189 IN VIVO ELONGATION OF TOPICALLY APPLIED FATTY ACIDS
the skin.2 This is essential to the skin barrier function. The lipid classes that dominate
the human SC are ceramides, cholesterol and free fatty acids (FFAs), which are organized
in lamellae.3–5 These are integral to skin barrier function as they maintain skin moisture,
limit the movement of material through the skin and prevent microbes and allergens from
entering the tissues.6
In healthy adults, most SC lipids adopt a dense orthorhombic packing.7,8 They can also
adopt a less dense, hexagonal packing that has been shown to increase permeability in
lipid model systems mimicking the human SC. Hexagonal packing is more abundant in
the skin of patients with atopic dermatitis (AD) than in healthy adults.9 In patients with
AD, SC FFA chain lengths are reduced compared with healthy adults in both non-lesional
and lesional skin, which is thought to contribute to reduced repeat distance of the lamellar
phases, reduced ceramide chain length, less dense lipid organization and decreased barrier
function.10–12 These findings indicate that both lipid organization and composition are
integral to the function of the skin barrier.
The composition and organization of SC lipids are not only affected by disease, but also
by external factors and behaviours. During the winter season, levels of SC lipids are
dramatically depleted compared with spring and summer.13,14 In particular, the ceramide
profile during winter season strongly correlate with indicators of dry skin condition.15 This
is likely to disrupt skin barrier function, influencing the activity of SC proteases involved in
desquamation and interfering with the production of natural moisturizing factors, leaving
the skin more prone to dryness.13 Likewise, regular cleansing with surfactant-containing
products is associated with symptoms of dryness, irritation, erythema, and post-wash
tightness.16 Such effects may be a result of the removal of SC lipids, exposing corneocytes
to the stresses of cleansing.17
Restoring SC lipid composition and organization, and therefore skin barrier function,
may be achieved through the application of topical formulations advancing the range of
benefits offered to the skin.4 Syndet bars and liquid cleanser formulations have evolved
from simply cleansing the skin to delivering moisturizing benefits, along with employing
milder surfactants helping to minimize skin damage.16 Moisturizers containing FFAs
and ceramides are also increasingly used for promoting barrier restoration and reducing
water loss.16,17 However, ex vivo evidence reveals that topically applied ceramides remain
predominantly on the SC surface, rather than penetrating deeper into the skin.18 In
previous work, we have shown significant increases in ceramides, FFAs, and cholesterol
in vivo following application of an FFA-containing leave-on lotion to cosmetically dry
skin correlated with improvements in the skin dryness.19 Levels of elongated FFAs
were also increased, suggesting increased synthesis and elongation from shorter-chain
FFA precursors.19 However, further investigation into the specific FFAs responsible for
influencing these beneficial changes is still needed. It is long established that FFAs are
deposited from lotions and cleansers to replenish the endogenous fatty acids lost during
the cleansing process.20 Investigation into the fate of FFAs from topically applied products
has been conducted in an ex vivo model of skin barrier repair to predict the in vivo
response.4 The topical application of both d31-PA and deuterated stearic acid resulted in
the elongation of fatty acids ex vivo, which were abundant up to C26:0.4 These findings
have been used as the foundation for in vitro studies using 3D human living skin equivalent
models.21 We hypothesize that the fatty acid elongation and incorporation into ceramides
demonstrated in vitro/ ex vivo can also take place in vivo from shorter-chain fatty acids
provided via topical formulations.

190 JOURNAL OF COSMETIC SCIENCE
Here, we demonstrate the first clinical evidence of the deposition of d31-PA into the SC of
human skin in vivo from a body wash, leave-on lotion, and a syndet bar formulation, and its
direct conversion into longer-chain FFAs and ceramides.
METHODS
STUDY DESIGN
We conducted two separate Institutional Review Board (IRB)-approved, randomized,
double-blind product application studies. The first study assessed a syndet bar formulation
and a leave-on lotion (Study 1), while the second study assessed body wash formulations
(Study 2). The data from each study were analyzed separately and conducted at Hill Top
Research, Winnipeg, Canada. Ethical approval was granted by IRB Services (Study 1:
MOD00194725, approval granted 29th January 2017 Study 2: MOD00244849, approval
granted 12th January 2018), and complied with the requirements defined in Health Canada
regulations (21 Code of Federal Regulations [CFR] parts 56 and 312.3 and US Department
of Health and Human Services [DHHS] regulations 45 CFR part 46). The IRB adheres
to good clinical practice (GCP) guidelines (e.g., International Council for Harmonisation
of Technical Requirements for Pharmaceuticals for Human Use GCP Guidelines), Health
Canada regulations, and is in compliance with 21 CFR parts 50 and 56, DHHS 45 CFR
part 46 and the Tri-Council Policy Statement for Ethical Conduct of Research Involving
Humans as appropriate to the research. Prior to inclusion, all participants gave their
informed consent to participate.
Skin dryness was graded by an expert assessor in both studies using a 0–6 scale of visual
dryness (“0 =none” to “6 =most severe”) with ½ point increments allowed. Study 1
included 12 healthy female participants aged between 35 to 50 years of age (mean age:
43.5 ± 5.1 years) with no-to-minimal dry skin on their forearms (Grade 0–1). Study 2
included 11 healthy female participants aged between 32 to 50 years of age (mean age:
41.7 ± 7.2 years) with no-to-minimal dry skin on their forearms (Grade 0–1) and moderate
dry skin on their lower legs (Grade 2–3).
Both studies included twice-daily controlled product application by trained researchers.
Applications were approximately three to four hours apart. Participants were instructed not
to use other cleansers during daily bathing or apply lotion to the test sites for the duration
of the studies.
In Study 1, after 3 days of conditioning with a mild syndet cleansing bar, one of the
following fully formulated products was applied to participants’ volar forearms twice daily
under controlled conditions:
• A mild syndet bar formulation containing FFAs and isethionate-based surfactant,
in which FFAs were partially replaced by d31-PA C16:0 (14% final). This bar
was dissolved in a 1:4 dilution with distilled water and 0.3 mL was dispensed onto
the designated pre-wet volar forearm. The solution was rubbed on the whole volar
forearm for 10 seconds, left for 30 seconds, and then rinsed off with tap water for 30
seconds.
• A leave-on lotion (0.3 mL) containing glycerine, petroleum jelly and d31-PA C16:0
(3.65% final). This was applied to the entire volar forearm and rubbed-in. Solvent
extracts and tape strips were collected at baseline and Day 28.