Original Contributions

Idebenone: a new antioxidant – Part I. Relative assessment of oxidative

Blackwell Publishing, Ltd.

stress protection capacity compared to commonly known

antioxidants
D H McDaniel,1 B A Neudecker,2 J C DiNardo,3 J A Lewis, II3 & H I Maibach2
1
Institute of Anti-Aging Research, Virginia Beach, VA, USA, Eastern Virginia Medical School, Norfolk, VA, USA
2
Department of Dermatology, University of California San Francisco, San Francisco, CA, USA
3
Pharma Cosmetix Research, LLC, Richmond, VA, USA

Summary Topical applications of skin care products containing antioxidants have become
increasingly popular. Numerous studies have elucidated the biological effects of these
substances. General antiaging effects, anti-inflammatory properties, photoprotective
properties, and prevention of ultraviolet (UV) immunosuppression have been docu-
mented. However, a standardized method to characterize and compare the properties
and oxidative stress protection capacity of antioxidants was lacking. A multistep in vitro
process utilizing a variety of biochemical and cell biological methods combined with in
vivo studies was designed to compare the oxidative stress protective capacity of com-
monly used antioxidants. Data were presented for L-ascorbic acid, dl-alpha-tocopherol,
kinetin, dl-alpha lipoic acid, ubiquinone, and idebenone. Methods included using UV-
induced radical trapping/scavenging capacity measured by photochemiluminescence,
pro-oxidative systems (LDL-CuSO4, microsome-NADPH/ADP/Fe3+) with measurement
of primary and secondary oxidation products, UVB irradiation of human keratinocytes,
and in vivo evaluation, using the human sunburn cell (SBC) assay. Correlation and trends
between in vitro and in vivo results were established, and the standardized test protocol
was used to quantify oxidative stress protection capacity of antioxidants. Summarizing
and totaling the data equally weighted for each oxidative stress study, the overall oxida-
tive protection capacity scores of 95, 80, 68, 55, 52, and 41 were obtained for idebenone,
dl-alpha tocopherol, kinetin, ubiquinone, L-ascorbic acid, and dl-alpha lipoic acid,
respectively. The higher the score, the more effective the overall oxidative stress protec-
tion capacity of the antioxidant became. This multistep protocol may serve as a standard
in investigating and comparing new putative antioxidants for topical use as well as a
valuable tool to assess the anti-inflammatory properties, photoprotective properties, and
prevention of UV immunosuppression of topical antioxidants.
Keywords: idebenone, antioxidant, aging

Introduction
Ultraviolet radiation (UVR)-generated reactive oxygen
Correspondence: David H. McDaniel, MD, Institute of Anti-Aging Research, species (ROS) and DNA photodamage play a critical
LLC, 933 First Colonial Road, Suite 205, Virginia Beach, VA 23454. E-mail: role in the process of extrinsic environmentally ind-
[email protected] uced aging (photoaging) and photocarcinogenesis.1 In
Accepted for publication January 26, 2005 addition to the well-known long-term effects such as

10 © 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 10– 17

 Idebenone • D H McDaniel et al.

immunosuppression and skin cancer, photo-oxidative Q-10 that showed potent radical scavenging capacity and
damage leads to alterations of cells and structural cell protection properties in previous studies.10 Five inde-
macromolecules of the dermal connective tissue pendent biochemical, cell-biologic, and in vivo methods
contributing significantly to photoaging with its clinical were combined to determine the antioxidant capacity of
appearance of wrinkle formation, laxity, and pigment the single substances under different conditions in order
dyschromias.2 The skin is constantly exposed to a pro- to demonstrate their overall performance. The studies
oxidative environment such as UVR and air pollutants. conducted were as follows:
The skin is equipped with various antioxidant defense Study 1. Radical scavaging capacity measured by
systems constituting a complex antioxidant network.1,3 photochemiluminescence: Utilizes Photochem®, a device
Whereas UVB can damage DNA, proteins, and lipids that offers fast and reliable chemiluminometric assess-
directly, UVA is believed to act largely via oxidative ment of the general antioxidative capacity of substances
processes.4 Increased exposure to exogenous sources in their ability to scavenge free radicals via the measure-
and/or endogenous production of ROS can provoke an ment of radicals generated (or lack thereof) through their
imbalance of the fragile pro-oxidant–antioxidant equilibrium, reaction with luminol and subsequent light emission.
resulting in oxidative damage of lipids, proteins, and Study 2. Low density lipoprotein (LDL) pro-oxidative
DNA.5,6 For example, the superoxide (O2) radical can system measuring primary oxidation by-products:
cause the “common deletion” mutation in mitochondrial assessment of antioxidant ability to protect LDL stressed
DNA, which can be found in high numbers in photo- with copper sulphate (CuSO4) oxidative system. The
damaged skin.7 According to the mitochondrial theory CuSO4-LDL system was used to evaluate the protection of
of aging, nonrepaired damage of mitochondrial DNA lipid bulks over time measuring the primary by-products
and unstable electron transfer cause an important loss of of lipid peroxidation – the highly reactive and cytotoxic
mitochondrial function in correlation with progression lipid hydroperoxides.
of age. Topical application of antioxidants is used to optimize Study 3. Microsome pro-oxidative system measuring
the cutaneous antioxidative capacity and to limit ROS- secondary oxidation by-products: assessment of antioxi-
induced skin damage. Numerous in vitro and in vivo dative ability to protect microsomal membrane stressed
studies have demonstrated specific antioxidative capacity with NADPH/ADP/Fe3+-oxidative system measuring
as well as their photoprotective properties.6,8 Antioxidants secondary oxidative by-products (malondialdehyde –
applied topically before UV-irradiation on animal and MDA equivalents) utilizing the thiobarbituric acid-reactive
human skin diminished UVA-induced polymorphous substances (TBARS) method.11 Antioxidants protecting
light eruption, psoralen + ultraviolet A (PUVA)-induced bulky lipids, such as LDL, are not necessarily good protec-
erythema, and sunburn cell formation.9 However, a stand- tors of cell membranes as a result of their hydrophilic/
ardized method to characterize and compare the complex lipophilic bilayer composition. Therefore, the pro-oxida-
properties and effects of topical antioxidants is lacking. In tive NADPH/ADP/Fe3+-microsome system was used as
this study, for the first time, a variety of biochemical and an in vitro model system more closely resembling natural
cell biologic methods are combined with in vivo studies in cellular biological systems. Oxidation of cell membranes
a protocol to compare protective capacity of commonly leads to serious consequences in altering cell membrane
used antioxidant ingredients. The in vivo method was fluidity and cell function.
included to assess real biological effects in living tissue, Study 4. UVB irradiation of keratinocytes measuring
as human skin itself contains lipophilic antioxidants such DNA damage: assessment of DNA damage in cell culture
as vitamin E (tocopherols and tocotrienols), ubiquinones experiments under pro-oxidative conditions (UVB irradiation
(coenzyme Q), carotenoids, and lipoic acid, as well as the of human keratinocytes) by measuring the positive cells
hydrophilic antioxidants, vitamin C (ascorbate), uric acid for antithymine dimer antibodies. This experiment is thought
(urate), superoxide dismutase (SOD), and glutathione. to reflect a direct correlation to the in vivo-occurring DNA
Reduction and oxidation (redox) cellular reactions cross linking damage following UVB exposure and the
couple these antioxidants in a network together through protection of such nuclear damage by antioxidants.
a complex concerted action in which the antioxidants are Study 5. UVB irradiation of human skin measuring
partly recycled by one another.5 damage by formation of sunburn cells (SBC): Exposure to
For this multistep protocol, the following compounds UVR can cause damage of epidermal cells, resulting in
were tested: L-ascorbic acid (vitamin C), kinetin (a plant the formation of sunburn cells.12 Because sunburn cells
derivative), dl-alpha tocopherol (vitamin E), dl-alpha can be enumerated, their formation provides a relatively
lipoic acid, ubiquinone (CoEnzyme Q-10), and ide- sensitive and quantitative measure of the extent of UVR
benone, a lower molecular weight analog of CoEnzyme damage to the epidermis.

© 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 10–17 11

Idebenone • D H McDaniel et al.

trifugation from pooled plasma of healthy donors using

Methods, materials, and results
an established protocol. After isolation, LDLs were
extensively dialyzed against a degassed and nitrogen-
Study 1. Radical scavenging capacity measured by
saturated tris-hydrochloric (HCl) buffer (5 mmol/L,
photochemiluminescence
pH 7.4) containing 1 mmol/L ethylenediaminetetraacetic
The individual antioxidant capacity of the putative acid (EDTA). Before oxidation by CuSO4, EDTA was
antioxidant substances was estimated by the Photo- removed from LDL by dialysis against a tris-HCl buffer
chem® system (Analytik Jena AG, Jena, Germany, and (5 mmol/L, pH 7.4) without added EDTA.
Analytik Jena USA, Inc., TX). The system combines the
generation of radicals through photochemical excitation Incubation LDL with the pro-oxidant system Ham’s F-10/
with highly sensitive luminometric detection via the CuSO4
radical reaction with luminol to produce measurable The LDL oxidation was achieved by incubating (37 °C,
light emission. Samples are diluted with premade buffers 95% O2, 5% CO2) 1 g of LDL protein/L with and without
(standardized kits) and applied to the device. The relative the putative antioxidant substances (at equivalent
antioxidative capacity is determined by comparison to 100 µmol concentrations) in 2 mL of serum-free Ham’s
a standardized blank (without antioxidants) and a F-10 medium (BioSource International, Camarillo, CA)
standard provided with the kit. The ACL-kit (integral in the presence of 20 µmol/L CuSO4 for the times
antioxidative capacity of lipid-soluble substances) and indicated in the figure legends.
the ACW-kit (integral antioxidative capacity of water-
soluble substances) were used. Antioxidant concen- Measurement of lipid hydroperoxides
trations effective to eliminate radical formulation were Lipid hydroperoxides were determined with the Cayman
established for each antioxidant as indicated in Table 1. Lipid Peroxidation (LPO) Assay Kit (Cayman Chemical,
Ann Arbor, MI) which measures the hydroperoxides
Results directly utilizing the redox reactions with ferrous ions to
Idebenone, dl-alpha-tocopherol, and L-ascorbic acid all produce ferric ions which can be detected using thiocy-
demonstrated effective neutralization of radicals as mea- anate ion as the chromogen. The antioxidative effect of
sured by photochemiluminescence at a relatively low con- the substances is shown as percentage compared to the
centration of 10 nmol/L. Ubiquinone was somewhat less Blank (incubation without addition of antioxidants).
effective, requiring 100 nmol/L for effective concentration,
kinetin required 1,000 nmol/L for effective concentration, Results
and alpha lipoic acid was ineffective in this system (see Kinetin and idebenone demonstrated a consistent protection
Table 1). against lipid peroxidation over 24 h. Other substances like
ubiquinone, lipoic acid, and ascorbic acid showed only a com-
paratively short-lasting protective efficiency (see Fig. 1).
Study 2. LDL pro-oxidative system measuring primary
oxidation by-products
Study 3. Microsome pro-oxidative system measuring
Isolation of LDL
secondary oxidation by-products
Low density lipoproteins (d = 1.019–1.063 kg/L) were
isolated in clean Beckman one-way Quick-Seal© Tubes Preparation of microsomes
(Beckman Coulter Inc., Palo Alto, CA) by ultracen- Livers were obtained from male Wistar rats weighing
between 250 and 400 g. Tissue was homogenized in
50 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulphonic
Table 1 Radical scavenging capacity measured by
photochemiluminescence. acid (HEPES), 250 mmol/L sucrose buffer, pH 7.4,
containing 150 mmol/L potassium chloride (KCl) and
Antioxidant substance Effective concentration (nmol/L) 500 µmol/L EDTA using a Kinematica Polytron PT3000
(Brinkmann Instruments, Westbury, NY) homogenizer.
Idebenone 10 Microsomal vesicles were isolated by removal of the
Ascorbic acid 10 nuclear fraction at 8,000 g for 10 min at 4 °C and
Tocopherol 10 removal of the mitochondrial fraction at 18,000 g for
Ubiquinone 100
10 min at 4 °C using a Beckman L8-55 ultracentrifuge
Kinetin 1000
and a 50Ti-13 rotor. The microsomal fraction was sedi-
Lipoic acid > 1000 (not detectable)
mented at 105,000 g for 60 min at 4 °C. The pellet was

12 © 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 10– 17

 Idebenone • D H McDaniel et al.

Figure 1 Low density lipoprotein (LDL)

pro-oxidative system measuring primary
oxidation by-products (lipid
hydroperoxides).

Figure 2 Microsome pro-oxidation system

measurement of secondary oxidative by-
products (MDA equivalents).

washed once in 50 mmol/L HEPES and 150 mmol/L KCl, Measurement of secondary oxidation products, MDA
pH 7.4, and collected again at 105 000 g for 30 min. The equivalents or TBARS method
resulting microsomal pellet was resuspended in HEPES/ Microsomal preparations (500 µL) were mixed with
KCl, pH 7.4, by careful sonication in ice and stored in 1 mL of thiobarbituric acid (0.67 g/100 mL, 0.05 mol/L
portions (10 mg protein/mL) at − 80 °C until use. sodium hydroxide – NaOH). After the addition of
trichloroacetic acid (50% w/v), the samples were heated
Incubation of microsomes with the pro-oxidant system to 90 °C for 30 min. After cooling and extraction of the
NADPH/ADP/Fe3+ samples with 1 mL of butanol, the absorbance of the
The microsomal preparations were incubated in the butanol phase was determined spectrophotometrically at
presence of the pro-oxidant system NADPH/ADP/Fe3+, 532 nm. For quantification, an external standard curve
consisting of 0.20 mmol/L NADPH, 50 mmol/L ADP, and was prepared using 1,1,3,3-tetraethoxypropane, which
0.25 mmol/L FeCl3 in HEPES/KCl buffer (150 mmol/L yields MDA. The antioxidative effect of the substances is
KCl, 50 mmol/L HEPES) with and without the putative shown as percentage compared to the control (incu-
antioxidant substances. Oxidation of 1-mL aliquots con- bation without addition of antioxidants).
taining 1 mg of protein was started at 37 °C by the addi-
tion of NADPH and was stopped with EDTA (10 mmol/L) Results
after the times indicated in the figure legends. Control Lipoic acid and idebenone showed the most effective
incubations without the pro-oxidant system were per- protection against oxidation of the cell membrane lipids.
formed at 37 °C in the presence of EDTA. All antioxidants Kinetin, which showed favorable results in protecting bulky
were dissolved in water or ethanol and added to the lipids (LDL), showed only a weak protective effect in the
incubations at equivalent 100 µmol concentrations. ability to protect microsomal membranes (see Fig. 2).

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Idebenone • D H McDaniel et al.

Table 2 UVB irradiation of keratinocytes measuring DNA damage.

Study 4. UVB irradiation of keratinocytes measuring DNA
damage Inhibition of
Positive cells photoproduct
Keratinocyte collection after UVB generation
Human primary foreskin keratinocytes (second passage) Antioxidant substance radiation* (protective effect)
were grown in 6-well plates containing cover slips to 60%
confluence in serum free medium (KGM, Clonetics; No radiation control 0% 100%
Cambrex Cooperation, E. Rutherford, NJ) containing Idebenone 29% 45%
0.07 mm calcium chloride (CaCl2). Six hours before UVB- Ascorbic acid 34% 36%
radiation, the medium was removed and replaced by Kinetin 34% 36%
Tocopherol 35% 34%
fresh growth medium with or without the antioxidative
Ubiquinone 51% 4%
substances. Each antioxidant concentration was 10 µmol
Lipoic acid 53% 0%
per well. 200 mJ/cm2 UVB radiation 53% 0%

Ultraviolet light (UVB) irradiation of keratinocytes *Percentage of positive cells (above threshold) in three fields
Keratinocyte cultures were irradiated with a single dose (counted cell number ∼120 –150).
of 200 mJ/cm2 UVB, using FS-20/T-12 bulbs (emission
range: 280–340 nm; 305 nm max.). Immediately prior
Study 5. UVB irradiation of human skin measuring
to irradiation, the medium was replaced with 1 mL sterile
damage by formation of sunburn cells (SBC)
phosphate buffered saline (PBS) (pH 7.4, 37 °C), and
after irradiation, PBS was replaced with fresh growth Treatment
medium without antioxidants. The UVB exposure was All applications were made to a 5 × 10 cm area site over
quantified using a Goldilux™ Ultraviolet Radiometer (Oriel the mid-back region once a day for 2 weeks. Each putative
Instruments, Stratford, CT). Cells were maintained at antioxidant was applied to five (n = 5) healthy adult
37 °C (5% CO2) for 1 h until fixation with paraform- volunteers between the ages of 18 and 60. All antioxidants
aldehyde (PFA). were dissolved in ethanol/water at 0.5% w/w concen-
trations. Additionally, one test site was left untreated and
Fixation and nuclear thymine–dimer staining of keratinocytes served as a control. Approximately 10 min after the last
Cells were fixed with 4% PFA in PBS for 30 min at application, test sites were irradiated to 1.5 minimal
room temperature (RT), washed with PBS and per- erythema dose (MED) of UVB light, a shave biopsy taken
meabilized by incubation with EtOH/PBS (90/10; v%/v%) and prepared histologically, and the number of sun burn
for 30 min at ∼10 °C. After fixation and permeabilization, cells (SBC) evaluated microscopically per high power field.
cells were washed twice with PBS containing 1% of
bovine serum albumin (BSA). They were then incubated Light source
for 30 min with 10 µg/mL antithymine dimer Ab (clone The light source used was a 150-W xenon arc solar
KTM53; Kamiya Biomedical Company, Seattle, WA) at simulator equipped with a UV reflecting dichromic
RT. After the incubation period, the cells were washed mirror and a 1-mm thick Schott WG-320 (BES Optics
twice with PBS-BSA and incubated for 30 min with Inc., W. Warwick, RI) filter to produce simulation of the
20 µg/mL secondary fluorescein-isothiocyanate (FITC)- solar spectrum. A 1-mm thick UG5 filter was added to
conjugated antimouse immunoglobulin G (IgG) at RT. remove reflected heat and remaining visible radiation.
After the incubation with the secondary antibody, cells
were washed twice with PBS-BSA and fixed again with Minimal Erythema Dose (MED) determination
4% PFA for 15 min at RT. Slides were analyzed by The MED for each subject was determined by exposing
confocal microscopy. a circle 1 cm in diameter to untreated areas to a series
of exposures in 25% dose increments from the solar
Results simulator. The MED was defined as the time of exposure
This experiment is thought to reflect the in vivo occurring required to produce a minimally perceptible erythema
DNA damage following UVB exposure and the protection 20 ± 4 h after exposure.
of such nuclear damage by antioxidants. The results (see
Table 2) have to be seen as approximate estimations of the Biopsies
occurrence of nuclear thymine dimer photo products. Approximately 10 min after the last topical application of
Idebenone provided the highest level of inhibition. the putative antioxidant, a circular area measuring 1 cm

14 © 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 10– 17

 Idebenone • D H McDaniel et al.

Figure 3 UVB irradiation of human skin

measuring DNA damage by formation of
SBC.

in diameter was exposed to a single dose of 1.5 MED using antioxidative capacities. In this study, a multistep pro-
the solar simulator. Approximately 20 h later, a shave tocol is presented to allow the comparison of different
biopsy (∼4 × 4 mm) was obtained from each irradiated antioxidants regarding their usefulness in topical appli-
and untreated control site following injections of a local cations. This combination of biochemical, cell-biologic,
anesthetic (lidocaine). The skin specimens were imm- and in vivo methods allows the determination of various
ediately fixed in 10% buffered formalin. independent aspects of antioxidant substances, such as
anti-inflammatory properties, photoprotective properties,
Histology or protection of cell membranes. The results demonstrate
The fixed specimens were processed routinely, embedded the diversity of actions and the value of utilizing a com-
in paraffin, and then sectioned and stained with bination of entirely different methods when comparing
hematoxylin-eosin. The numbers of SBC were deter- the relative efficacy of antioxidant activity. Kinetin for
mined in at least 12 sections at 50-µ intervals. A example showed a very weak antioxidant effect when
minimum of 70 high power fields (HPF) was counted evaluated by chemiluminometric detection of antioxidative
from each biopsy, and the average number of SBCs per capacity yet showed the strongest effect in the LDL/
HPF determined. All specimens were counted in a blinded CuSO4-oxidation system. Another example is lipoic acid
manner. which showed a strong effect in the microsome-NADPH/
ADP/Fe3+-system while showing minimal response in
Results every other method employed. Even tocopherol, which
Figure 3 expresses the photoprotective benefits of the showed good results in most experiments, revealed a
antioxidants tested based on the percent change over weakness regarding lipid protection over time (LDL/
baseline (delta percent) for the number of SBC per high CuSO4-oxidation system). This is most likely because its
power field. Idebenone was the most effective antioxidant pro-oxidative metabolites appear through reduction of
in the study in its ability to protect human skin from radicals by hydrogen donation. The implementation of an
sunburn cell formation post-UVR exposure. in vivo approach employing the human SBC assay gave
crucial additional clinical information, as the capacity of
each compound to penetrate the upper skin layers may
Discussion
vary. These results confirm that favorable in vitro results
Previous studies comparing different antioxidants for use do not necessarily reflect the in vivo situation. An example
in topical applications primarily focused on certain thereof is ascorbic acid which surprisingly had no
biochemical or cell-biologic aspects of those substances. protective effect (at the concentration tested) on in vivo
Because the currently popular topical antioxidants are SBC formation. Idebenone, while to date had demonstrated
of very heterogeneous structure and origin (vitamins, only the ability to protect against ROS-mediated damage
flavonoids, coenzymes, etc.), a protocol to compare their in organ preservation solutions and to treat Alzheimer’s
properties should consist of a variety of methods aiming disease,13 showed a strong overall performance throughout
to elucidate the overall picture regarding their specific all experiments conducted.

© 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 10–17 15

Idebenone • D H McDaniel et al.

Table 3 Global relative antioxidant activity: total oxidative stress protection capacity scores (environmental protection factor; EPF of
common antioxidants).

Test Idebenone Tocopherol Kinetin Ubiquinone Ascorbic acid Lipoic acid

Sun burn cell assay 20 16 11 6 0 5

Photochemiluminescence 20 20 10 15 20 5
Primary oxidative products 16 10 20 5 3 4
Secondary oxidative products 19 17 10 12 12 20
UVB irradiated keratinocytes 20 17 17 17 17 7
Total points (EPF score) 95 80 68 55 52 41

damage) and in vivo (study 5; UVB irradiation of human

Establishing a standardized way to summarize results
skin measuring damage by formation of SBC) test
The scoring system introduced in this study is designed to methods. Therefore, overall, equal weighting of study
provide, for the first time, a standardized comparison results seems to be most appropriate at this time. To
of the protective capacity of different antioxidant sub- assign values, the active antioxidant that demonstrates
stances used in topical applications against oxidative the greatest benefit for the test conducted became
stress. Five tests, which were equally weighted at 20 the standard for the study, and received 20 points. The
points each, allowed for the maximum highest possible remaining antioxidants were assigned a percentage of
score of 100 points. Using this scale, the higher score the 20 points based on their efficacy relationship to the
indicates higher oxidative stress protection capacity highest scoring antioxidant in each independent study.
of the antioxidant. Equal weight was given to the Example: idebenone produced the greatest benefit in
antioxidant’s performance in each study because each the SBC assay, a 38% reduction in SBCs. Therefore, it
study tested the antioxidant’s ability to protect against a became the standard and received 20 points. Tocopherol
unique different set of oxidative stress parameters. was second, producing a 31% reduction. To determine the
Without knowing the direct correlation of each study to relative activity (efficacy) of tocopherol to the standard,
actual living biological systems (with one exception, the idebenone, one calculates 31/38 × 100 = 82% relative
SBC study was conducted in vivo), the equal weight activity. Therefore, tocopherol would receive 82% of
approach was selected for the initial introduction of this the 20 points or 16 points (rounded to the nearest whole
concept. Future experiments and clinical correlation number).
should allow refinement of this concept and the Because the photochemiluminescence assay results
weighting may be adjusted, if data warrant, for optimal are expressed as the lowest effective concentration, and a
clinical correlation. For example, it is known that the base 10 serial dilution was used, the antioxidant scores
superoxide radical, a natural by-product of metabolic were assigned as follows: 10 nmol/L = 20; 100 nmol/
energy production, causes serious deleterious effects L = 15; 1000 nmol/L = 10; > 1000 nmol/L = 5.
to living cells if not quenched, neutralized, or reduced The “relative value” of each of the five tests was arbi-
almost immediately after production. In study 1, we trarily assigned equal weight. That is, each test con-
tested the ability of the antioxidant to suppress super- tributed equally to the antioxidant scale. Further clinical
oxide radical formation. It is also known that lipid testing in the future may allow additional refinement of
peroxidation is a major problem in biological systems. In this scoring system and weight. The overall scores, and
study 2 we assessed the antioxidants’ ability to suppress thus relative oxidative stress protection capacity of the
lipid oxidation. Protecting cell membrane oxidation is respective antioxidants tested, are summarized in Table 3.
of paramount importance to living biological systems
because the cell membrane is the cell’s first line of defense
Conclusion
against oxidation. In study 3, we assessed this protec-
tive parameter. Because UV light is known to be the These studies compared the protective capacity of five
predominant cause of premature aging of the skin, commonly used antioxidant ingredients and one novel
the antioxidants’ ability to protect against UV-induced new antioxidant for skin care, idebenone, in both in vitro
oxidative stress was tested under both in vitro (study and in vivo methods. A standardized testing protocol that
4; UVB irradiation of keratinocytes measuring DNA quantifies the oxidative stress protection capacity of the

16 © 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 10– 17

 Idebenone • D H McDaniel et al.

substances studied was developed, and a scoring system 5 Berneburg MH, Plettenberg J, Krutmann J. Photoaging of
to compare relative activity was presented. human skin. Photodermatol Photoimmunol Photomed 2000;
This quantitative scoring system to assess the oxidative 16: 239 – 44.
stress protection capacity of an antioxidant could be the 6 Scharffetter-Kochanek K, Brenneisen P, Wenk J et al.
Photoaging of the skin from phenotype to mechanisms.
basis for an antioxidant ingredient performance rating
Exp Gerontol 2000; 35: 307–16.
system that consumers could easily understand. Similar
7 Berneburg M, Grether-Beck S, Kurten V et al. Singlet oxygen
in analogy to sun protection factor (SPF) or immune pro- mediates the UVA-induced generation of the photoaging-
tection factor (IPF),14 the antioxidant protection factor or associated mitochondrial common deletion. J Biol Chem
environmental protection factor (EPF) could be used to 1999; 274: 15345–9.
assess the relative strength of antioxidants in their ability 8 Wenk J, Brenneisen P, Meewes C et al. UV-induced oxidative
to protect against oxidative stress. Reviewing the summa- stress and photoaging. Curr Probl Dermatol 2001; 29:
tion of all study results presented (Table 3), one com- 83 –94.
pound, idebenone, appears as a powerful antioxidant 9 Darr D, Pinell SR. Reactive oxygen species and antioxidant
most consistently throughout all experiments. Although protection in photodermatology. In: NJ Lowe, NA Shaath,
this potent antioxidant is relatively unknown to derma- MA Oathak, eds. Sunscreens – Development, Evaluation, and
Regulatory Aspects, 2nd ed. New York: Marcel Dekker, Inc.;
tology today, idebenone may represent a promising new
1997: pp. 155–73.
agent for topical skin care protection. Further research is
10 Fuchs J, Parler L. Oxidative Stress in Dermatology, Marcel
currently being conducted to establish its in vivo ability to Dekker, New York: Basel; 1993: pp. 55–73.
protect human skin from oxidative stress and assess its 11 Wieland E, Schutz E, Armstrong VW, Kuthe F, Heller C,
efficacy in the treatment of photodamaged skin. Oellerich M. Idebenone protects hepatic microsomes
against oxygen radical-mediated damage in organ
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