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© 2001 by the American Society for Dermatologic Surgery, Inc. • Published by Blackwell Science, Inc.
ISSN: 1076-0512/01/$15.00/0 • Dermatol Surg 2001;27:137–142
Topical L-Ascorbic Acid: Percutaneous Absorption Studies
Sheldon R. Pinnell, MD,* Huanshu Yang, MD,
‡
Mostafa Omar, PhD,
†
Nancy Monteiro Riviere, PhD,
‡
Holly V. DeBuys, MD,* Linda C. Walker,*
Yaohui Wang, MD,
§
and Mark Levine, MD
§
*
Duke University Medical Center, Durham, North Carolina,
†
PhytoCeuticals, Elmwood Park, New Jersey,
‡
College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, and
§
National Institute of Diabetes & Digestive & Kidney Diseases, NIH, Bethesda, Maryland
background.
Reactive oxygen species generated by ultraviolet
light result in photocarcinogenic and photoaging changes in the
skin. Antioxidants protect skin from these insults.
objective.
This study defines formulation characteristics for
delivering L-ascorbic acid into the skin to supplement the skin’s
natural antioxidant reservoir.
methods.
L-ascorbic acid or its derivatives were applied to pig
skin. Skin levels of L-ascorbic acid were measured to determine
percutaneous delivery.
results.
L-ascorbic acid must be formulated at pH levels less
than 3.5 to enter the skin. Maximal concentration for optimal
percutaneous absorption was 20%. Tissue levels were saturated
after three daily applications; the half-life of tissue disappear-
ance was about 4 days. Derivatives of ascorbic acid including
magnesium ascorbyl phosphate, ascorbyl-6-palmitate, and dehy-
droascorbic acid did not increase skin levels of L-ascorbic acid.
conclusions.
Delivery of topical L-ascorbic acid into the skin
is critically dependent on formulation characteristics.
IN THE PRESENCE of our oxygen-rich atmosphere,
ultraviolet light generates reactive oxygen species in
skin. In addition to sunlight, other inflammatory in-
sults including smoking and pollution generate reac-
tive oxygen species. Reactive oxygen species, in turn,
cause oxidation of nucleic acids, proteins, and lipids.
Reactive oxygen species alter DNA,
1–7
as well as its
repair,
8
and trigger cytokine cascades that result in
photoaging
9,10
and photocarcinogenesis.
11
The body protects itself naturally from reactive ox-
ygen species by using antioxidants to neutralize them
before they cause damage to the skin and its compo-
nents. Vitamin C, or L-ascorbic acid, is the most
abundant antioxidant in skin.
12
Although most plants
and animals synthesize L-ascorbic acid to protect
themselves from free radical attack, a gene necessary
for its synthesis, L-gulono-
␥
-lactone oxidase, has been
mutated in humans.
13
As a result, humans rely on di-
etary intake for their supply.
14
L-ascorbic acid is quite
water soluble and serves as the major aqueous phase
reductant in the body.
15
Since skin relies on antioxidants for protection
against reactive oxygen species, and since skin pre-
dominantly receives and must deal with the free radi-
cal assault resulting from UV light, increasing the anti-
oxidant defense of skin becomes an attractive strategy
for increased photoprotection.
16
If antioxidants could
be delivered in high concentration through the stratum
corneum barrier into the skin, then the antioxidant
protective reservoir could be increased and photopro-
tection might be enhanced. Indeed, our laboratory has
described a stable aqueous formulation of L-ascorbic
acid that gets into skin and provides photoprotection
against both UVB and UVA-psoralen phototoxicity by
a mechanism that is clearly not a sunscreen effect.
17
Moreover, we have demonstrated that topical L-ascor-
bic acid protected against UV immunosuppression and
tolerance to contact antigen in mice.
18
In order to maximize the protective effects of topi-
cal L-ascorbic acid in skin, we have undertaken this
study of formulation composition and kinetics so that
we can maximize the amount of L-ascorbic acid deliv-
ered into the skin.
Materials and Methods
L-ascorbic acid (pharmaceutical grade) was purchased from
Roche (Nutley, NJ). All concentrations of L-ascorbic acid
were made fresh and stabilized in 2% ZnSO
4
, 0.5% biofla-
vonoids, 1% hyaluronic acid, 0.1% citrate in glass-distilled
water. pH was adjusted with triethanolamine. Commercial
formulations of 13% magnesium ascorbyl phosphate (Vivi-
H. Yang, MD, N. Monteiro-Riviere, PhD, H.V. DeBuys, MD, L.C.
Walker, Y. Wang, MD, and M. Levine, MD have indicated no signifi-
cant interest with commercial supporters. S.R. Pinnell, MD is a consult-
ant for Skinceuticals (Dallas, TX). M. Omar, PhD is president of Phyto-
Ceuticals (Elmwood Park, NJ).
Address correspondence and reprint requests to: Sheldon R. Pinnell,
MD, Duke University Medical Center, Department of Medicine, Divi-
sion of Dermatology, P.O. Box 3135, Durham, NC 27707, or e-mail:
pinne002@mc.duke.edu.
138
pinnell et al.: topical l-ascorbic acid
Dermatol Surg 27:2:February 2001
fying Serum C, Dr. Mary Lupo Skin Care Products, New
Orleans, LA) and 10% ascorbyl-6-palmitate (C-Esta Serum,
Jan Marini Skin Research, San Jose, CA) were obtained
fresh from the manufacturer and opened just prior to test-
ing. Samples were tested as is; the content was not con-
firmed. Dehydroascorbic acid 1 M (17.4%) was prepared
by taking 1 ml of a 1 M ascorbate solution, adding bromine
55
l, vortexing vigorously for 30 seconds, and then imme-
diately bubbling with nitrogen for 10 minutes.
19
The institu-
tional review board of the College of Veterinary Medicine at
North Carolina State University approved the animal exper-
iments.
Experiments were conducted in white Yorkshire pigs.
The skin was shaved with an electric shaver 24 hours before
the experiment began to allow healing of any skin nicks.
Two hundred
l of formulation, the maximal volume of the
chamber, was placed under a Hill Top Chamber (Hill Top
Co., Cincinnati, OH) for 22–24 hours. The chamber is semi-
occlusive and protects the material from smearing. Although
the chamber may enhance percutaneous absorption, in sev-
eral cases in the experimental results, the baseline skin levels
of L-ascorbic acid were not increased. In some experiments
the chamber was replaced with fresh solution and changed
each day. In clinical practice, vitamin C solutions are usually
applied daily. At the end of the experiment, the formulation
was washed vigorously from the skin with water. Washed
skin was tape stripped 15 times to remove surface contami-
nation and stratum corneum. The tape strips were dis-
carded. Studies have shown that tape stripping removes the
stratum corneum layers (data not shown) and removes sur-
face radioactivity of topically applied substances bound to
the stratum corneum.
20
,
21
Full-thickness 6 mm punch biopsy
specimens of skin were taken and placed immediately into
liquid nitrogen. Tissue was shattered in liquid nitrogen and
weighed aliquots extracted and stabilized in 60% methanol,
1 mM EDTA in water, centrifuged, and kept at
⫺
70
⬚
C until
analyzed. Samples were analyzed in a blinded manner for
vitamin C by high-performance liquid chromatography
(HPLC) with coulometric electrochemical detection.
22
,
23
The
method is specific for L-ascorbic acid and has a sensitivity of
50 fmol. The results are expressed as mean
⫾
standard devi-
ation. Unless otherwise noted,
n
⫽
3. The
P
values were cal-
culated by two-tailed Student’s
t
-test with equal variance.
Results
pH
Fifteen percent formulations of L-ascorbic acid were
tested at pH levels between 2.0 and 5.0 (Figure 1). Tis-
sue levels of L-ascorbic acid were enhanced only at for-
mulation pH levels less than 3.5. The pKa for L-ascorbic
acid is 4.2. Apparently the molecule must be un-ionized
for percutaneous absorption to occur. Low pH is es-
sential for absorption and delivery is enhanced as the
pH is reduced to 2.0. The effect of the solutions on
skin pH is unknown.
Concentration
L-ascorbic acid concentrations were tested from 5 to
30.0% (Figure 2). pH was adjusted to 3.2. Tissue lev-
els of L-ascorbic acid increased and were maximal at
20%. For unknown reasons, concentration levels
higher than 20% resulted in decreased tissue levels.
Kinetics
Fifteen percent L-ascorbic acid at pH 3.2 was applied
daily for 1–5 days (Figure 3). After 3 days, tissue levels
were apparently saturated. Levels achieved were ap-
proximately 20 times normal tissue levels.
Washout
Fifteen percent L-ascorbic acid at pH 3.2 was applied
daily for 5 days to saturate skin levels (Figure 4). Skin
levels of L-ascorbic acid were then measured at daily
intervals, with no further topical application of L-ascor-
bic acid, to measure L-ascorbic acid remaining in the
tissues. Half-life of L-ascorbic acid in tissues was found
to be approximately 4 days.
Figure 1. Effect of pH on percutaneous absorption. 15% L-ascorbic
acid at different pHs were applied to pig skin for 24 hours. Skin
levels of L-ascorbic acid are expressed as mean ⫾ SD (n ⫽ 3). *Av-
erage (n ⫽ 2).
Figure 2. Effect of concentration on percutaneous absorption.
Varying concentrations of L-ascorbic acid pH 3.2 were applied to
pig skin for 24 hours. Skin levels of L-ascorbic acid are expressed as
mean ⫾ SD (n ⫽ 3).
Dermatol Surg 27:2:February 2001
pinnell et al.: topical l-ascorbic acid
139
Ascorbic Acid Derivatives
Because L-ascorbic acid is an unstable molecule to
formulate for topical use, more stable derivatives of
L-ascorbic acid have been utilized in topical formula-
tions. Although esters of ascorbic acid are more stable
and readily converted to L-ascorbic acid after oral in-
gestion, it is not clear that derivatives, after topical ap-
plication, are absorbed into the skin or converted to
L-ascorbic acid after penetration. We have tested com-
mercially available high concentration formulations of
magnesium ascorbyl phosphate and ascorbyl-6-palmi-
tate to see if topical application resulted in elevated
skin levels of L-ascorbic acid (Figure 5). Neither ester
significantly increased L-ascorbic acid skin levels.
Dehydroascorbic Acid
Since dehydroascorbic acid can be enzymatically con-
verted to L-ascorbic acid in the body, we asked
whether topical dehydroascorbic acid could preferen-
tially raise skin L-ascorbic acid levels. Neither 20 mM
nor 1 M solutions of dehydroascorbic acid were effec-
tive. Skin levels of L-ascorbic acid were 7.51
⫾
3.34
pmol/mg for 20 mM dehydroascorbic acid and 8.70
⫾
2.13 pmol/mg for 1 M dehydroascorbic acid (
n
⫽
4)
and 9.24
⫾
3.55 for control skin.
Discussion
L-ascorbic acid is the most plentiful antioxidant in
body fluids
15
and in the skin.
12
It efficiently neutralizes
reactive oxygen species including superoxide anion,
24
hydroxyl radical,
25
singlet oxygen,
26
and peroxyni-
trite.
27
It is a particularly efficient antioxidant because
in one electron transfer reaction, its free radical inter-
mediate, ascorbic acid free radical, has low pro-oxi-
dant activity
28
and is enzymatically regenerated back
to L-ascorbic acid. Moreover, L-ascorbic acid’s effi-
ciency extends to lipophilic antioxidants as well; it re-
generates oxidized vitamin E molecules.
29,30
Maxi-
mum skin levels of L-ascorbic acid from ingestion are
regulated by active transport mechanisms that pre-
clude increasing levels by further ingestion.
31
In this
study we identify formulation characteristics that al-
low us to bypass these controls and increase the skin
reservoir by direct topical application.
This study reveals the critical importance of formu-
lation pH for percutaneous absorption of L-ascorbic
acid. Not until the pH was 3.5 or lower were cutane-
ous levels increased. Since the pKa of L-ascorbic acid
is 4.2, the molecule apparently must be un-ionized for
delivery across the stratum corneum barrier.
32
Percu-
taneous absorption of varying L-ascorbic acid concen-
trations formulated at acid pH increased steadily to a
maximum of 20%. Higher concentrations were less ef-
fective for unknown reasons. Daily application for 3
days of 15% L-ascorbic acid formulated at pH 3.2 re-
sulted in saturating skin concentrations of L-ascorbic
acid at more than 20 times control values. After satu-
rating the skin reservoir, the L-ascorbic acid was ap-
Figure 3. Time course of percutaneous absorption. 15% L-ascorbic
acid pH 3.2 was applied daily to pig skin for varying times. Skin
levels of L-ascorbic acid are expressed as mean ⫾ SD (n ⫽ 3).
Figure 4. Washout of skin L-ascorbic acid. Skin levels of L-ascorbic
acid were saturated by five daily applications to pig skin of 15%
L-ascorbic acid pH 3.2. After varying intervals skin levels of L-ascor-
bic acid were determined. Skin levels of L-ascorbic acid are ex-
pressed as mean ⫾ SD (n ⫽ 3).
Figure 5. Percutaneous absorption of ascorbic acid derivatives.
15% L-ascorbic acid pH 3.2 (VC-15), 10% ascorbyl-6-palmitate
(C-Esta), and 12% magnesium ascorbyl phosphate (Mag-C) were
applied to pig skin for 24 hours. Skin levels of L-ascorbic acid are
expressed as mean ⫾ SD (n ⫽ 10). P values are expressed versus
control. The P value of VC-15 versus each product is also 0.0005.
140
pinnell et al.: topical l-ascorbic acid
Dermatol Surg 27:2:February 2001
parently stabilized and remained in the tissue with a
half-life approaching 4 days. We have no data about
the relative distribution of ascorbic acid in the skin. A
persistent reservoir of antioxidant provides an impor-
tant and attractive photoprotection strategy when
contrasted to sunscreens which must be applied daily.
In the body both L-ascorbic acid and dehydroascor-
bic acid can be transported into cells, the latter con-
verted efficiently into L-ascorbic acid by glutathione
and enzymatic reduction.
33
L-ascorbic acid requires a
specific protein to be transported into cells. Hexose
transporters transport dehydroascorbic acid. Indeed
dehydroascorbic acid is preferentially accumulated in
comparison to L-ascorbic acid in HaCaT, a human
keratinocyte cell line.
34
Our experiments to deter-
mine whether dehydroascorbic acid was preferable to
L-ascorbic acid for topical use failed to reveal any in-
crease in skin levels of L-ascorbic acid with dehydro-
ascorbic acid.
Topical magnesium ascorbyl phosphate and ascor-
byl-6-palmitate in the tested formulations failed to in-
crease skin levels of L-ascorbic acid. Previous studies
have documented the marginal percutaneous absorp-
tion of magnesium ascorbyl phosphate;
35
as a charged
molecule, it would not be expected to traverse the stra-
tum corneum. Previous studies of ascorbyl-6-palmitate
failed to demonstrate protection against photoaging in
mouse skin;
36
in comparison, L-ascorbic acid was pro-
tective even though the formulation used was not op-
timal for percutaneous delivery. Although ascorbyl-
6-palmitate appears to readily enter skin,
36
its conversion
to L-ascorbic acid may be inefficient. Ascorbyl-6-pal-
mitate appears to remain on the extracellular surface
of cells and may not be readily converted to L-ascorbic
acid.
37
Indeed in human skin fibroblast culture 10
⫺
5
M
L-ascorbic acid, which is the physiologic concentration
in humans, stimulated cell growth, whereas similar lev-
els of ascorbyl-6-palmitate were toxic.
38
Topical antioxidants have been previously demon-
strated to be photoprotective for skin. Topical L-ascor-
bic acid has been shown to decrease UVB erythema in
pig
17
and human skin.
39
It also lessened UVA-psoralen
phototoxic injury in pig skin.
17
In hairless mice, topi-
cal L-ascorbic acid decreased photoaging changes in
human
40
and hairless mouse skin.
36
In addition, it pre-
vented UVB immunosuppression and tolerance to di-
nitrochlorobenzene (DNCB).
18
In persons with either
basal cell carcinoma or squamous cell carcinoma, se-
rum levels of L-ascorbic acid were below control lev-
els.
41
Topical
␣
-tocopherol decreased photoinjury in
skin
36,42–47
and prevented UV immunosuppression.
45,48,49
Herbal antioxidants, including silymarin, a flavonoid
present in the thistle plant,
50
and grape seed polyphe-
nols
51
have been shown to prevent UV-induced squa-
mous cell skin cancer in mice.
Although all UV light can produce oxidative stress
in skin, UVA is more efficient. The peak UV spectrum
for generation of singlet oxygen from trans-urocanic
acid, a known photoreceptor in skin, is about 350
nm.52 The UV spectrum is similar to that previously
demonstrated to generate photoaging changes in mouse
skin.53 UVA from artificial light sources has been dem-
onstrated to generate photoaging changes in sun-pro-
tected skin.54,55 Similar changes have been demonstrated
using only long-wave UVA (340–400 nm).56 Studies in
skin cells,57,58 as well as human skin,10 implicate acti-
vation of matrix metalloproteinase by a mechanism in-
volving singlet oxygen, AP-1, and NF-B. In prelimi-
nary studies, antioxidants reverse activation of AP-1.58,59
Previous studies have demonstrated that photoaging
changes are even more pronounced in smokers than
sunbathers, and the combination of smoking and sun
exposure was most damaging of all.60 Presumably smok-
ing and UV exposure are both damaging to skin by
generating reactive oxygen species. In smokers, serum
ascorbic acid levels were reduced;61 they required an
elevated minimum daily dose of L-ascorbic acid to
keep body stores saturated.26
In addition to its antioxidant effects, L-ascorbic
acid is important for wound healing.62,63 It is essential
for collagen synthesis; in addition to its cofactor re-
quirements for lysyl hydroxylase and prolyl hydroxy-
lase,64 it stimulates transcription of collagen genes.65 It
has been used as a skin lightener; it inhibits tyrosi-
nase.66 Topical L-ascorbic acid has been reported to
be useful for healing of skin resurfaced by CO2 laser;
it reduced postlaser erythema.67 Topical L-ascorbic
acid together with 20% glycolic acid used for 3
months improved striae alba.68
Topical L-ascorbic acid provides a safe and effec-
tive supplement to normal tissue stores to enhance
photoprotection, improve wound healing, and in-
crease antioxidant defenses. Details of formulation are
essential if it is to be maximally effective. It must be
formulated at high concentration and at a pH lower
than 3.5 to be effective. After being delivered into the
skin, L-ascorbic acid is stabilized and remains in the
tissue for a period of days. Magnesium ascorbyl phos-
phate and ascorbyl-6-palmitate are not effective sub-
stitutes for L-ascorbic acid in topical formulations. Al-
though they are effective vitamin C derivatives for oral
use, they are apparently ineffective for increasing tis-
sue vitamin C levels when applied to the skin.
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