. Statistics

Standard deviations were calculated for all % of LPO inhibitionmean values. Analysis of variance (ANOVA) with a one-way layoutwas applied for group comparisons. The software used was theSTATGRAPHICS plus 5. Significant differences in the mean valueswere evaluated by the F-test. A p value below 0.05 was consideredsignificant.

3. Results and discussion

Several in vitro methodologies have been described to deter-mine the amount of LPO species formed due to photooxidativedamage [19–25]. Some others authors have reported in vivo meth-odologies to demonstrate the efficacy of several antioxidant com-pounds [28,29]. All these in vivo methodologies are invasivelytechniques for volunteers.

In the ex vivo methodology proposed in this work, following anidea previously described by Brown [31], to assess the lipid perox-idation in SC, the study was performed using human volunteers.Unlike other methods the volunteers are not directly UV irradiated.Only the outermost layers of the human skin, after being obtainedby the stripping methodology, were irradiated avoiding inductionsany kind of erythema in the volunteers. This is a simple methodnon-invasive for the volunteers.

The experimental procedure has been previously optimized tak-ing account both the amount of lipids extracted per strip and theintensity of UV irradiation. Afterwards, the LPO inhibition (%) hasbeen determined, using the most external layers of SC by stripping,when two different antioxidant formulations were topically ap-plied on human volunteers.

3.1. Optimization of the methodology used for the determination ofLPO

One critical experimental factor of the protocol used was toknow the quantity of lipid extracted for a given strip to obtain sig-nificant results by spectrophotometry. In order to determine thenumber of collected strips to extract the amount of LPO speciesfor the TBA assay, a preliminary experiment was performed withthe antioxidant formulation (A). Formulation was applied on oneforearm of six volunteers during a period of 7 days and the outer-most layers of SC were obtained in tapes following the protocolindicated above. After UV irradiation of 182.7 J/cm2 on half of thestrips containing SC layers, the lipids were extracted from a group

Table 3LPO values determined (P0 and A0 basal values without and with antioxidant, respectively,two different amounts of strips (three or five) irradiated at 182.7 J/cm2. Also, the % LPO in

Num. strips Volunteer LPO no antiox. L

Po (lM) Px (lM) A

3 2 0.0934 4.5377 03 0.1946 7.5353 04 0.0003 3.4748 0

5 1 0.0816 1.4794 05 0.0091 2.4511 06 0.1004 1.2525 0

a Not statistically significant.

of three or five tapes. The total amount of stratum corneum fromfive strips was 1.81 ± 0.72 mg SC/five strips whereas total amountfrom three strips was 1.01 ± 0.24 mg SC/three strips.

The LPO was measured for each group, using the linear equationpreviously obtained and the results are indicated in Table 3. Themean result on LPO inhibition (%) obtained from five strips was58.20 ± 11.74 whereas the mean result obtained from three stripswas 65.62 ± 29.26.

As it can be seen in Table 3, no significant differences werefound in the mean values obtained (p > 0.05). It can be deducedthat, in both cases, the results obtained for % LPO inhibition weresimilar. As a consequence, three strips of the SC were selected asan optimal number of adhesive tapes to be performed during theexperimental work.

Also, the experimental procedure was optimized taking into ac-count the UV irradiation intensity needed to form lipid peroxides,using again the antioxidant formulation (A). The UV irradiationintensity was modified as a function of the exposure time usingfour volunteers. After a week of application of the formulation onthe forearm volunteer, the outermost layers of SC were sampledin tape strips. The UV irradiation times on strips containing SCwere 30, 60, and 120 min with irradiation intensity associated to91.35, 182.7, and 365.4 J/cm2, respectively. The LPO inhibition re-sults (%) obtained using those irradiation intensities are shown inTable 4. A very low percentage of LPO inhibition was obtained(1.52 ± 1.25%) with the irradiation intensity of 91.35 J/cm2, whichwas significantly lower compared to the values obtained with theothers two intensities (p < 0.05). As a consequence, this irradiationintensity was discarded.

As it can be seen in Table 4, the LPO values obtained in the caseof irradiation intensities of 182.7 and 365.4 J/cm2 from the unpro-tected skin (Px–Po) were higher than the values from the protectedskin with antioxidant formulation (Ax–Ao). These results demon-strate a lower formation of lipid peroxides in the stratum corneumof the skin protected after UV irradiation when the antioxidant for-mulation was used. It seems that the antioxidant formulation usedhas a protector effect on human stratum corneum against the lipidperoxidation, obtaining a percentage of LPO inhibition of59.58 ± 19.09 and 20.86 ± 5.80 for irradiation intensities of182.7 J/cm2 and 365.4 J/cm2, respectively.

In order to better visualize the results, the LPO inhibition valuesobtained for the different UV intensities are graphically plotted inFig. 3. It can be seen that the samples irradiated with an intensityof 365.4 J/cm2 resulted in a lower % LPO inhibition values than thevalues obtained when an intensity of 182.7 J/cm2 was used withsignificant difference (p < 0.05).

An UV irradiation intensity of 365.4 J/cm2 promoted high for-mation of lipid peroxides in SC even when an antioxidant formula-tion was topically applied. On the other hand, a high UV radiationintensity provokes a major death of the SC cells. It seems that theantioxidant formulation has less protective effect against lipid per-oxidation when high UV irradiation is used for a long exposure

and Px and Ax final irradiated values without and with antioxidant, respectively) usinghibition values are indicated.

PO antiox. % LPO inhib. % LPO mean ± SD

o (lM) Ax (lM)

.3005 0.9390 85.60

.8666 2.3920 79.22 65.62 ± 29.26a

.9207 3.2822 32.03

.2065 0.6661 67.12

.5213 1.8668 44.90 58.20 ± 11.74a

.8053 1.2365 62.57

Table 4LPO values (P0 and A0 basal values without and with antioxidant, respectively, and Px and Ax final irradiated values without and with antioxidant, respectively) using thedifferent irradiation intensities of 91.35, 182.7, and 365.4 J/cm2. Also, the % LPO inhibition values are indicated.

Irradiation intensity (J/cm2) Volunteer LPO no antiox. LPO antiox. % LPO inhib. % LPO mean ± SD

Po (lM) Px (lM) Ao (lM) Ax (lM)

91.35 1 0.2742 0.7902 0.5895 1.0924 1.392 0.0911 0.4940 0.2981 0.6882 3.16 1.52 ± 1.25a

3 0.1968 0.3078 0.3666 0.4763 1.434 0.0003 0.3473 0.5843 0.9310 0.12

182.7 1 0.0003 0.9203 0.7949 1.1404 62.505 0.2842 0.8474 0.5085 0.6998 66.02 59.58 ± 19.09b

6 0.4760 1.2394 0.1280 0.6430 32.547 0.6520 1.5826 0.6277 0.8395 77.24

365.4 2 1.0505 4.6094 1.0187 3.9701 17.074 0.6138 3.4439 0.6867 2.7009 28.83 20.86 ± 5.80c

5 0.2842 3.2721 0.5085 3.0156 16.097 0.0520 2.4852 0.0277 1.9406 21.43

a/b = (p < 0.05); a/c = (p < 0.05); b/c = (p < 0.05).

Fig. 4. LPO inhibition results (%) with standard deviations obtained for eachvolunteer 1–10 and mean value, when formulation (A) was topically applied.

C. Alonso et al. / Journal of Photochemistry and Photobiology B: Biology 97 (2009) 71–76 75

time. An irradiation time of 60 min was chosen to be the optimal tostudy the antioxidant effect of a formulation.

3.2. Determination of % LPO inhibition of two antioxidant formulations

Once obtained the optimal experimental conditions, a furtherstudy with six more volunteers was undertaken by topically apply-ing the antioxidant formulation (A) for 7 days. Similarly, a secondstudy with 10 volunteers was done by topically applying the for-mulation (B) containing fish extract. The results obtained on themean weight of SC for both studies were similar and in the rangeof 1.01 ± 0.24 mg/three tape strips indicated in Section 3.1.

Half of the SC layers (from unprotected and protected skin) instrips of both formulations were irradiated with an intensity of182.7 J/cm2. LPO compounds were extracted from a group of threestrips (irradiated and non-irradiated). The LPO values and the per-centage of LPO inhibition due to the application of both formula-tions were determined for all volunteers. The results of the LPOinhibition (%) for the antioxidant formulation (A) are graphicallyplotted in Fig. 4.

On the other hand, Fig. 5 contains the results on LPO inhibition(%) obtained for fish extract formulation (B).

As it can be seen in Figs. 4 and 5, despite the variability of thepercentage of LPO inhibition values obtained, due to the influenceof several variables such as the amount of formulation applied, dif-ferent skin types of volunteers, amount of SC present in the sam-ples, etc., the results obtained indicate a global % LPO inhibitionof 57.37 ± 20.70 for the formulation (A) and 41.86 ± 18.08% LPOfor the formulation (B). These results obtained were not statisti-cally significant for the two formulation tested (p < 0.05).

Assuming that a similar amount of stratum corneum is re-trieved on the three strips (1.10 ± 0.29 mg SC/three strips) the per-

Fig. 3. Percentage of LPO inhibition values obtained with the different UVintensities applied. �p < 0.05.

centage of LPO inhibition was calculated as it was previouslydescribed in Section 2.5 with the Po, Px, Ao and Ax as the totalamount of lipid peroxides on the stratum corneum treated or notwith the antioxidant with or without UV irradiation. However, tak-ing into account the amount of SC retrieved in each case, all theseparameters Po, Px, Ao, and Ax can be calculated as the total amountof lipid peroxides per mg of SC. Calculations were performed forthe two formulations applied and a higher % LPO inhibition was

Fig. 5. LPO inhibition results (%) with standard deviations obtained for eachvolunteer 11–20 and mean value, when formulation (B) was topically applied.

76 C. Alonso et al. / Journal of Photochemistry and Photobiology B: Biology 97 (2009) 71–76

found in each case, namely 68.39 ± 20.92 for the first formulation(A) and 51.84 ± 32.99 for the second formulation (B).

The content of Vitamin A acetate, Vitamin E, and Vitamin C inFormulation A represent a 6% of vitamin present in formulationwhereas the fish extract is used in Formulation B at a 2%. Thoughthe content of compounds with antioxidant properties is minorin Formulation B, it is important to remark that both have a similarcapacity against the lipid peroxidation in human stratum corneum(p < 0.05).

From the results obtained, it can be deduced that the ex vivomethodology designed, and conveniently optimized, in this workto assess the lipid peroxidation in SC stripped from unprotectedor protected skin using two antioxidant formulations may be suit-able enough to evaluate the beneficial effect of a given antioxidanttopically applied against UV radiation.

4. Conclusions

The study presents a new methodology with the particularitythat allows the evaluation of the antioxidant effect of a given for-mulation after an in vivo topical application followed by anex vivo UV irradiation of the outermost layers of the SC. After somepreliminary experiments to optimize the methodology, an UV irra-diation with intensity of 182.7 J/cm2 was selected to determine theLPO inhibition of SC layers extracted from a group of three strips ofthe outermost layers of SC of volunteers.

This experimental protocol was used to evaluate the LPO inhibi-tion capacity of two different antioxidant formulations. The LPOinhibition calculated from the results obtained was57.37 ± 20.70% (68.39 ± 20.92% considering the amount of SC ex-tracted) for the antioxidant formulation (A) with Vitamins A ace-tate, E and C, and 41.86 ± 18.08% (51.84 ± 32.99% considering theamount of SC extracted) for the base crème containing fish extract(B).

It has been confirmed the different capacity of a tested antiox-idant formulation to reduce lipid peroxidation in the outermostlayers of the skin. This ex vivo methodology, suitably optimized,can be useful to test the antioxidant capacity of a given formula-tion to be topically applied.

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