skin res tec

Non-invasive monitoring of oxidative skin stress by
ultraweak photon emission measurement.
II:biological validation on ultraviolet A-stressed skin
Ralf Hagens 1,Faryar Khabiri 1,2,Volker Schreiner 1,Horst Wenck 1,Klaus-Peter Wittern 1,
Hans-Ju ¨rgen Duchstein 2and Weiping Mei 1
1
F&E cosmed,Beiersdorf AG,Hamburg,Germany and
2
Institut fu
¨r Pharmazie der Universita ¨t Hamburg,Hamburg,Germany Background/purpose:Several physical or chemical envir-onmental stressors generate reactive oxygen species,
which trigger oxidation reactions of cells or tissues and thereby induce a correlated ultraweak photon emission (UPE)signal.The present study was designed to qualify and validate UPE measurement following ultraviolet (UV)excitation of porcine and human skin as an analytical method to assess the potency of topical antioxidants in vivo .
Methods:UPE of porcine skin in vitro and human skin in vivo following excitation with UVA was recorded using sensitive photomultiplier systems.For validation purposes,the effects of variation of extrinsic and intrinsic parameters encompassing skin thickness,humidity,temperature,pH,and composition of the surrounding atmosphere were as-sessed.Signals were analyzed with regard to overall signal intensity and spectral distribution.In two clinical trials en-rolling 20volunteers each,the effects of topical antioxidant treatment on UVA-induced UPE were validated.
Results:Different stressors encompassing exposition to ozone,UVA irradiation,or even cigarette smoke induced UPE of skin.Critical parameters affecting the quality and quantity of the UPE signal were the spectral composition of
the exciting UV light,skin temperature,skin humidity,and the O 2concentration of the surrounding atmosphere.Gen-erally,UVA-induced UPE decreased with increasing tem-perature,humidity,and O 2concentration.Skin pH had no significant effect on UPE with regard to signal quality and quantity over a pH range of 2.8–8.2.In a clinical study UPE measurement following UVA excitation could precisely re-flect a dose-dependent antioxidant effect of topically applied vitamin C and a -glucosylrutin.
Conclusion:Our data indicate that UVA irradiation induces UPE especially in deeper (living)skin layers,where antiox-idants must be active in order to interfere with accelerated skin ageing.Based on the clinical data,and with knowledge of modulating external variables,UPE measurement follow-ing UV excitation can be qualified as a reliable and valid method for the non-invasive measurement of antioxidant efficacy on the skin.
Key words:UPE –antioxidants –in vivo measurements –oxidative stress
&Blackwell Munksgaard,2007
Accepted for publication 14July 2006
T
HE SKIN as the outermost barrier of the body is exposed to exogenous oxidative damage by reactive oxygen species (ROS)resulting from UV irradiation,ozone,or air pollutants.It is further-more constantly exposed to oxidative effects resulting from endogenous metabolic sources of ROS.A subtle network of enzymatic and non-enzymatic antioxidants allows the skin to detox-ify ROS (1).Increased generation of ROS,how-ever,exhausts the antioxidative capacity of the skin and causes damage to a variety of biomole-cules,including DNA (2),lipids (3),and proteins (4).The results are accelerated skin ageing with a loss of elasticity and wrinkle formation (5),light
dermatoses in susceptible subjects such as poly-morphous light eruption,(PLE)and,due to an accumulation of genetic damage,an increased risk of skin cancer (6).
Ultraviolet (UV)irradiation of cells or tissues generates ROS that trigger oxidation reactions and thereby induce UPE as a by-product of these reactions.This process requires endogen-ous chromophores (photosensitizers),such as porphyrins (protoporphyrin),flavins (riboflavin),quinones (ubiquinone),or co-enzymes (NADH and NADPH)that either transfer radiation en-ergy to 3O 2,yielding ROS (especially 1O 2)(7,8),or directly induce the generation of free radicals.
112
Skin Research and Technology 2008;14:112–120&2007The Authors
Journal compilation &2007Blackwell Munksgaard
Printed in Singapore ÁAll rights reserved Skin Research and Technology
doi:10.1111/j.1600-0846.2007.00207.x
ROS and resulting oxidation products like pro-tein carbonyls could be detected after UV irradia-tion of structural proteins of the dermis(9,10). Measuring techniques to quantify the extent of oxidative skin stress are needed for establishing successful treatment strategies to combat skin ageing and other processes of skin degeneration. Most contemporary techniques are based on the chemical analysis and quantification of lipid or protein oxidation products in skin samples or biopsies.Among them,determination of protein carbonyl compounds is the most common method for detecting oxidative protein modifica-tions in biological samples.Such measurements are hampered by the time-consuming sample preparation and the short half-life of the reaction products and intermediates.Furthermore,due to legal restrictions and limited acceptance from volunteers,such invasive procedures are largely restricted to animal studies,which are forbidden for cosmetics in the European Union(EU)in future.Methods that allow non-invasive investi-gation of oxidative processes would therefore be preferred in case of volunteer studies.
The problem of invasiveness may be overcome by measurement of ultraweak photon emission (UPE).The term UPE stands for luminescence phenomena(UV,VIS,or IR spectral range)of very weak intensity(0.01–104h/s),which occur in biological systems or samples without the use of light-enhancing substances(11).Recent studies have shown a clear correlation and dependence of the UPE signal on oxidative processes.The factors with the strongest inducing effect on UPE are those exogenous stressors,which lead to oxidation of biomolecules like lipids,DNA,and proteins.A reduction of UVA-induced UPE of the skin could be determined after topical applica-tion of several antioxidants(12–15).It must be pointed out that UPE is clearly distinct from the ATP-consuming enzymatic process of luciferin-based bioluminescence,which has an intensity of 103–1012h/s and a quantum yield of sometimes over95%,and that is often visible to the naked eye.
Owing to the biological complexity of the skin, the quantitative contribution of the many sources of UPE to the overall quantum emission of oxidation stressed skin cannot be determined currently.However,the fact that the oxidation of proteins and amino acids is associated with generation of UPE(16,17),and that a higher level of protein carbonyl compounds is found in the skin after exposure to oxidants like UV,benzoyl peroxide(BPO),HOCl,or ozone(10,18),suggests that protein oxidation plays a key role in the generation of stress-induced UPE on the skin.In a previous report,we could demonstrate that oxi-dation-induced UPE of the skin is dominated by proteins as the major source of emitted photons. Intensity and spectral distribution of oxidation induced UPE in this case are mainly determined by energy transfer and photon emission from oxidized amino acids.Among the amino acids involved,Trp functions as a terminal emitter in this case,which accepts energy from other acti-vated amino acid species.The UPE spectrum of oxidation-stressed skin is therefore dominated by the Trp UPE emission spectrum(19). Materials and Methods
UPE measuring systems
UPE system(m.u.t.GmbH,Wedel,Germany): UPE was recorded in a dark room using a photo-multiplier system.The detector was equipped with an Electron Tube9558QD(Electron Tubes Ltd.,Ruislip,UK),having a spectral sensitivity of 200–800nm,and maximum sensitivity at approxi-mately450nm.The technical set-up has already been described in an earlier publication(13). SRUPE system(m.u.t.GmbH):For spectral analyses,the detector(Electron Tube9635QD, spectral sensitivity:200–650nm,and maximum sensitivity atapproximately450nm)was equipp-ed with a synchronized rotating(10Hz)filter disk containing six long-passfilters encompassing a wavelength range of420–550nm.
Proprietary technical details and specifications of the applied UPE recording systems will not be disclosed in this report,but may be requested from the corresponding author under certain circumstances.
UV A irradiation
UV light was generated using a300W xenon lamp(Oriel-lamp,L.O.T.-Oriel GmbH,Darm-stadt,Germany)with a spectral range of 4250nm and an intensity maximum between 350and475nm.UVA light was generated by in-sertion of two opticalfilters(WG335and UG11, SCHOTT AG,Mainz,Germany)into the optical path of the xenon lamp.The light source was coupled to the UPE and SRUPE measuring sys-tems via liquid light guides and a light shutter.
113 Biological validation on ultraviolet A-stressed skin
Porcine skin samples for UPE measurement Unscalded skin of the back of pigs selected for slaughter(females,approximately130days old) was obtained from a local butcher and processed and measured on the same day as the slaughter. The skin was shaved and rinsed with tap water after removal of the subcutaneous fatty tissue. Afterwards,round pieces(d55cm)were punched out,packed in an airtight plastic bag, and stored at41C until measurement.Porcine skin is a useful ex vivo model for human skin because it is readily available and closely resem-bles human skin with regard to anatomical and biochemical properties(20–22).
Study populations and treatments
Vitamin C study
For UVA-induced UPE studies on intact human skin following topical treatment with vitamin C, 20female volunteers with an age range of18–65 years were recruited from the volunteer pool of Beiersdorf AG(Hamburg,Germany)according to defined eligibility criteria.All volunteers had given written informed consent to the study. The study subjects were topically treated with three antioxidant standard formulations(0.25%, 0.5%,and1%vitamin C)and the respective vehicle control on their inner forearms twice daily for3consecutive days.Application sites were randomized for each individual test subject. The vehicle-controlled UPE measurements took place approximately16h after the last applica-tion.The measurements(measuring time:40s) started500ms after irradiation using a dose of 86mJ/cm2UVA(exposure time15s).
a-glucosylrutin study
For UVA-induced UPE studies on intact human skin following topical treatment with a-glucosyl-rutin,23male and female volunteers with an age range of22–60years were recruited from the volunteer pool of Beiersdorf AG according to defined eligibility criteria.Nineteen patients (18w11m)completed the study and were in-cluded in the analysis.All volunteers had given written informed consent to the study.The anti-oxidant and vehicle control were applied twice daily for7consecutive days.Test sites as well as untreated reference sites on both inner forearms were randomly assigned for each individual test subject.Measurements of UPE took place imme-diately before thefirst application(t0)and approximately14h after the last application(t1). The measurements(measuring time:40s)started 500ms after irradiation using a dose of20mJ/ cm2UVA(exposure time8s).
All study subjects acclimatized at least7min before UPE measurement in the dark room (21Æ11C;50Æ5%relative humidity)at reduced red lightning.
Experimental procedures and study details
Owing to the high number of different experi-mental procedures used in this report,most experimental procedures are described with the respectivefigure legends.
Statistical analysis
Data for statistical analysis were found to be normally distributed using either the Kolmo-gorov–Smirnov test or the Shapiro–Wilk test for normality.Differences of means of independent samples were analyzed using the two-sided t-test for unpaired samples.Differences of means of dependent samples(e.g.data derived from multi-ple UPE measurements on the same volunteers) were analyzed using the two-sided t-test for paired samples.In case of non-normally distrib-uted data,the Mann–Whitney U-test and the Wilcoxon matched-pairs test were used.Differ-ences between means were regarded statistically significant on a P o0.05level.
Results
UPE following oxidative stress of human skin in vivo and a porcine skin model in vitro
The SRUPE system is designed to allow quantita-tion of UPE intensities over time,as well as an analysis of the spectral distribution of the UPE signal.Human skin in vivo shows a significant increase of UPE following oxidative ing the2D UPE system,developed by Beiersdorf AG, UPE can be visualized as depicted in Fig.1for different stressors encompassing exposition to ozone,UVA irradiation,or even cigarette smoke. Similar increases of UPE of human skin can also be measured using other topically applied che-mical oxidants like BPO(data not shown).
As the effects of many physical parameters on induced UPE of skin cannot be analyzed using human volunteers,an in vitro model based on freshly resected porcine skin was used.Porcine skin is a useful experimental model due to its
114 Hagens et al.
similarity to human skin with regard to anato-mical and biochemical characteristics.As with human skin in vivo ,different stressors encom-passing chemical oxidants such as H 2O 2and BPO,as well as physical stressors like UV irradia-tion,induce UPE of porcine skin in vitro .As exemplified for UVA irradiation in Fig.2,the spectral distribution of UPE of porcine skin in vitro is indistinguishable from that of human skin in vivo .
Afferent parameters affecting light-induced UPE in an ex vivo skin model
UVA radiation is a frequently used stressor in human studies on skin care products,which is associated with generation of ROS in biological materials and subsequent oxidative damage of biomolecules.UVA exposition has therefore been chosen as the stressor of choice to validate UPE measuring as a tool for the assessment of efficacy of antioxidative treatment regimes.As shown in
Fig.3,UV irradiation of porcine skin causes a two-phased UPE signal.The first phase is char-acterized by a rapid decline of the induced UPE signal over several orders of magnitude within the first 20s following UV irradiation.This first phase is followed by a second phase of weak but constant UPE that is stable over a period of several minutes.The same congruent kinetics of the UPE signal can be observed when porcine skin is exposed to filtered UVA radiation,although the signal intensity is by a factor of 410lower in this case (Table 1).In contrast
to
Fig.1.Ultraweak photon emission (UPE)signals of human skin stressed by ozone (a,b),ultravioletA (UV A)irradiation (c),and cigarette smoke (d).(a,b)A defined area of skin was treated for 14days with 2%a –tocopherol acetate (right picture half)or was left untreated (left picture half).The pictures were recorded during perfusion of the measurement chamber with ozone (a),or after replacement of ozone by normal air (b),using identical recording times.(c)A defined area of skin was treated for 14days with 0.15%a -glucosylrutin (right picture half)or was left untreated (left picture half).The picture was recorded immediately following UV A exposition for 30s.(d)A defined area of skin was sealed with an impermeable tape (right picture half)or was left uncovered (left picture half).The picture was recorded immediately following exposition of the respective skin areas for 45s to cigarette smoke.All pictures were recorded with a highly sensitive UPE camera (2D -UPE System;Photek HRPCS-2,Photek Ltd.,St Leonards on Sea,UK);the photon counting system incorporates a three-stage MCP (micro-channel plate)coupled to a Philips FTM800CCD camera (Philips Image Technologies,The Netherlands)with a spectral response of 350–800
nm).
Fig.2.Spectral distribution of induced ultraweak photon emission (UPE)in human skin in vivo and a porcine in vitro skin model.Ultraviolet A (UV A)-induced UPE was recorded immediately after irradiation of either human skin of volunteers (})or porcine skin ( ),and integrated over 40s.The filtered UV A radiation using a WG335and a UG 11filter had a dose of 59mJ/cm 2at an exposure time of 15s.UPE intensity of distinct spectral areas was normalized to the total recorded UPE intensity (4420nm).Data represent means of distinct spectral areas from 7to 10independent
measurements.
exposition to UVA or UVB radiation,exposition of porcine skin to visible light causes almost no UPE signal(Table1).
No clear dose dependency exists between the cumulative doses of UVA exposition and the resulting intensity of the UPE signal.UPE of porcine and human skin generally increases with the intensity of UVA exposition.However, with prolonged exposition times,the signal in-tensity may decrease again after30–180s(de-pending on the exposition intensity used).In a series of pre-tests,an exposition time to UVA of 8–20s at5mW/cm2,corresponding to a dose of 40–100mW/cm2,could be identified to give the best signal dynamics(data not shown).This mode of exposition was used for the following experiments.
Efferent parameters affecting light-induced UPE in an ex vivo skin model
The intensity of induced UPE of porcine skin following UVA exposition is strongly dependent on the thickness of the excited skin ing skin layers of defined surface area and variable thickness(up to3mm),which were prepared using a dermatome,UPE following UVA excita-tion was recorded.As shown in Fig.4,the UVA-induced UPE increases with increasing skin layer thickness,indicating that deeper skin layers(stra-tum corneum:20–25m m,epidermis:40–45m m, dermis:2–3mm)contribute substantially to the overall UPE signal.The apparent non-linearity of the UPE signal with increasing layer thickness is due to the fact that the emitted UPE from deeper layers is itself absorbed by the skin tissue.Ex-periments using piled layers of porcine skin and H2O2-induced UPE revealed that about60%of UPE is absorbed when passing a skin layer of 3mm thickness(data not shown).
Skin temperature has a major effect on the light-induced UPE signal.As shown in Fig.5, UVA-induced UPE drastically declines with in-creasing skin temperature.The difference of UPE signals is still highly significant between the temperature ranges of17–201C and35–391C, both of which fall in the range of local skin temperatures that may be encountered during UPE measurement.A proper acclimatization and control of skin temperatures is therefore pivotal for the recording of UPE data in a test series.As a further dermatologically relevant variable,UV-induced UPE is strongly affected by the degree of skin ing porcine skin dried for different periods of time by means of an exsicca-tor,a substantial increase of UV-induced UPE can be measured with decreasing skin hydration(Fig. 6a).A substantial decrease of the UVA-induced UPE intensity following topical application of glycerol as a humidifier on skin areas can also be verified in human volunteers(Fig.6b).Mod-ulation of the superficial pH by topical applica-tion of phosphate buffers to porcine skin, followed by an equilibration for60min,had no substantial effect on UV-induced UPE over a pH range of2.8–8.2.Skin pH can thus be disregarded
TABLE1.Effects of different radiation qualities on the intensity of the induced UPE signal
Excitation Opticalfilter UVB intensity(mW/cm2)UVA intensity(mW/cm2)VIS intensity(mW/cm2)UPE intensity(countsÂ103) Mean SD
UV radiation None 1.8 6.948.54602249 UVA radiation WG335– 5.944.5345110 VIS radiation GG400––38.582 UVA,ultraviolet A;UPE,ultraweak photon emission;VIS,
visible.
Fig.4.Dependence of light-induced ultraweak proton emission(UPE)
on skin thickness.Porcine skin layers of defined thickness(0.5,1.0,and
3.0mm)were prepared using a dermatome.The layers were excited
from the upper side(stratum corneum)using a ultraviolet A dose of
360mJ/cm2.UPE was integrated over60s immediately after excita-
tion using the SRUPE system in stationary mode.Data represent
means of10independent measurementsÆSD.Statistics:two-sided t-
test for independent samples;*P o0.05;***P o0.001.
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Hagens et al.
as a parameter significantly affecting the accu-racy of UPE measurement (data not shown).The strength of the UPE signal is strongly affected by the concentration of O 2in the surrounding atmo-sphere.Following an equilibration of porcine skin layers with either pure N 2(0%O 2),pres-sured air (20%O 2),or pure O 2(100%O 2),for 5min,an elevation of the UVA-induced UPE signal by a factor of 8.3is seen in the absence of O 2as compared with the signals obtained under a normal (20%O 2)atmosphere (Fig.7).Elevation of the O 2concentration from 20%to 100%leads to a slight,yet statistically significant,reduction of the UPE signal.The spectral distribution of UVA-induced UPE is not affected by either of the applied testing atmospheres (data not shown).Validation of UV-induced UPE measurement on human skin
To test the validity of UVA-induced UPE mea-surement on human skin following topical treat-ment with vitamin C,20female volunteers were topically treated with three antioxidant standard formulations (0.25%,0.5%,and 1%vitamin C)and the respective vehicle control on their inner forearms twice daily for 3consecutive days.The vehicle-controlled UPE measurements took place approximately 16h after the last application.As
shown in Fig.8,topical vitamin C treatment of skin reduces the UVA-induced UPE in a dose-dependent way.Reduction of the UPE signal,as compared with the vehicle control,was statisti-cally significant for formulations containing 0.5%and 1%vitamin C.
Reduction of UVA-induced UPE could be con-firmed in a second human study,enrolling 19volunteers topically treated for 7consecutive days with a formulation containing 0.25%a -glucosylrutin as an antioxidant,or the respective vehicle control.As shown in Fig.9,topical treat-ment of skin with a formulation containing 0.25%a -glucosylrutin significantly reduces UVA-induced UPE.
Accordingly,it can be assumed that a reduc-tion of UVA-induced UPE due to a previous topical treatment with an antioxidant
indicates
Fig.5.Dependence of light-induced ultraweak photon emission (UPE)on skin temperature.Porcine skin was placed on a tempera-ture-adjustable surface and allowed to acclimatize before irradiation and UPE measurement.The temperature on the skin surface was controlled before and after measurement using a radiation pyrometer.The skin was ultraviolet A (UV A)irradiated for 15s using a filtered Oriel lamp emitting a UV A dose of 88.5mJ/cm 2,followed by recording and integration of UPE for 1min using the SRUPE system in stationary mode.Data represent means ÆSD of four to six indepen-dent measurements.Statistics:two-sided t-test for independent sam-ples;*P o 0.05;**P o
0.01.
Fig.6.Dependence of ultraviolet (UV)-induced ultraweak photon emission UPE on skin hydration using porcine skin in vitro (a)and human skin in vivo (b).(a)Porcine skin layers were dried for defined periods of time in an exsiccator.Skin layers having different degree of dryness were irradiated for 30s with a spectrally distributed UV dose
of 63mJ/cm 2UVB 1202mJ/cm 2UV
A,followed by the recording and integration of ultraweak photon emission (UPE)for 1min using the SRUPE system in stationary mode.Data represent means ÆSD of three independent measurements.(b)Skin areas of the inner forearm of 10volunteers were irradiated for 8s with filtered UV A of 47.2mJ/cm 2.UPE was recorded and integrated for 1min using the SRUPE system in stationary mode.Data represent means ÆSD of 10independent measurements.
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Biological validation on ultraviolet A-stressed skin
a protection of living skin layers from pro-oxida-tive stress.
Discussion
In a previous report,we could demonstrate that oxidation-induced UPE of the skin is dominated by proteins as the major source of emitted
photons.The intensity and spectral distribution of oxidation-induced UPE in this case are mainly determined by energy transfer and photon emis-sion from oxidized amino acids,with Trp func-tioning as a terminal emitter and energy acceptor from other activated amino acid species (19).Compared with other methods used for the study of oxidative processes like electron spin resonance (ESR)spectroscopy (23)or determina-tion of oxidation markers such as protein carbo-nyl compounds,the UPE measuring technique offers several advantages.As in ESR spectro-scopy,the UPE method detects reactive inter-mediates that can form during the oxidation processes and therefore allows rapid recording of reaction kinetics.No complicated analytical workup of samples is necessary like that required for chemical detection of oxidation markers.Un-like in the ESR measurement,it is possible to detect reactive intermediates directly even at room temperature without adding reagents such as spin traps,which are not evenly distributed in the skin and that may themselves interfere in radical processes by capturing radicals (23).
As shown in this report,the UPE measuring technique following UVA induction of oxidative skin stress is a simple non-invasive technique for routine studies,especially on the efficacy of topically applied antioxidants on human skin.Our data obtained with skin samples of different thickness indicate that UV irradiation
induces
Fig.7.Dependence of ultraviolet A (UV A)-induced ultraweak photon emission (UPE)on the O 2concentration of the testing atmosphere.Porcine skin layers were equilibrated with pure nitrogen (0%O 2;N 2mode),normal air (20%O 2),or pure oxygen (100%O 2;O 2mode)for 5min before UV A irradiation and recording of UPE under the same atmospheric conditions.Skin layers were irradiated for 60s with a UV A dose of 360mJ/cm 2,followed by the recording and integration of UPE for 1min using the SRUPE system.For data normalization,UPE induced in the presence of normal air was set 100%.Data represent means ÆSD of three independent measurements.Statistics:two-sided t-test for independent samples;*P o 0.05;***P o
0.001.
Fig.9.Dependence of ultraviolet A (UV A)-induced ultraweak photon emission (UPE)of human skin on antioxidant pretreatment [a -glucosylrutin (AGR)].Male and female volunteers (n 519)were treated with antioxidant formulations containing 0.25%AGR,as well as the respective vehicle (V)on their inner forearms twice daily for 7consecutive days.Reference areas on the forearm were left untreated.Measurement of UPE took place immediately before the first applica-tion (t 0)and approximately 14h after the last application (t 1).The measurements (measuring time:40s)started 500ms after irradiation
using a dose of 20mJ/cm 2UV
A (exposure time 8s).Data represent means ÆSD of 19volunteers.Statistics:Wilcoxon ’s matched pairs test;**P o 0.01;***P o 0.001.
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Hagens et al.
UPE especially in deeper(living)skin layers, where oxidative processes contribute most to accelerated skin ageing.These deeper layers are the sites where antioxidants must be active in order to interfere with accelerated skin ageing. Parallel studies based on the topical application of chemical oxidative stressors like H2O2,ozone, or BPO could also demonstrate the induction of substantial UPE signals on porcine and human skin.These signals,however,must be assumed to be generated in the upper(dead)layers of the skin,which are of little relevance for dermato-logical and cosmetic studies on antioxidative compounds(data submitted for publication else-where).
We and others(15)could demonstrate that the UVA-induced UPE signal of skin is composed of two phases:afirst phase,characterized by a rapid decline of the UPE signal over several orders of magnitude within seconds,and a second UPE phase,which is stable over minutes.The data generated during this validation study clearly demonstrate that the UPE signal induced by UVA irradiation of skin is strongly affected by external and internal physical and chemical para-meters.From a purely physical point of view,the most critical parameters are the spectral compo-sition of the exciting UV light,skin temperature, and the O2concentration of the surrounding atmosphere.Less critical parameters in this case are the humidity and pH of the skin area to be examined.From a clinical point of view,external and internal parameters like the spectral compo-sition of the exciting UV light,O2concentration of the surrounding atmosphere,and skin tempera-ture can be well controlled during the generation and recording of UPE signals.In contrast,biolo-gically determined internal parameters such as skin humidity and skin pH must be assumed to show a substantial individual variation in human trials and are largely not adaptable.These biolo-gical parameters,especially the actual skin hu-midity,should be recorded separately and factored in during data analysis.
In clinical studies,UPE measurement follow-ing UVA excitation could precisely reflect an antioxidant effect of topically applied vitamin C as well as a-glucosylrutin even14–16h after the last administration of the antioxidant.
Based on the clinical data,and with knowledge of modulating external variables,UPE measure-ment following UV excitation can be qualified as a reliable and valuable method for the non-invasive measurement of antioxidant efficacy on the skin.In summary,our studies clearly demon-strate the potential of the UPE measuring techni-que following UV excitation of the skin for in vivo evaluation of the antioxidant capacity of derma-tologically and cosmetically active substances. References
1.Kohen R.Skin antioxidants:their role in aging and in
oxidative stress–new approaches for their evaluation.
Biomed Pharmacother1999;53:181–192.
2.Marnett LJ.Oxyradicals and DNA damage.Carcinogen-
esis2000;21:361–370.
3.Shindo Y,Witt E,Han D,Packer L.Dose-response effects
of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis.J Invest Dermatol1994;102:470–475.
4.Stadtman ER,Levine RL.Free radical-mediated oxida-
tion of free amino acids and amino acid residues in proteins.Amino Acids2003;25:207–218.
5.Benedetto AV.The environment and skin aging.Clin
Dermatol1998;16:129–139.
6.Armstrong BK,Kricker A.The epidemiology of UV
induced skin cancer.J Photochem Photobiol B2001;63: 8–18.
7.Dalle CM,Pathak MA.Skin photosensitizing agents and
the role of reactive oxygen species in photoaging.J Photochem Photobiol B1992;14:105–124.
8.Young AR.Chromophores in human skin.Phys Med
Biol1997;42:789–802.
9.Wondrak GT,Roberts MJ,Cervantes-Laurean D,Jacob-
son MK,Jacobson EL.Proteins of the extracellular matrix are sensitizers of photo-oxidative stress in human skin cells.J Invest Dermatol2003;121:578–586.
10.Sander CS,Chang H,Salzmann S et al.Photoaging is
associated with protein oxidation in human skin in vivo.
J Invest Dermatol2002;118:618–625.
11.Mei WP.About the nature of biophotons.J Biol Syst1994;
2:25–42.
12.Evelson P,Ordonez CP,Llesuy S,Boveris A.Oxidative
stress and in vivo chemiluminescence in mouse skin exposed to UVA radiation.J Photochem Photobiol B 1997;38:215–219.
13.Sauermann G,Mei WP,Hoppe U,Stab F.Ultraweak
photon emission of human skin in vivo:influence of topically applied antioxidants on human skin.Methods Enzymol1999;300:419–428.
14.Yasui H,Sakurai H.Chemiluminescent detection and
imaging of reactive oxygen species in live mouse skin exposed to UVA.Biochem Biophys Res Commun2000;
269:131–136.
15.Ou-Yang H,Stamatas G,Saliou C,Kollias N.A chemi-
luminescence study of UVA-induced oxidative stress in human skin in vivo.J Invest Dermatol2004;122:1020–1029.
16.Barnard ML,Gurdian S,Diep D,Ladd M,Turrens JF.
Protein and amino acid oxidation is associated with increased chemiluminescence.Arch Biochem Biophys 1993;300:651–656.
17.Watts BP Jr.,Barnard M,Turrens JF.Peroxynitrite-de-
pendent chemiluminescence of amino acids,proteins, and intact cells.Arch Biochem Biophys1995;317:324–330.
119 Biological validation on ultraviolet A-stressed skin。