The Roles of p H and
Concentration in Lactic
Acid-in duced Stimulation
of Epidermal Turnover
DAVID 0. THUESON, PHD
ELAINE K. CHAN, PHARMD
LAUREN M. OECHSLI, BA
GARY S. HAHN, MD
BACKGROUND. Alpha hydroxy acids such as lactic and glycolic
acids (AHAS)accelerate epidermal turnover and exfoliate the
stratum corneum. The roles of pH and concentration in these
antiaging effects of AHAs is unclear, but a lower (more acidic)
pH and higher concentration of acid are thought to be more
effective.
OBJECTIVE. To examine the efects on skin renewal rates of lactic
acid 10% at pHs of 2.0, 3.0,and 4.0, and of 5%, 10% and 15%
lactic acid at a pH of 3.0.
METHODS. Twenty-six female subjects participated in this dou-
ble-blind, randomized, placebo-controlled study. The dansyl
M
uch attention has been focused on alpha hy-
droxy acids (AHAs), a group of organic acids
found naturally in many fruits, plants, and
foods, which, when applied to the skin, have been
shown to induce exfoliation of the stratum corneum. In
addition, AHAs can stimulate skin cell renewal and
reduce fine lines and wrinkles, thereby providing a
more youthful appearance. AHAs (lactic and glycolic
acids in particular) have been used in cosmetics for
decades, and were discovered to have effects on kera-
tinization by Van Scott and Yu in 1974.' Since then,
research into the physiologic actions of these com-
pounds on specific skin compartments has shown that
the effects of AHAs extend beyond the stratum cor-
neum to the epidermal and dermal layers as ell.^-^ At
the level of the stratum corneum, topically applied
AHAs reduce corneocyte adhesion at the lowest levels
of the stratum corneum, which results in desquamation
of both normal and diseased skin, the latter resulting in
normalization of retention hyperkeratosis.' The exact
mechanism of desquamation is still unknown, but spec-
From Cosmederm Technologies, lnc. (DOT, EKC, LMO,GSH), La lolla;
and the Immunology b Allergy Division, Department of Pediatrics (GSH),
University of California, Sun Diego, California.
Elaine K. Chun, PhurmD is now at Lakeport Hospital, Lakeport, CA.
This work was supported by Cosmederm Technologies, lnc.
Address correspondence and reprint requests to: G a y S. Hahn, MD,
Cosmederm Technologies, 3252 Holiday Court, La lolla, C A 92037.
0 1998 by the American Society for Dermutologic Surgery, Inc. Published by Elsevier Science Inc.
10764512/98/$19.00 PI1 S1076-0512(98)00041-7
chloride stain technique was used to measure epidermal renewal
times.
RESULTS. Both pH and concentration are critical in the lactic
acid effect. A t aFxed lactic acid concentration, the desquamative
effect was highly pH dependent. A t afixed pH, the turnover rate
of skin was concentration dependent.
CONCLUSION. The desquamative and proliferation-stimulating
effects of lactic acid are very pH and concentration dependent,
suggesting the "flee acid" concentration is the active moiety. 0
1998 by the American Society f i r Dermatologic Surgery, Inc.
Dermatol Surg 1998;24:641- 645.
ulation has centered on sulfate transferases, phospho-
transferases, and b a s e s , which are the enzymes re-
sponsible for the sulfation and phosphorylation of
proteins, which form the ionic bonds between corneo-
cytes.6 At the epidermal level, AHAs stimulate basal
cell mitosis, with resultant epidermal proliferation? At
the dermal level, AHA treatment in clinical trials has
demonstrated increased skin thickness that is due at
least in part to the stimulation of glycosaminoglycan
production! The discovery of these AHA effects has led
to the clinical use of these acids in ichthyosis, actinic
keratosis, warts, acne, and wrinkle reduction.7t8
While the mechanisms behind the AHAs' "antiag-
ing" effects remain controversial, they have been shown
clinically to increase skin firmness, improve smooth-
ness, and reduce fine lines and wrinkles.' Part of the
antiaging effects of AHAs has been attributed to their
ability to exfoliate the skin and induce epidermal pro-
liferation. The fact that skin renewal rates naturally de-
cline with age has been demonstrated in numerous
~tudies.'~-'~ The ability of the AHAs, therefore, to
speed skin renewal is consistent with clinical observa-
tions that A H A s counteract many visible signs of skin
aging.
The role of AHA concentration and the pH of the
product in influencing the antiaging effects of AHAs is
still controversial, but a lower (more acidic) pH and
higher concentrations are purported to produce a faster
611
642 THUESON ET AL
ORIGINAL ARTICLES
onset of effects and to be more effective, but are also
more irritating to the skin.7To date, however, there has
been very little objective data on the role of pH in
AHA-induced skin renewal.
In these studies, we examined the effect on skin re-
newal rates of a representative AHA (lactic acid, 10%) at
a very low pH (2.0), a moderate pH (3.0), and a high pH
(4.0). The dansyl chloride labeling technique was used
to measure stratum corneum renewal time under con-
ditions of different pHs at a fixed lactic acid concentra-
tion compared with a control lotion. In another series of
experiments, we examined the role of different concen-
trations of lactic acid held at a constant pH on the ability
to stimulate skin cell turnover. Lactic acid concentra-
tions of 5%, lo%, and 15%were compared a t a pH of 3.0
to compare efficacy under controlled conditions.
Materials and Methods
Materials
Dansyl chloride (1-dimethylaminonapthalene-5-sulphonyl
chloride) was obtained from Curtin Matheson Scientific Inc.
and was finely triturated at 5% (wt/wt) into white petrola-
tum, USP. A long-wave ultraviolet lamp (Black Ray, Solar
Light Company, Philadelphia, PA) was used to visualize the
fluorescent dansyl stain. Baby Magic Baby Lotion & Aloe (The
Mermen Company, Morristown, NJ) was used as the lotion
vehicle since it is formulated to be nonirritating for sensitive,
baby skin. Lactic acid at 5%, lo%, or 15%by weight (85% lactic
acid; Spectrum Chemical Mfg. Corp., Gardena, CA) was
added to the lotion and different samples were then partially
neutralized using NaOH to adjust the pHs to 2.0, 3.0, and 4.0
as specified for the study. The unadulterated lotion vehicle
(pH 4.0) served as the placebo (vehicle) control.
Subjects
Nine healthy female volunteers, 29-52 years of age (mean, 35
years) participated in the first study. Informed consent was
obtained. For the second study, 17 female volunteers, 34-64
years of age (mean, 47 years), were studied.
Stratum Corneum Staining
Each subject had two areas on each of the right and left volar
forearms stained with dansyl chloride (5%wt/wt in petrola-
tum) as per the method outlined by Jansen et al.I4 The petro-
latum containing dansyl chloride was liberally smeared over
Hill Top Chambers (25 nun in diameter) and fastened to the
forearm using occlusive tape. After 24 hours, the patches were
removed and the excess ointment was removed. Fluorescence
was scored according to a subjective visual scale of 0-4 (0 =
no fluorescence remaining; 1 := only small areas of fluores-
cence remaining; 2 = moderate areas of fluorescence remain-
ing with noticeable areas of fading; 3 = large areas of fluo-
rescence remaining with small areas of fading present; 4 =
whole, even areas of fluorescence present throughout) to
quantitate and confirm good stain intensity.
Derrnatol Surg
1998;24;641- 645
lnitial (Acute) Treatment: Weeks 1-3
Following staining, subjects were provided tubes of the lotion
vehicle containing the appropriate lactic acid concentration, at
the specified pH (pH 2.0, pH 3.0, or pH 4.0). Another tube
contained the placebo lotion. Labeling of the tubes maintained
the double-blind design of the treatment assignments. Sites on
the right and left volar forearms of each subject were random-
ized so that each site was assigned one of the four treatments.
Subjects were instructed to apply a 1/8th-inch bead of the
designated lotion to each of the sites twice daily (morning and
evening). Sites were examined under long-wave ultraviolet
lamp illumination on days 1 (baseline), 7,9,11,14,16,18, and
21. Fluorescence intensity was scored on the scale of 0-4 as
described above. The endpoint was designated as the day on
which the complete disappearance of the fluorescent stain
occurred or by the peeling away of the stained skin area,
leaving no stain.
Prolonged (Chronic) Treatment: Weeks 5-7
During weeks 3-5, subjects continued to use each lotion twice
daily after the stained spots disappeared. At the beginning of
week 5, all four sites on the forearms of all subjects were
restained with dansyl chloride. The same protocol as was
used initially was followed. After the restaining, subjects re-
sumed lotion treatments twice daily to the same designated
areas of the forearms through to the second endpoint of loss
of stain. The same examiner scored all sites and both the
examiner and the subjects remained blinded throughout the
entire study.
Data Analysis
Because the subjects returned only every second or third day
(over weekends), the exact day of stain disappearance was
estimated using the following method: on the day when the
dansyl stain(s) was(were) judged to be completely gone, the
day of the last score greater than zero was noted and the
"disappearance day" was determined by adding the last flu-
orescence score (either 1,2, or 3) to that visit day number. For
example, if on day 13 the score was "1" and on day 15 the
score was "0," day 14 was the "disappearance day" (ie, 13 +
1 = 14).
The mean epidermal turnover time for each of the treat-
ment groups was calculated along with the standard error of
the mean. Statistical analyses using the repeated measures
analysis of variance (ANOVA) was performed, and statisti-
cally significant individual treatment differences were deter-
mined using the Student's Newman-Keuls test. The paired t
test was then used to determine statistically sigruficant differ-
ences (P < 0.05) between selected treated and control turnover
times.
Results
Acute Treatment: Weeks 1-3
The mean time for disappearance of fluorescence (ie,
epidermal turnover time) was calculated for the nine
subjects for each of the four different pH treatment
groups. The mean and standard error of the mean (2 +-
SEM) for the acute treatment phase (weeks 1-3) are
shown in Figure 1. All three lactic acid-containing lo-
tions at pH 2.0, 3.0, and 4.0 decreased epidermal turn-
Dermatol Surg
1998;24;641-645
Figure 1. Mean epidermal turnover times (2 t SEM) of volar
arm skin sites treated BID with lactic acid, 10% at pH 2.0, pH
3.0, or pH 4.0, or vehicle for 1-3 weeks. For placebo us pH 2, 3,
and 4, P C 0.05; for pH 2 us pH 3 and 4, P < 0.05;for pH 3 us
pH 4, P > 0.05.
over time significantly (P < 0.05) compared with pla-
cebo, with the pH 2.0-treated site (X = 12.4 ? 1.0 days,
P < 0.01) showing the fastest rate of epidermal turn-
over. The pH 3.0-treated site (%= 15.4 ? 0.8 days) was
not significantly different from the pH 4.0 site (X =
15.6 t 0.8 days), but both were different compared with
the placebo treated site (X = 18.2 ? 1.6 days, P < 0.05).
Visible peeling (desquamation of large flakes of epider-
mis) of at least one treatment site was noted in seven
subjects, with all seven showing peeling at their pH
2.0-treated site, and two of nine subjects displayed
peeling at their pH 3.0-treated site. No peeling was
observed at the pH 4.0- or placebo-treated sites.
Chronic Treatment: Weeks 5-7
The mean epidermal turnover times (X ? SEM) for each
of the treatments during the chronic treatment phase
Figure 2. Mean epidermal turnover times (X ? SEM) of volar
arm skin sites treated BID with lactic acid, 10% at pH 2.0, 3.0,
or 4.0 or with vehicle for 5-7 weeks. For placebo us pH 2 and 3,
P < 0.05; for placebo us pH 4, P > 0.05;for pH 2 us pH 4, P <
0.05;for p H 2 us pH 3, P > 0.05;for pH 3 us pH 4, P > 0.05.
THUESON ET AL 643
CONCENTRATION AND pH EFFECTS OF LACTIC ACID
Figure 3. Comparison of mean epidermal turnover times (X 2
SEM) for sites treated with lactic acid, 10% at pH 2.0, 3.0, 4.0,
or placebo during acute and chronic treatment phases. The paired
t test (see P values) was used to determine statistical differences
between acute and chronic turnover times.
(weeks 5-7) are shown in Figure 2. The pH 4.0 lactic
acid 10% treatment did not achieve a statistically dif-
ferent turnover time compared with the placebo lotion
(nonlactic acid, pH 4.0). The pH 3.0-treated site (X =
10.1 t 0.9 days), however, did produce a significantly
faster turnover time compared with both pH 4.0 (X =
13.1 ? 1.0 days, P < 0.01) and placebo (X = 14.7 ? 1.1
days, P < 0.01) lotions. In fact, the pH 3.0-treated site
did not differ sigruficantly from the pH 2.0-treated site
(X = 9.8 t 0.7 days), which was also significantly faster
than the control and pH 4.0 sites. No peeling was ob-
served at any time at any of the treatment sites on the
subjects during this extended phase of the study.
Comparison of Epidermal Turnover Time during
Acute Treatment (Weeks 1-3) and Chronic Treatment
(Weeks 5-7)
The mean epidermal turnover times for each treatment
site during acute and chronic treatment are presented
together in Figure 3. All four of the treatments resulted
in faster turnover times during the chronic treatment
phase compared with the acute treatment phase. This
result implies an increase in cell renewal rate following
4 weeks of twice daily moisturizer treatment even with-
out AHA. The epidermal turnover times for the pH
3.0-treated site showed the most pronounced increase
in effect between acute and chronic use and was highly
statistically sigruficant (P = 0.001) compared with con-
trol.
Efect of AHA Concentration on Treatment and
Turnover Time
The mean epidermal turnover time (X 2 SEM) for each
of the three different concentrations of lactic studied is
644 THUESON ET AL
ORIGINAL ARTICLES
Figure 4. Mean epidermal turnover times (a 2 SEM) of volar
arm skin sites treated BID with lactic acid, 5%, lo%, or 15% all
at pH 3.0 or with vehicle (0%)for 5-7 weeks. For 0% vs 5%,
lo%, and 15%, P < 0.05;for 5% vs 10% and 15%, P < 0.05;
for 10% 'us 15%, P > 0.05.
shown in Figure 4. Each of the treatments was adjusted
to pH = 3.0 so that the relative concentration of "free
acid" was proportional at the lactic concentrations of
5%, lo%, and 15%studied. Figure 4 shows that the skin
turnover rate increased with increasing concentration
of lactic acid and that all three concentrations induced
an apparent increase in skin turnover rate compared
with the vehicle control. The increase in turnover rate
was relatively greater (and statistically significant) for
the 5% to 10% concentration change than for the 10%to
15% concentration increase, suggesting that a maximal,
plateau effect may have been reached by the 15% level.
Discussion
Under normal conditions of homeostasis, the epidermis
is in a state of equilibrium where the rate of cell forma-
tion in the basal layers equals the rate of transit of cells
from the granular layer to the stratum corneum, where
desquamation takes place. Topically applied AHAs can
disturb this steady state through exfoliative and other
actions on the stratum corneum.
The time required for dansyl chloride to disappear
from the fully stained stratum corneum equals the tran-
sit time of epidermal cells from the deepest layers of the
stratum corneum to surface desquamation. Although
the measured epidermal transit time has been used as
an indicator of mitotic activity, Ridge et all5 have
shown that under conditions where a state of disturbed
equilibrium is produced, the epidermis must be al-
lowed to reestablish a steady state prior to the measure-
ment of transit time if the results are to reflect the full
effects of the treatments. Ridge et a1 postulated that the
initial effects seen on transit time reflect a change in the
Dermatol Surg
1998;24;641- 645
desquamation process only, and that later effects reflect
changes in the mitotic rate of the epidermis.
In our studies, the acute effects seen during weeks
1-3 are most prominent for the pH 2.0, lactic acid 10Y0-
treated site. The effect is related to the large number of
subjects (seven of nine) who experienced visible peeling
at this site. Little if any of the apparent early increase in
turnover rate seen for this treatment could be due to
increased epidermal proliferation, but rather must be
attributed to strong desquamation effects of the pH
being 2.0. The later (5-7 weeks of treatment) increase in
skin turnover is no doubt due to true increased prolif-
eration.
While the pH 3.0 treatment showed a somewhat
shorter (faster) turnover time compared with placebo
during the acute treatment, the effects were not fully
appreciated until after chronic treatment (7 weeks) to
establish the new steady state. With continued treat-
ment, the pH 3.0 lactic acid 10% treatment site demon-
strated a dramatic increase in turnover rate, eventually
approximately equaling the pH 2.0 results. This "de-
layed" onset of action in the pH 3.0 group is probably a
true reflection of increased epidermal proliferative ac-
tivity. Unlike the pH 2.0 treatment, this treatment group
did not show substantial peeling (only two of nine sub-
jects displayed visible peeling and only early in the
treatment).
Although the pH 4.0 treatment demonstrated an ap-
parent slightly increased turnover rate compared with
placebo during the initial treatment, the effect is not
significantly different from the placebo after continued
treatment. These results imply a largely desquamative
(acute) effect induced by 10% lactic acid at pH 4.0 treat-
ment with a relatively minor induction of proliferative
(chronic) effects. It has been shown by others that pla-
cebo treatment can indeed accelerate apparent e ider-
ma1 turnover compared with untreated sites?," Our
findings confirm these reports. Placebo treatment alone
with a moisturizing lotion decreased epidermal turn-
over time from 18.2 days initial1 to 14.7 days after 5-7
weeks of treatment. Ridge et alz have speculated that
such an increase in turnover rate may be due to an
immediate desquamative effect induced by surfactants
within oil/ water emulsions coupled with mechanical
forces applied to the skin during treatment. It is also
possible that skin hydration from moisturizers may
loosen intercorneocyte binding within the stratum cor-
neum and cause desquamation. Van Scott and Yu6 pos-
tulated that in a highly hydrated stratum corneum, the
distances between corneocytes is increased, resulting in
a decrease in cohesive force and more rapid desquama-
tion.
Epidermal turnover times for the stratum corneum
on untreated volar forearms reported by different au-
thors range from 13.3 to 18.5 days.'*'' Our placebo
Dermatol Surg
1998;24;641-645 CONCENTRATION AND pH EFFECTS OF LACTIC ACID
data agree with these reported values, and once again
confirms that the normal renewal time of the skin is
approximately 2-3 weeks. Additionally, we have deter-
mined the epidermal turnover times following repeated
treatment with a single concentration of AHA (lactic
acid 10%) at three different pHs and for three concen-
trations at one pH, to elucidate the roles of pH and
concentration in AHA-induced skin turnover.
Compared with placebo, 5%, lo%, and 15% lactic
acid in the lotion vehicle increased the skin turnover
rate significantly and in a dose-related manner, with an
increased skin turnover rate for each increase in con-
centration. The effect seems to begin to plateau at the
highest concentration tested, 15%.
Although the mechanisms by which A H A s act to
exfoliate and increase turnover rate for the skin are not
well understood, some investigators suggest that it is
directly related to the concentration of "free acid" ap-
plied. The free acid concentration depends on the
amount (concentration) of acid applied and the pH of
the formulation used according to the Henderson-Has-
selbalch equation. Most commercial AHA products to-
day are formulated at pHs of approximately 4 or
higher. The pKa values of the most commonly used
AHAs are typically 3.2-3.8, so less than half of the total
AHA present, and frequently less than 30% of the acid
in the formulation (at pH L 4.2), is in the "free acid"
form. Other studies have suggested that the efficacy of
AHAs to substantially enhance skin turnover is appar-
ent only at pHs of 3.5 or below. Our studies confirm this
concept and suggest that for a lactic acid 10% lotion, a
pH of approximately 3.0 induces optimal epidermal
turnover which, with continued treatment, eventually
equals that of the same acid concentration at a pH of 2.0.
Lactic acid 10% lotion at pH 4.0, however, is no more
effective at stimulating epidermal renewal than a non-
AHA moisturizing lotion (pH 4.0), which by its mois-
turizing actions alone should increase epidermal turn-
over for untreated skin.
It is widely accepted that topically applied A H A s
increase epidermal turnover and cause desquamation.
In our study, we have found that lactic acid-induced
stimulation of epidermal turnover is strongly pH and
concentration dependent. As demonstrated by the pH
2.0-treated site, the more acidic the pH of the prepara-
tion, the faster the onset and more dramatic the effect of
desquamation. Although 10%lactic acid at pH 3.0 has a
slower onset of action, the epidermal turnover rate at
pH 3.0 equals that of pH 2.0 after prolonged treatment,
while pH 4.0 appears to be no more effective than a
THUESON ET AL 645
non-AHA moisturizer in accelerating epidermal turn-
over. The concentration of "free acid" seems to be the
main determinant of the effect on skin turnover rate, as
shown in the present studies. When the product pH is
4.0 or more, very little of the AHA is in the free acid
form and the efficacy of the AHA is lost. To optimize
lactic acid-induced skin renewal, our data suggest that
the product pH should range between 3.0 and 3.5 to
provide 50-75% of the AHA in the free acid form,
which is the effective entity for maximal epidermal re-
newal.
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