Content uploaded by David Counts
Author content
All content in this area was uploaded by David Counts on Aug 12, 2014
Content may be subject to copyright.
j. Soc. Cosmet. Chem., 39, 235-240 (July/August 1988)
The effect of retinyl palmitate on skin composition
and rnorphornetry
DAVID F. COUNTS, FRANK SKREKO, JULIANNE McBEE,
and A. G. WICH, The Lilly Research Laboratories, Eli Lilly and
Company, Lilly Corporate Center, Indianapolis, IN 46285.
Received January 21, 1988.
Synopsis
Topical administration of increasing doses (0.1%-5% (w/w)) of retinyl palmirate (RP) for 14 days, in a
suitable cosmetic vehicle, caused significant dose-related changes in skin composition and morphometry of
the hairless mouse. There was a maximum 32% increase of protein per unit of skin surface area and a
maximum of 128% increase of collagen per unit of skin surface area in response to RP administration when
compared to control vehicle. In addition, there was an increase of DNA content in response to RP adminis-
tration. There was significant thickening of the epidermis in response to the increasing dose of RP. Al-
though the total thickness of skin was not significantly increased by RP application, the total skin thick-
ness was greater than the untreated control or the control-vehicle-treated animals at every dose of RP tested.
These results indicate that the topical application of RP (in an active form) can alter the composition and
morphometry of the skin in the hairless mouse.
INTRODUCTION
Vitamin A or retinol is essential for normal skin development. Vitamin A is an impor-
tant regulator of keratinocyte terminal differentiation (1). An excess of vitamin A in-
hibits keratinization (1), whereas a deficiency results in squamous metaplasia and kera-
tinization of epithelial tissue (2). Thus, epidermal development is, in part, regulated by
vitamin A. It is also known that vitamin A can alter or modulate total collagen syn-
thesis (3-5). Also, retinoic acid has been demonstrated to alter the type of collagen
synthesized (6).
Retino! has the potential to alter the expression of protein molecules in both the epi-
dermis and dermis. Although retinoic acid can alter both keratinocyte as well as fibro-
blast (7) protein metabolism, the precise effect of retino! on intact skin composition has
not been determined. Several cosmetic formulations contain vitamin A. The present
study was undertaken to determine whether or not vitamin A, if present in an active
form in cosmetic formulations, can alter skin composition and morphology.
MATERIALS AND METHODS
ANIMALS
Female hairless mice (HRS/J) (six to eight weeks old) were obtained from Jackson Labo-
235
236 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
ratories (Bar Harbor, Maine) and maintained for one week prior to all studies. The mice
were housed five animals per cage in standard "shoe box"-type cages (6 in x 10 in x 5
in) and given food and water ad libitum. Each animal received 0.1 ml of a test cosmetic
formulation applied to the dorsal skin surface and rubbed into the skin (approximately
5-10 sec), using a gloved finger as an applicator. This treatment was continued each
day for 13 additional days.
BIOCHEMICAL AND HISTOLOGICAL ANALYSES
On Day 15, the animals were terminated by cervical dislocation and the skin removed
from the dorsal, treated surface of the animals. One section of the skin was taken for
histological evaluation and a second piece was scraped free of subcutaneous fat. Two
circular punch biopsies (6 mm in diameter) were taken from the latter, blotted dry, and
weighed. These biopsies were then pulverized in liquid nitrogen and homogenized in
phosphate-buffered saline using the Polytron-ST homogenization system. This homoge-
nate was used to determine the protein content, DNA content, and collagen content (as
measured by proteinaceous hydroxyproline content of the tissue) (8). An equal volume
of 10% (w/v) trichloroacetic acid (TCA) was added to the homogenate, and the suspen-
sion mixed and allowed to equilibrate for five minutes (3-5øC). The suspension was
centrifuged at 10,000 x g for ten minutes. The pellet was suspended in 0.5 ml of 5%
(w/v) TCA and centrifuged at 10,000 x g for ten minutes. The pellet was suspended in
0.2 ml of 10% (w/v) TCA and heated at 90øC for 20 minutes. The suspension was then
set in an ice bath for 30 minutes. The suspension was then centrifuged at 10,000 x g
for three minutes. The supernatant was analyzed for DNA as described by Schneider
(9). The precipitate was suspended in 0.5 ml of 0.5M NaOH, heated for one hour at
55øC, and an aliquot was removed for protein determination (10). The remainder of the
sample was sealed in a glass ampule in vacuuo with an equal volume of 12 N HC1 and
hydrolyzed for 24 hours at ! !0øC. After hydrolysis the sample was dried in a desiccator
and the dried material was assayed for hydroxyproline (! 1).
The skin samples taken for micromorphometric analyses were fixed in buffered neutral
formalin solution, dehydrated, and embedded in paraffin blocks which were then cut
into 7-1•m-thick sections. These sections were stained with hematoxylin and eosin for
later examination. Thickness measurements were based on the cross-sectional thickness
of the skin. Several (six to eight) sections were stained and used for evaluation per
animal. Thickness measurements (eight to ten) per animal were made by selecting the
measurement site at random. The average of these measurements was considered as the
measurement for one animal. Total skin thickness was considered the thickness of the
skin from the outer stratum corneum to the panniculus carnosus. Dermal thickness was
taken as the distance from the dermal epidermal junction to the top of the dermal
adipose deposits.
COSMETIC FORMULATION
All cosmetic formulations used in this study were oil-in-water emulsions and were
manufactured under a nitrogen blanket. The water phase of the emulsion systems
ranged from 79-$4%, the oil phase was !6%, and the retinyl palmitate (Roche) con-
centration ranged from 0-5 %. Water was removed from the emulsion to accommodate
EFFECT OF RETINYL PALMITATE ON SKIN 237
the additional retinyl palmitate. The concentration of the retinyl palmitate in each
formulation was determined by HPLC on a Lichrosorb Si 60/5 micron column (25 cm
X 4.5 mm id) (Alltech Assoc.) using 0.4% (v/v) methanol / 0.4% (v/v) isopropanol in
99.2% (v/v) isooctane as the mobile phase after separation and saponification according
to a modified USP XXI procedure (12). The determinations of the retinyl palmitate
concentration were made both before and after the animal treatment period. Results
indicated no significant degradation of the retinyl palmitate throughout the two-week
treatment period and were found to vary by less than 20% of the theoretical amount of
retinyl palmitate.
RESULTS
Skin tissue per surface area, as determined by punch biopsy weight, did not change
appreciably (Table I). This finding correlates well with the little or no change in total
skin thickness determined by micromorphometry (Table II). Although there was a 21%
increase in total skin thickness in animals treated with 0.1% retinyl palmitate when
compared to untreated control mouse skin, this change was not noted at any of the
other doses of retinyl palmitate. This increase was noted to be due to changes in both
the dermal thickness (Table II) and the adipose layer in the dermis. Although there was
a dramatic and dose-related increase in skin collagen content when up to 1.5 % retinyl
palmitate (Table I) was administered, there was no concomitant increase in dermal
thickness (Table II).
The most dramatic change in skin composition was noted in the collagen content. The
Table I
Effect of Treatment With Increasing Doses of Retinyl Palmitate in Visible Difference on Skin Composition
Collagen
Punch Protein DNA content
Treatment weight content content (nmole HYP)
group (mg/punch) (mg/punch) (Ixg/punch) (punch)
Control 11.8 - 0.5 1.28 _ 0.09 44.2 - 3.7 82 _ 20
(no treatment) (8) (8) (8) (7)
Vehicle q- 12.1 - 0.5 1.37 - 0.07 51.3 - 2.3 67 - 8
0% retinyl palmitate (8) (8) (8) (8)
Vehicle q- 12.7 - 0.4 1.71 -+ 0.13"** 62.1 ñ 4.3*'** 126 - 18'*
0.1% retinyl palmitate (8) (8) (8) (8)
Vehicle q- 13.1 ñ 0.5 1.71 - 0.10"** 57.7 ñ 3.2* 135 ñ 27**
0.5 % retinyl palmitate (8) (7) (8) (8)
Vehicle + 12.5 - 0.5 1.81 - 0.10"** 54.2 ñ 4.4 153 - 15"**
1.5% retinyl palmitate (8) (8) (8) (8)
Vehicle q- 12.9 ñ 0.4 1.70 ñ 0.08*'** 66.5 ñ 3.4"** 134 ñ 14"**
5.0% retinyl palmitate (8) (8) (8) (8)
Values represent the mean ñ S.E.M. of the number of animals in parentheses. The diameter of each punch
was 6 mm.
* Indicates significantly different from untreated control at p • 0.05.
** Indicates significantly different from the control-vehicle-treated group at p • 0.05.
238 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Table II
Effect of Treatment With Increasing Doses of Retinyl Palmitate in Visible Difference on
Skin Morphometry
Epidermal Dermal Total
Treatment thickness thickness thickness
group (}xm) (}xm) (}xm)
Control 27.9 --- 1.8 171 --- 17 377 -+ 28
(no treatment) (8) (8) (8)
Vehicle + 42.5 - 4.3* 180 --- 15 411 --- 25
0% retinyl palmirate (8) (8) (8)
Vehicle + 63.5 --- 2.9"** 206 ___ 24 458 --- 25*
O. 1% retinyl palmirate (8) (8) (8)
Vehicle + 71.7 - 2.6*'** 201 --- 14 431 --- 15
O. 5% retinyl palmirate (8) (8) (8)
Vehicle + 74.7 - 3.7"** 209 + 19 430 --- 28
1.5% retinyl palmitate (8) (8) (8)
Vehicle + 86.0 --- 4.9"** 286 --- 13"** 424 - 17
5.0% retinyl palmirate (8) (8) (8)
Values represent the mean --- S.E.M. of the number of animals in parentheses.
* Indicates significantly different from untreated control at p •< 0.05.
** Indicates significantly different from the control-vehicle-treated group at p •< O. 05.
lowest administered retinyl palmitate dose maximally increased the amount of protein
present per unit of surface area. The DNA content was near maximal at 0.1% retinyl
palmitate and decreased to near normal at 1.5% retinyl palmitate but increased dramat-
ically at 5% retinyl palmitate.
The photomicrographs in Figure 1 demonstrate the dose-related increase in epidermal
thickness (Table II). Although there is an inflammatory cell infiltrate into the dermal
E
DEJ
NT 0% RP 0.1% RP 0.5% RP 1.5% RP 5% RP
Figure 1. Cross section of skin following 14 days of application of 0.1 ml of cosmetic vehicle. The
concentration of retinyl palmitate in each cosmetic vehicle is indicated under each skin cross section. E
indicates the epidermal tissue, DEJ indicates the dermal/epidermal junction, and D indicates the dermal
tissue. Magnification is 80 X.
EFFECT OF RETINYL PALMITATE ON SKIN 239
tissue, note that changes in skin composition and the epidermal proliferative changes
are also apparent.
DISCUSSION
Cosmetic materials are generally accepted to have a minimal effect on skin biology. The
proven benefits of the application of cosmetic skin treatment products have usually been
attributed to a moisturization effect. One particular material used in cosmetic formula-
tions, vitamin A, is known to have systemic physiological and biochemical effects
(7,13). All of the physiological functions of retinoids (such as the requirement for
normal vision and reproduction) cannot be satisfied by retinoic acid (7). Indeed, these
functions seem to be adequately satisfied by retinol. However, the role of retinoids in
the regulation of skin development seems to be best satisfied by retinoic acid. Thus,
part of the actions of retinyl palmitate in skin may depend on its conversion to retinoic
acid (13). This conversion depends on the enzymatic cleavage of the ester bond in the
retinyl palmitate. Nonspecific esterase enzyme activity exists within skin (14). In addi-
tion, the skin must be able to oxidize the retinol to retinoic acid. It has been demon-
strated that skin preparations can indeed convert retinyl to retinoic acid (15). Although
it has been known that retinyl palmitate has the potential to effect a change of skin
composition and physiology, this potential has never been examined.
Retinoids, in general, prevent connective tissue atrophy. This prevention of atrophy is
probably mediated by an inhibition of collagenolytic activity (16). In addition, dermal
repair in response to UV damage, which is accelerated by retinoic acid administration,
appears to involve a zone of regenerating connective tissue (17). Although retinoids
inhibit collagen synthesis (3,5,18), they do promote glycosaminoglycan synthesis
(5,19,20). Therefore, some of the effects of retinoids in terms of altering dermal com-
position can be thought of as due to an inhibition of connective tissue breakdown and a
promotion of the synthesis of glycosaminoglycans. This hypothesis is .supported by the
observed changes in skin composition following treatment with retinyl palmitate.
The increase of DNA content within the skin can be partially explained by the influx of
inflammatory cells into the skin. Although there was an increase of skin DNA content
following treatment with 0.1% retinyl palmitate, this was the maximal response. There
is then a decrease of DNA content with increasing concentrations of retinyl palmitate.
This may be due to the inhibitory effect of high concentrations of retinoids on both cell
mobility and cellular proliferative capacity (18,21). However, at the highest dose ad-
ministered (5 %), there was a tremendous increase in DNA content. This may be due to
an irritant/inflammatory response to the application of the retinoid. Of interest in this
study is that even with irritation present, there was little or no change in the punch
biopsy weight. This suggests that the response is not significantly mediated by an
edematous response. Finally, retinyl palmitate administration causes an accumulation of
collagen within the dermis. These changes indicate that although there is an irritation
response in the skin treated with RP, there is also a significant effect on the collagen
content within the dermis. This alteration of the collagen content is unlikely to be due
to an irritation response.
240 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Thus, cosmetic products which contain retinyl palmitate, in an active form, may indeed
bring about biochemical changes with both the epidermis and the dermis.
REFERENCES
(1) J. Kubilus, Modulation of differentiation by retinoids, J. Invest. Dermatol., 81, 55s-58s (1983).
(2) W. Wolbach and P. R. Howe, Tissue changes following deprication of fat soluble vitamin A, J. Exp.
Med., 42, 753-777 (1925).
(3) H. Oikarinen, A. Oikarinen, E. Tan, R. Abergel, C. Meeker, M. Chu, D. Prockop, and J. Uitto,
Modulation of procollagen gene expression by retinoids, J. Clin. Invest., 75, 1545-1553 (1985).
(4) R. Beach and C. Kenney, Vitamin A augments collagen production by corneal endothelial cells,
Blochem. Biophys. Res. Commun., 114, 395-402 (1983).
(5) M. Kenney, L. Shih, U. Labermeir, and D. Satterfield, Modulation of rabbit keratocyte production of
collagen, sulfated glycosaminoglycans and fibronectin by retinyl and retinoic acid, Biochim. Biophys.
Acta, 889, 156-162 (1986).
(6) P. Benya and S. Padilla, Modulation of the rabbit chondrocyte phenotype by retinoic acid terminates
type II collagen synthesis without inducing type I collagen: The modulated phenotype differs from
that produced by subculture, Develop. Bid., 118, 296-305 (1986).
(7) F. Chytil, Retinoic acid: Biochemistry and metabolism, J. Am. Acad. Dermatol., 15, 741-747
(1986).
(8) D. Counts, P. Knighten, and G. Hegreberg, Biochemical changes in the skin of mink affected with
Ehlers-Danlos syndrome: Increased collagen biosynthesis in the dermis of affected mink, J. Invest.
Dermatol., 69, 521-526 (1977).
W. Schneider, Determination of nucleic acids in tissues by pentase analysis, Methods Enzymol., 111,
680-684 (1956).
O. H. Lowry, N. Rosebrough, A. Farr, and R. Randall, Protein measurement with the Folin phenol
reagent, J. Biol. Chem., 193, 265-275 (1951).
D. Prockop and S. Udenfriend, A specific method for the analysis of hydroxyproline in tissues and
urine, Anal. Biochem., 1, 228-239 (1960).
The United States Pharmacopeia, XXIth ed. (United States Pharmacopeial Convention, Inc., Rockville,
MD, 1985), p. 1215.
S. Shapiro, "Retinoids and Epithelial Differentiation," in Retinoids and Cell DifJSrentiation, Michael
Sherman, Ed. (CRC Press, Inc. Boca Raton, FL, 1986), pp. 25-59.
Y. Igoshin, Histochemical study of nonspecific esterases, lipase and cholinesterase in normal skin,
Vestnik Dermatologii Vernerologii, 12, 13-16 (1976).
M. Connor and M. H. Smit, The formation of all-trans retinyl in hairless mouse skin, Blochem.
Pharmacol., 36, 919-924 (1987).
E. Bauer, J. Seltzer, and A. Eisen, Retinoic acid inhibition of collagenase and gelatinase expression in
human skin fibroblast cultures. Evidence for dual mechanism, J. Invest. Dermatol., 81, 162-169
(1983).
L. Kligman, Effects of all-trans-retinoic acid on the dermis of hairless mice, J. Am. Acad. Dermatol.,
15, 779-785 (1986).
R. Hein, H. Mensing, P. Muller, O. Braun-Falco, and T. Krieg, Effect of vitamin A and its deriva-
tives on collagen production and chemotactic response of fibroblasts, Br. J. Dermatol,, 1 ! 1, 37-44
(1984).
I. King, Increased epidermal hyaluronic acid synthesis caused by four retinoids, Br. J. Dermatol.,
110, 607-608 (1984).
I. King and F. Pope, Synthesis of cellular and extracellular glycoproteins by cultured human keratin-
ocytes and their response to retinoids, Biochim. Biophys. Acta, 887, 263-274 (1986).
C. Marcelo and K. Madison, Regulation of the expression of epidermal keratinocyte proliferation and
differentiation by vitamin A analogs, Arch. Dermatol. Res., 276, 381-389 (1984).
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
