Arch Dermatol Res (1997) 289 : 404–409
© Springer-Verlag 1997
O R I G I N A L PA P E R
M. Fartasch · J. Teal · G. K. Menon
Mode of action of glycolic acid on human stratum corneum: ultrastructural and functional evaluation of the epidermal barrier
Received: 20 November 1996
Abstract Alpha-hydroxy acids (AHA) such as glycolic acid have recently been used extensively in cosmetic and dermatological formulas. In low concentration (2– 5%) glycolic acid is believed to facilitate progressive weakening of cohesion of the intercellular material of the stratum corneum (SC), resulting in uniform exfoliation of its outermost layers (the stratum disjunctum). Since thinning of the SC as well as changes of intercellular lipids could theoretically compromise the barrier functions of the skin, we investigated the mode of AHA action on the SC to determine whether enhanced desquamation compromises the barrier structures of the SC and changes transepidermal water loss (TEWL) values. Electron microscopy of the epidermis biopsied from the volar forearm of human volunteers after 3 weeks of treatment with a 4% glycolic acid formulation twice daily was employed to evaluate 1) epidermal morphology and thickness of the SC, (2) the lamellar body and SC lipid bilayer organization, and (3) desquamative events based on degradation of desmosomes. TEWL values and SC hydration were recorded prior to and at the end of the study. Electron microscopy revealed no ultrastructural changes in the nucleated layers of the epidermis. The lamellar body (LB) secretory system in the stratum granulosum (SG), and intercellular lipid lamellae in the SC in both vehicleand glycolic acid-treated samples were comparable to normal human SC. Within the SC, enhanced desmoso-
This work was presented in part at the annual meeting of The Society For Investigative Dermatology, Baltimore, Maryland, 1994 M. Fartasch (Y) Department of Dermatology, University of Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany Tel. +49-9131-853638; Fax +49-9131-853850; e-mail:
[email protected] J. Teal · G. K. Menon R&D Division, Avon Products, Inc., Division Street, Suffern, NY 10901, USA
mal breakdown, promoting loss of cohesion and desquamation, was restricted to the stratum disjunctum while desmosomes of the stratum compactum were unaffected. Treated areas displayed histologically, a more compact appearing SC. TEWL values remained unchanged in glycolic acid- and vehicle-treated skin. Our findings indicate that the barrier structures of the SC are not disrupted by glycolic acid formulations at the concentration used. One of the mechanism of action of AHA on the SC seemed to be a „targeted“ desmosomal (corneosomal) action without compromising the barrier structures of the skin. Key words Alpha hydroxy acid · Epidermal barrier · Stratum corneum · Lipid structures · Desmosomes
Introduction Alpha-hydroxy acids (AHA) are organic acids present in natural sources such as fruits, wine and milk. Such sources of AHA have been used as cosmetic material for several centuries. Since reports [4, 37–39] that AHA are effective in treating hyperkeratotic skin conditions such as ichthyosis, and in the treatment of acne [17, 19] and pseudofolliculitis barbae [26], their use in cosmetic dermatology has increased dramatically [9, 14, 28, 30]. The popularity of facial peels employing fairly high concentrations of AHA (20–70%) has soared as part of ‘anti-aging or wrinkle treatment regimens. However, the greatest impact of AHA has been in skin care and beauty regimens, with AHA-containing cosmetic products for daily use proliferating about 100-fold [2, 18, 28, 30]. Compared to the popularity of these products worldwide, information on the mode of action as well as real and perceived effects on the skin is scanty [32, 34] and sometimes controversial. There have been generalizations on the modes of action of AHA [31], as well as several claims of functionalities based on cell culture studies [29]. Well-defined and controlled human studies with a specific focus are required to delineate facts from broad generalizations that are often made,
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which disregard concentration, pH and the specific AHA in question. The importance of the pH, as well as the formulation, on AHA effects on various skin compartments has been stressed by VanScott and Yu [40]. One crucial issue is the site of action of cosmetic concentrations of AHA within the stratum corneum (SC), and whether their effects are confined to the most superficial layers or affect the deeper SC as often assumed [37, 39]. Since the majority of marketed glycolic products for regular use (skin care products) contain low concentration of glycolic acid (2–5%), the aim of the present study was to address specifically: (1) whether 4% glycolic acid in a cosmetic formulation (pH 3.8) affects the deep SC via a decrease in corneocyte cohesion (as reported by Van Scott and Yu [37–39]) leading to detrimental effects on cutaneous water barrier function, (2) which structural entities in the SC are targets for the action of AHA, and (3) whether the lipid lamellae and other extracellular structures, such as desmosomes, of the SC and stratum granulosum (SG) are altered by its daily applications for up to 3 weeks.
Two punch biopsies were obtained from the right and left ventral forearms of all four subjects. To preserve an intact full-thickness SC during further processing the modified embedding technique of Holbrook and Odland [16] was used, described briefly as follows. Prior to biopsy the skin surface was marked with pyoctanin blue to ensure that the biopsying and the process of dividing did not remove the outer layer of SC. Biopsies were divided into two parts. Then small sheets of lens paper were placed above and below the tissue. This preparation was then placed onto a hollow plastic cylinder and secured in place with an open-top cap. Half of the biopsy was processed for routine electron microscopy following aldehyde fixation and postfixation with osmium tetroxide. The other half was postfixed with 0.5% ruthenium tetroxide (Polyscience, Warrington, Pa.) and 0.25% potassium ferrocyanide for 1 h in darkness at 4° C. Samples were routinely dehydrated and embedded in plastic. Thick sections were stained with 1% methylene blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a Jeol 100 CX transmission electron microscope. For the purpose of evaluating the number of corneocytes before or after treatment the SC was photographed, a line was drawn perpendicular to the skin surface on each micrograph. The number of cells intersected by that line were then counted.
Results Materials and methods
TEWL, capacitance and colorimetry
Four healthy subjects (one male and three females, age range 27–45 years) with no history of atopic dermatitis or other skin diseases were recruited in the study. Informed consent was obtained from all subjects. The volar forearm was chosen as the test region because transepidermal water loss (TEWL) values at various volar forearm sites have been well studied and characterized [1, 4, 25, 36, 37]. An area on each arm, 10 cm above the wrist skinfolds, was marked in the middle part of the forearm, and designated as the test site. Basic values were measured on normal skin at the two test sites on each arm prior to the initiation of the treatment and after 3 weeks of treatment at least 6–8 h after the last application. Additionally, pre- and posttreatment skin capacitance measurements were performed. A 4% glycolic acid formulation (pH 3.8) or the vehicle formulation as a cosmetic lotion were applied to the volar forearm twice daily for 3 weeks. The formulation was an ‘essence’ type (clear, slightly viscous fluid) containing in addition to glycolic acid, water, a mixture of glycols, thickeners and preservatives. To minimize subjective influences on quantitative assessment, the letters A and B were allotted to the glycolic acid and vehicle. The application of the glycolic acid or the vehicle formulation to either the left or right arm was randomized among subjects. In three of the four subjects additionally TEWL and skin capacitance measurements were carried out in an air-conditioned room after the subjects had acclimatized to the room conditions for 30 min (room temperature 20–22° C, relative humidity 30–45%). Skin temperature (which was between 28.3 and 30.9° C) was measured at all test sites using a skin thermometer (° C H11 S, Digimed). TEWL measurements were performed using a TEWAMETER (Tewameter 2180, Courage and Khazaka, Cologne, Germany) under standardized environmental conditions [1, 27, 36]. The probe rested on the skin and the TEWL was continuously recorded for a period of 3 min. Mean values were obtained from three successive recordings for every test site. Skin hydration was investigated by measurement of skin capacitance (CORNEOMETER CM 820, Courage and Khazaka). Skin surface color was quantified using the standard tristimulus system suggested by the commission Internationale de l’ Eclairage (CIE) using a Minolta Chroma Meter CR200 colorimeter according to the manufacturer × s instructions. The color is expressed in using three-dimensional coordinate system. The color of the skin is expressed as an admixture of a*, b* and L* values. Luminance (L*) expresses the brightness. a* represents the color range from green to red, and b* the color range from blue to yellow.
Measurements of vehicle and glycolic acid-treated skin showed no marked increase in TEWL after the 3 weeks of treatment (Table 1). Monitoring of capacitance did not show evidence of increased or decreased water content of the SC after either treatment (Table 1). With colorimetry neither the L* values nor the a* values gave evidence of erythema at either test site (data not shown). Morphology Light microscopy Light microscopic examination of the thick sections did not reveal any structural differences in the nucleated layers between the glycolic acid-treated and the vehicletreated skin. In contrast, the horny layer of glycolic acidtreated skin appeared more compact. Electron microscopy Glycolic acid-treated and vehicle-treated epidermis were similar and showed no abnormalities in their ultrastrucTable 1 Measurements of TEWL and capacitance in glycolic acidand vehicle-treated skin Sub- TEWL (g/m2 h) ject no. Glycolic acid Vehicle
1 2 3
Capacitance (arbitary units) Glycolic acid Vehicle
Before After
Before After
Before After
Before After
9.0 10.9 7.5
8.4 9.3 8.1
73 92 68
76 90 70
10.0 10.0 6.4
8.0 9.4 6.8
85 79 69
85 84 68
406 Fig. 1A, B Low magnification electron micrographs of vehicle- versus glycolic acid-treated epidermis. The glycolic acid-treated site (B) shows a normal stratum compactum. There was no evidence of loss of cohesion between the corneocytes of the stratum compactum (lower SC); osmium tetroxide staining, subject no. 2 (bar 10 µm) Fig. 2 A Normal lamellar body (LB) structures and secretory system in glycolic acidtreated sites. The glycolic acidtreated samples show intact corneosomes (arrows) in the stratum compactum (lower SC) osmium tetroxide staining, subject no. (3 bar: 1 µm). B Ruthenium tetroxide staining reveals normal, unchanged lipid bilayers of the intercellular spaces in the lower SC of the glycolic acid-treated sites (arrow); subject no. 3 (bar: 0.1 µm)
1A
2A tural features. In the SC the keratohyalin granules displayed a normal stellate appearance and normal stratum compactum (Fig. 1A, B). No noticeable differences in cell layers of the SC or SG (stratum granulosum) were seen in glycolic acid-treated skin (Table 2).
B
B Epidermal lamellar bodies (LB) displayed normal morphology and cytosolic distribution, appearing identical in both glycolic acid-treated and vehicle-treated skin. The typical pattern of LB fusion with the apical membrane of keratinocytes was seen at the SG/SC interface (Fig. 2A).
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The extracellular domains appeared compact and filled with extruded LB contents. There was no evidence of loss of cohesion between desmosomes at the level of the stratum compactum (lower SC, Fig. 2A). Comparison of the
Table 2 Number of SC layers in glycolic acid- and vehicletreated skin
Number of SC layers Subject no.
Glycolic acid
Vehicle
1 2 3 4
17 18–20 18–20 21–22
17–18 24 17–19 20–22
Fig. 3A, B Ruthenium tetroxide staining reveals enhanced corneosome degradation (arrows) within the stratum disjunctum of glycolic acidtreated sites (B) compared with a vehicle-treated site (A) with more corneosome retention (arrows) subject no. 1 (bar 1 µm)
A
B
intercellular (Fig. 2B) lipid-enriched lamellae of glycolic acid-treated sites with vehicle-treated sites showed no differences in the bilayer pattern and were comparable to normal human SC profiles as described in earlier studies [13]. Desmosomes (corneosomes) in the lower SC (stratum compactum) in the glycolic acid-treated sites were normal and unaltered (Fig. 2A, B), their electron-dense plugs of desmosomes spanning the entire width of the intercellular space. In the outermost SC (stratum disjunctum) degradation of desmosomes herald normal desquamatory events. The desmosomal plug showed mostly separation of the central core and formed spindle-like bodies with pointed edges [13] in locally enlarged intercellular spaces (ICS) (arrow, Fig. 3B), followed by the degeneration of the opaque middle portion of plugs, with electron-
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dense areas still preserved at their periphery. In comparison to the vehicle-treated sites (Fig. 3A) this process seemed more advanced in the glycolic acid-treated sites (Fig. 3B). The effect was limited to three or four superficial layers (stratum disjunctum) of the SC, and as seen in Fig. 2A, desmosomes in the lower layers (stratum compactum) in glycolic acid-AHA treated skin appeared normal and unaffected.
Discussion Known beneficial effects of AHA (glycolic, lactic, gluconolactone) at low concentrations (4 to 8%) include improvement in xerosis [24] and synergy/compatibility with retinoids or steroids in treating photodamage [21]. For the assessment of the effects of AHA, it is essential to identify the type, the concentration and pH employed, as well as to identify the conditions of use (therapeutic/cosmetic peels vs. daily use in skin care products). The present study showed that a 4% glycolic acid formulation (pH 3.8), applied twice daily, neither increases the TEWL values as an indicator of a barrier disruption as discussed previously [32] nor decreases the thickness of the human SC. Furthermore, our ultrastructural studies show that the barrier lipid structural organization of human SC was not altered. The site of action of glycolic acid at this concentration and under these conditions of application appeared to be the desmosomes (corneosomes). Normally, progressive stages of degradation of the desmosomal plug are seen as a function of their precise location in the different SC layers [13]. The normal degradation in the stratum disjunctum was accelerated in the glcolic acid-treated skin compared with vehicle-treated skin. It is possible only to speculate about the mechanism(s) by which AHA enhance the normal process of corneosome dissolution. Enhanced desquamation may be achieved via preferential acidification of these polar domains embedded within the hydrophilic lipid bilayers, or by activating by other means the acid proteases crucial for desmosomal degradation [7, 33]. However, it is important to note that the morphology of desmosomes in the lower SC (stratum compactum, Fig. 2A) and the cohesiveness of the layer remained intact. This observation is not in accordance with generalizations made previously that low concentrations of AHA diminish corneocyte cohesion in the lower levels of the SC [37, 39]. In the qualitative morphological part of the study, no striking differences in the number of SC cell layers were apparent between the glycolic acid-treated and vehicletreated sites. This may reflect the fact that in clinically normal skin, the effects of AHA (at the low concentration tested) is to smooth the skin surface by removing the randomly retained squames of the stratum disjunctum. The effect of AHA on the SC has also been investigated by photon tunneling microscopy, a novel imaging technique, and further validated by scanning electron microscopy [15]. Although it has been reported previously [28], an increase in SC hydration following a 3-week AHA treat-
ment was not evident in our study. In a previous study [6], in which 12% glycolic acid was applied for 2 weeks, the hydration of the SC was even shown to be reduced after day 11. Another aspect could be the observation of Takahashi and Machida [35] that the increased flexibility of the SC after topical application of AHA is not related to an increased water content of the SC [3, 22]. This apparent discrepancy may be because our subjects had normal skin to begin with, whereas in subjects with xerosis, improved hydration may be readily seen [22]. In addition to the fact that the stratum compactum cohesion remains unaltered, the lipid bilayer structures of the SC did not reveal any abnormal changes following repeated, daily glycolic acid application. Additionally, focusing on the stratum compactum, the glycolic acidtreated skin showed a normal secreted content of epidermal LB [5, 8, 12, 13, 20, 23], as well as the regular transformation of their secreted lipids into intercelluar lipid bilayers. Disturbances of the LB secretory mechanism, as observed in noninvolved dry atopic skin [12], or an alteration in the transformation of LB-derived lipids into bilayers as found in irritated skin [10, 11, 23] were not observed. Thus, there were no indications, histologically, ultrastructurally or from TEWL data, of altered epidermal barrier function after daily applications of a 4% glycolic acid formulation.
References 1. Barel AO, Clarys P (1995) Study of stratum corneum barrier function by transepidermal water loss measurements: comparison between two commercial instruments: evaporimeter and tewameter. Skin Pharmacol 8:186–195 2. Beradesca E, Distante F (1994) AHA: biological effects on skin function. Tenth Symposium of the Belgian Association of Dermato-Cosmetic Sceinces (BADECOS), Brussels. proceedings pp 11–23 3. Berardesca E, Maibach H (1995) AHA mechanisms of aciton. Cosmetics Toiletries 110:30–31 4. De Boer EM, Bezemer PD, Bruynzeel DP (1989) A standard method for repeated recording of skin blood flow using laser doppler flowmetry. Dermatosen 37:58–62 5. Downing DT (1991) Lipids; their role in epidermal structure and function. Cosmetics Toiletries 106:63–69 6. Effendy I, Kawangsukstith C, Lee JY, Maibach HI (1995) Functional changes in human stratum corneum induced by topical glycolic acid: comparison with all-trans retinoic acid. Acta Derm Venereol (Stockh) 75:455–458 7. Egelrud T, Lundstrom A (1991) A chymotrypsin-like proteinase that may be Involved in desquamation in plantar stratum corneum. Arch Dermatol Res 283:108–112 8. Elias PM (1983) Epidermal lipids, barrier function and desquamation. J Invest Dermatol 80:44–49 9. Elson ML (1992) The utilization of glycolic acid in photoaging. Cosmetic Dermatol 5 :12–15 10. Fartasch M (1995) Human barrier formation and reaction to irritation. Curr Probl Dermatol 23:95–103 11. Fartasch M (1997) Ultrastructure of the epidermal barrier after irritation. Microsc Res Tech 37 : 193–199 12. Fartasch M, Bassukas ID, Diepgen TL (1992) Disturbed extruding mechanism of lamellar bodies in dry non-eczematous skin of atopics. Br J Dermtol 127:221–277
409 13. Fartasch M, Bassukas, ID, Diepgen TL (1993) Structural relationship between epidermal lipids, lamellar bodies and desmosomes in human epidermis. An ultrastructural study. Br J Dermatol 128:1–9 14. Franchimont PC, Mauhin DF, Dubois A, Gofifn V, Viatour M, Pierard GE (1994) Rides et microrelief cutane modificatins par un α-hydroxy acide. Rev Med Liege XLIX:268–273 15. Guerra J, Menon GK (1996) Photontunneling microscopy: a new technique for imaging stratum coreneum: Intl. Soc. Bioeng Skin. 20th Anniversary Symposium, International Society of Bioengineering of the skin, 15–17 February, Miami, Florida 16. Holbrook KA, Odland GF (1974) Regional differences in the thickness (cell layers) of the human stratum corneum: an ultrastructural analysis. J Invest Dermatol 62:15–22 17. Hunt MJ, Barnetson RS (1992) A comparative study of gluconolactone versus benzoyl peroxide in the treatment of acne. Australas J Dermatol 33: 131-134 18. Jackson EM (1994) Update on AHA-containing products. Cosmetic Dermatol 7 :29–30 19. Kligman AM (1995) The compatibility of combinations of glycolic acid and tretinoin in acne and in photodamaged facial skin. J Geriatr Dermatol 3:25A–28A 20. Landmann L (1986) Epidermal permeability barrier: transformation of lamellar granule-discs into intercellular sheets by a membrane-fusion process, a freeze-fracture study. J Invest Dermatol 87:202–209 21. Lavker RM, Kaidbey K, Leyden JJ (1992) Effects of Topical ammonium lactate on cutaneous atrophy from a potent topical corticosteroid. J Am Acad Dermatol 26:535–544 22. Leyden JJ, Lavker RM, Grove G, Kaidby K (1995) Alpha hydroxy acids are more than moisturizers. J Geriatr Dermatol 3:33A–37A 23. Menon GK, Feingold KR, Elias, PM (1992) Lamellar body secretory response to barrier disrupton. J Invest Dermatol 98: 279–289 24. Morganti P, Persechino S, Bruno C (1994) Effects of topical alpha hydroxy acids on skin xerosis. J Appl Cosmetol 12:85–90 25. Panisset F, Treffel P, Faivre B, Lecomte PB, Agache P (1992) Transepidermal water loss related to volar forearm sites in humans. Acta Derm Venereol 72:4–5 26. Pericone NV (1993) Treatment of pseudofolliculitis barbae with topical glycolic acid: a report of two studies. Cutis 52: 232–235
27. Pinnagoda J, Tupker RA, Agner T, Serup J (1990) Guidlines for transepidermal water loss (TEWL) measurement. Contact Dermatitis 22:164–178 28. Rubin MG (1994) Therapeutics: personal practice. The clinical use of alpha hydroxy acids. Australas J Dermatol 35:29–33 29. Sakaki S, Oyama T, Okano Y, Masaki H (1993) Application of MTT test for the screening of cell growth activities – evaluation of α hydroxy acids. J Soc Cosmet Chem J 27:166–169 30. Sexton CR, Rubin MG (1994) An overview of alpha hydroxy acids. Dermatology Nursing 6:17–22 31. Smith WP (1994) Hydroxy acids and skin aging. Cosmetics Toiletries 109:41–44 32. Smith WP (1996) Epidermal and dermal effects of topical lactic acid. J Am Acad Dermatol 35:388–391 33. Sondell B, Thornell LE, Egelrud T (1995) Evidence that stratum corneum chymotryptic enzyme is transported to the stratum corneum extracelluar space via lamellar bodies. J Invest Dermatol 104:819–823 34. Stiller MJ, Bartolone J, Stern R, Smith S, Kollias N, Gillies R, Drake LA (1996)Topical 8% glycolic acid and 8% L-lactic acid creams for the treatment of photodamaged skin. Arch Dermatol 132:631–636 35. Takahashi M, Machida Y (1985) The influence of hydroxyacids on the rheological properties of the stratum corneum. J Soc Cosmet Chem 36:177–187 36. Van der Valk PGM, Mailbach HI (1989) Potential for irritation increases from the wrist to the cubital fossa. Br J Dermatol 121:709–712 37. Van Scott E, Yu RJ (1974) Hyperkeratinization, corneocyte cohesion and alpha hydroxy acids. J Am Acad Dermatol 11:867– 879 38. Van Scott E, Yu RJ (1982) Substances that modify the stratum corneum by modulation of its formation. In: Frost P, Horwitz SN (eds) Principles of cosmetic for the dermatologist. Mosby, St. Louis, pp 70–74 39. Van Scott EJ, Yu RJ (1989) Alpha hydroxy acids: therapeutic potential. Can J Dermatol 1:108–112 40. VanScott EJ, Yu RJ (1995) Actions of alpha hydroxy acids on skin components. J Geriatr Dermatol 3:[Suppl A] 19A–25A
