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J. Cosmet. Sci., 74.3, 188–199 (May/June 2023)
*Address all correspondence to Galina Yarova, galina.yarova@unilever.com
In Vivo Elongation of Topically Applied Fatty Acids From a
Range of Different Topical Skincare Formulations
GALINA YAROVA, WILLIAM F. LATHROP, ANDREW E. MAYES, STACY HAWKINS,
BRIAN DOBKOWSKI, BIVASH R. DASGUPTA AND JOHN S. BAJOR
Department of Human Biology, Unilever U.S. Research &Development, Trumbull, Connecticut, USA
(G.Y., W.L. J.B.)
Unilever U.S. Research &Development, Clinicals, Trumbull, Connecticut, USA (S.H.)
Unilever U.S. Research &Development, Product Development, Trumbull, Connecticut, USA (B.D., B.D.)
Department of Human Biology, Unilever U.K. Research &Development, Colworth Science Park,
Sharnbrook, Bedford, United Kingdom (A.M.)
Accepted for publication August 05, 2023.
Synopsis
The lipid matrix of the stratum corneum (SC) is essential to skin barrier function, and detrimental structural
changes may occur in response to human behaviour, disease, and external factors. The application of topical
formulations is a common approach to repairing the SC and has been investigated ex vivo and in vitro. We
aim to demonstrate the first clinical evidence of deuterated palmitic acid (d31-PA) deposition from topical
formulations into the SC of human skin in vivo and its conversion into longer-chain free fatty acids (FFAs) and
ceramides. Healthy female participants aged 30 to 50 years old participated in two separate product application
studies. Participants were randomized to an application of a leave-on lotion or a syndet bar containing d31-PA
in the first study, with the second study having an option of a body wash containing different levels of d31-PA.
Skin tapes and solvent extracts were collected at baseline and Day 28. Lipid composition and the presence
of deuterated sphingosine were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS).
After four weeks, d31-PA was sufficiently delivered into the SC by all topical formulations. LC/MS/MS data
show that FFAs are elongated up to C26:0 and used in the formation of ceramides and free sphingosine. These
data provide the first in vivo evidence that FFAs deposited from a range of different topical formulations are
elongated in the SC and provide the building blocks for ceramide synthesis. Together, the evidence shows
that commercially available topical formulations can nourish the skin which may contribute to maintaining
a healthy skin barrier.
INTRODUCTION
The skin is the largest organ of the human body, with a surface area of 1.5 m2 in adults,
and acts as a barrier between the body’s interior and external environment.1 The uppermost
epidermal layer of the skin, the stratum corneum (SC), comprises corneocyte layers embedded
in a lipid matrix, which acts as the main pathway for the diffusion of substances through

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.