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Keywords:

  • ceramides;
  • desmosomes;
  • desmosomes;
  • natural moisturiser factor;
  • stratum corneum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References

Synopsis

The process leading to the loss of corneocytes form the skin surface is termed desquamation. In healthy skin it is an orderly and essentially invisible process whereby individual or small groups of corneocytes detach from neighbouring cells to be lost to the environment and replaced by younger cells from the deeper layers. Desquamation is carefully controlled to ensure that corneum cohesion and integrity, and hence tissue thickness, is maintained.

The most important components of the corneocytes contributing towards intercellular cohesion are the corneodesmosomes and lipids. Corneodesmosomes are proteinaceous complexes which effectively rivet corneocytes together. The intercellular lipids, primarily responsible for the water barrier, also provide part of the extracellular cement. In addition, the shape of the corneocyte itself plays a role in stratum corneum cohesion. Through interdigitation along their peripheral edges, adjacent corneocytes become physically locked together, a process which reinforces the integrity of the tissue.

For effective desquamation to occur corneodesmosomes must be degraded: a process catalysed by serine proteases present within the intercellular space and facilitated by subtle changes in lipid composition and phase behaviour. Ultimately, it is the availability of free water which controls corneodesmolysis. In healthy skin this proteolytic process leaves relatively few corneodesmosomes intact in the most superficial layers.

By contrast, in chronic and acute dry skin conditions, corneodesmosomal degradation and hence the final stages of desquamation are perturbed, leading to the characteristic formation of visible, powdery flakes on the skin surface. The inability to degrade these structures ultimately reflects a decreased hydrolytic activity of the desquamatory enzymes, either through reduced synthesis of the enzymes, inherent loss of activity, leaching from the surface layers of the corneum or changes in the surrounding lipid-rich microenvironment, which may indirectly reduce enzyme functionality.

Increased understanding of the desquamation process is providing new insights into the mode of action of current moisturizing ingredients and is offering opportunities to develop novel therapies for preventing and correcting dry skin.

Résumé

Le processus conduisant à la perte des cornéocytes à la surface de la peau est appelé desquamation. Sur une peau saine c’est un processus normal et quasiment invisible par lequel des cornéocytes individuels ou par petits groupes se détachent des cellules voisines pour se perdre dans l’environnement et être remplacés par des cellules plus jeunes provenant des couches plus profondes. La desquamation est soigneusement contrôlée pour assurer le maintien de la cohésion cornée et son intégrité, et par conséquent l’épaisseur du tissu.

Les composants les plus importants des cornéocytes contribuant à la cohésion intercellulaire sont les cornéodesmosomes et les lipides. Les cornéodesmosomes sont des complexes protéinés qui fixent efficacement les cornéocytes entre eux. Les lipides intercellulaires, principalement responsables de la barrière aqueuse, apportent aussi une partie du ciment extracellulaire. En outre, la forme du cornéocyte lui-même joue un rôle dans la cohésion de la couche cornée. Par une interdigitation le long de leurs extrémités périphériques, les cornéocytes adjacents se bloquent physiquement les uns les autres, un processus qui renforce l’intégrité du tissu.

Pour qu’une desquamation efficace se produise les cornéodesmosomes doivent être dégradés: un procédé catalysé par les protéases de la sérine présentes dans l’espace intercellulaire et facilité par de subtils changements dans la composition des lipides et le comportement de la phase. Enfin, c’est la disponibilité de l’eau libre qui contrôle la cornéodesmolyse. Chez les peaux saines ce procédé protéolytique laisse relativement peu de cornéodesmosomes intacts dans la plupart des couches superficielles.

Au contraire, dans les cas de peau sèche aigus et chroniques, la dégradation cornéodesmosomale et donc les étapes finales de la desquamation sont perturbées, conduisant à la formation caractéristique de particules floconneuses, visibles à la surface de la peau. L’incapacitéà dégrader ces structures reflète finalement une activité hydrolytique diminuée des enzymes de desquamation, soit par synthèse réduite des enzymes, par perte inhérente d’activité, par lessivage des couches de surface de la cornée ou par modifications du milieu riche en lipides environnant, ce qui peut indirectement réduire la fonction enzymatique.

Une meilleure compréhension du processus de desquamation apporte un éclairage nouveau sur le mode d’action des ingrédients hydratants actuels et donne des opportunités de développement de nouvelles thérapies pour prévenir ou traiter la peau sèche.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References

The skin is the major barrier tissue preventing desiccation. To achieve this, the outermost layer of the skin, the epidermis, undergoes a process of terminal differentiation to produce a thin, inert water-retaining layer, the stratum corneum. Morphologically, the epidermis is comprised of four layers: the basal layer; the spinous layer; the granular layer; and the outermost cornified layer or stratum corneum ( Fig. 1) [1]. The lower basal layer contains the undifferentiated epidermal stem cells, which upon commitment to differentiation detach from the basement membrane and migrate upwards, undergoing a series of biochemical changes as they progress through the tissue. The purpose of this progressive cellular specialization is to prime the keratinocytes for the final phase of differentiation, i.e. cornification, which produces the inert water barrier, the stratum corneum [2, 3].

The stratum corneum is unlike any other tissue in the skin, being comprised of anucleated, non-viable cells, called corneocytes. Structurally it has been likened to a brick wall, in which the corneocytes represent the bricks and the intercellular matrix represents the mortar [4]. The corneocytes are composed essentially of disulphide cross-linked keratin filaments surrounded by a cornified envelope formed from highly cross-linked isopeptide bonded proteins [5]. In healthy skin the mature corneocytes contain high concentrations of the Natural Moisturizing Factor (NMF) [6], low molecular weight, water-soluble compounds, which effectively bind water against the desiccating action of the environment. The intercellular matrix surrounding the corneocyte is composed primarily of highly structured lipid lamellae, which provide an effective barrier to water loss. Indeed, the stratum corneum is the only tissue of the body in which the cells exist in such a predominantly hydrophobic environment. To allow interaction of the hydrophilic protein surface of the corneocyte with the hydrophobic extracellular lipid lamellae, the outer surface of the cornified envelope is coated with covalently bound lipids [7, 8]. Ultimately, the overall integrity of the stratum corneum depends upon the cohesion provided by specialized intercellular protein structures called desmosomes; in the stratum corneum these structures are modified specifically and called corneodesmosomes [9, 10].

However, the initial process of cornification is not the end of epidermal terminal differentiation, only the start of stratum corneum maturation. The deepest layers of the stratum corneum represent an immature form of this tissue and a maturation process takes place leading to the eventual loss of the peripheral corneocytes. During maturation the intercellular lipids are enzymatically modified to decrease their polarity, and the NMFs are produced at an early stage by the degradation of their precursor protein filaggrin [11]. Finally, the cohesive forces holding the corneocytes together are progressively degraded to allow cell shedding at the surface of the skin, a process known as desquamation.

Under most circumstances the stratum corneum provides a highly efficient barrier against water loss. Water retention within the tissue is essential to maintain its flexibility and to provide the necessary hydration for the enzymes involved in various aspects of stratum corneum maturation to function. However, owing to its proximity to the environment, this barrier is always under a major desiccation stress and, moreover, continually prone to damage by external forces. With failure of the barrier and tissue water loss, the major perceivable symptom is that of dry flaky, scaly skin, or xerosis.

Traditionally, treatments for dry skin have consisted of moisturizing agents, although without any detailed understanding of the underlying problem. With the realization that a critical level of hydration was required to allow the stratum corneum to maintain its flexibility [12] came the dawn of moisturization research, and cosmetic scientists began to investigate ingredients that could improve the ability of the stratum corneum to retain water. Early theories on dry skin concentrated on the effect of stratum corneum hydration on tissue flexibility and it was believed that dry skin was due to the simple mechanical cracking of a dry and brittle stratum corneum. Hence, moisturizers were thought to simply hydrate the tissue and increase flexibility.

In recent years, our understanding of the biochemistry of the stratum corneum has advanced enormously. Although, by definition, the stratum corneum cannot be considered a viable tissue, it is nevertheless a dynamic structure in which many enzymatic reactions are carefully regulated to ensure full functionality. With this increased knowledge has come the realization that moisturizing agents act in more subtle ways than simply increasing the hydration of the stratum corneum structural elements. It has become apparent that skin scaling is the result of a perturbation of the stratum corneum maturation process, and especially that of desquamation. Many factors may contribute to this process, although in winter xerosis this is primarily a result of environmental stresses. In this review article, we will describe the stratum corneum biochemical processes that are involved in tissue water retention and cell shedding and consider how perturbation of these processes can precipitate the formation of dry skin. Additionally, we will discuss how moisturization can ameliorate the condition and consider how new understanding of stratum corneum biology and the xerotic condition can aid in the development of novel moisturization therapies.

Stratum corneum and desquamation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References

Stratum corneum cohesion

In normal skin, stratum corneum maturation is a carefully balanced process, in which the loss of surface corneocytes is precisely balanced by the underlying rate of proliferation. For desquamation to occur there must be a degradation of the cohesive forces binding the corneocytes to their neighbours in the stratum corneum and, moreover, this process must occur in a carefully controlled manner so as to maintain the integrity, and thus barrier func-tion, of the tissue. As a result, desquamation is a progressive process of corneocyte dyshesion.

The main cohesive force within the stratum corneum is the corneodesmosome (or corneosome) [9], a specialized desmosome. The cohesion of the classical desmosome structure is provided by two heterogeneous families of demosomal cadherins (desmogleins or dsgs and desmocollins or dscs), each of which occur as three distinct isoforms [13, 14]. The predominant dsg and dsc isotypes in the corneodesmosme appear to be dsg1 and dsc1, which are specifically modified for their specialized role within the lipid-rich intercellular spaces. Dsc1 and dsg1 span the corneocyte envelopes and bind homophilically, in the intercellular space, to their counterparts on adjacent cells. A potentially critical difference between epidermal desmosomes and corneodesmosomes is the association of the latter with the protein corneodesmosin [15, 16]. This protein, recently identified as S protein [17], is a late differentiation antigen, which colocalizes with the extracellular domains of the corneodesmosomes. The glycoprotein nature of this protein and its location have suggested that it is involved in cohesion, although this remains to be confirmed [18]. Alternatively, corneodesmosin may protect the extracellular portions of the cadherins from premature proteolysis [19]. Corneodesmosomes also differ from desmosomes in their extensive cross-linking of their transmembrane region into the cornified envelope during late differentiation; hence, the structure becomes locked into position. This modification has two major implications for both tissue structure and desquamation. Firstly, it increases the overall mechanical strength of the stratum corneum, and secondly it dictates that corneodesmosomal degradation must occur in situ[20, 21].

Other cell surface glycoproteins have also been implicated in stratum corneum cohesion and desquamation [22], most notably the lectin-like desquamin [23]. This cell adhesion molecule has recently been shown to have both trypsin-like serine protease [24] and ribonuclease [25] activity. The precise role of desquamin in desquamation, and its relative contribution to tissue cohesion, remains to be established.

The weakest of the binding elements holding together the lipid lamellae and the adjoining corneocytes are the van der Waal’s forces. At the corneocyte wall these lipid lamellae interact with the covalently bound lipid of the cornified envelope. Although these intercorneocyte van der Waal’s forces are weak, the extent of the lipid interactions provides a significant cohesive component [26[27]–28]. There is evidence, both chemical and structural, that these lipid lamellae are modified during the maturation process, leading to reduced cohesion in the peripheral layers [29[30]–31]. This is best illustrated by electron microscope studies, which demonstrate that the classical lipid lamellar organization is lost in the outer layers, which no longer contribute to tissue cohesion [21]. In addition, it has been shown that the peripheral layer lipids are more fluid than those in the deeper stratum corneum [32]. Although the precise nature of the processes underlying these changes remains to be established, alterations in the lipid lamellae organization may reflect a critical change in the ratio of fatty acids, sterols and ceramides. Ceramidases present in the stratum corneum could modify the lipid lamellae by the degradation of ceramide to fatty acid and sphingosine [33], and loss of ceramide 1 by this process may be significant, given the putative role of this ceramide as a lipid phase modulator [34]. Alternatively, surfactant-like sebaceous fatty acids may perturb the lipid ratio of the lamellae close to the skin surface and facilitate bilayer disruption [35]. What role, if any, the covalently bound lipids play in desquamation is not yet known.

The final and least appreciated aspect of stratum corneum cohesion is that of corneocyte interdigitation. Although the ‘plate-like’ corneocytes have planar contact surfaces, the peripheral edge regions are interdigitated, physically interlocking the cells [36]. Interestingly, these regions of interdigitation appear to be where corneodesmosomes are retained in the superficial layers of the stratum corneum (see below). Indeed, following lipid disorganization and essentially complete corneodesmolysis, the major cohesive element at the skin surface may be these ‘lip and groove’ interdigitations. Ultimately, only mild frictional forces are required to disrupt this last cohesive element.

Corneodesmosome degradation

A striking feature of stratum corneum maturation is the rapid degradation of many of the corneodesmosomes within the innermost layers, the so-called stratum compactum [10]. Indeed, this initial degradative phase was originally taken as evidence that corneodesmosomes did not play a significant role in overall stratum corneum cohesion [4]. However, a more considered evaluation of the electron microscopical data revealed a retention of corneo-desmosomes into the most superficial layers of the stratum corneum, providing residual cohesion for the superficial corneocytes in addition to that provided by the lipid lamellae and corneocyte interlocking. Biochemically, this is reflected in the degradation of dsg1, dsc1 and corneodesmosin [18, 37], with the reduction of intact proteins and an increase in their degradative fragments in more peripheral layers. Intact dsg1 and dsc1, which presumably are the effective binding components, are barely detectable in these peripheral layers.

The progressive and essentially complete degradation of these corneodesmosomal proteins during stratum corneum maturation points to the role of proteases in the desquamatory process. These hydrolases, along with specific lipases, are delivered to their site of action through the extrusion of lamellar bodies into the intercellular space [38].

The stratum corneum is an extremely rich repository of proteases ( Fig. 2), and the identification of specific desquamatory enzymes is complicated by the fact that much of the proteolytic activity extractable from this tissue is likely to represent redundant activity responsible for the intensely autolytic process of stratum corneum formation, rather than with specific aspects of its maturation. Nevertheless, although the definitive identification of the proteases involved in corneodesmosome hydrolysis remains a challenge, the studies of Egelrud and others [39[40]–41] have provided strong circumstantial evidence that the enzyme Stratum Corneum Chymotryptic Enzyme (SCCE) plays a critical role in desquamation. Protease inhibitor studies have revealed similar inhibition profiles for SCCE, corneodesmosome and dsg1 degradation, as well as corneocyte release in vitro[41, 42]. Moreover, immuno-localization studies demonstrate its occurrence within lamellar bodies in the stratum granulosum and in the intercellular spaces in the stratum corneum; localizations consistent with a role in desquamation [43]. Nevertheless, more recent studies have implied an additional, or possibly an alternative, physiological role for this enzyme in the processing of the pro-form of interleukin-1β within the stratum corneum [44].

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Figure 2. % casein and caseinolytic activity determined.

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Trypsin-like enzyme activity has also been implicated in desquamation; although the characterization of this activity, and whether it comprises intrinsic stratum corneum proteases, remains to be determined [45[46]–47]. Certainly, at least one trypsin-like enzyme is required to convert the inactive pro-SCCE form to that of active SCCE [44, 48]. Similarly, the identity of the enzyme which, by degrading corneodesmosin, may also control corneodesmosome proteolysis is unknown at this time.

In addition to the desquamatory proteases, glycosidases present within the stratum corneum may be required for corneodesmosome degradation and desquamation. Corneodesmosomal glycoproteins may require deglycosylation to deprotect the proteins, rendering them more susceptable to proteolysis [49]. However, as yet no precise desquamatory glycosidase has been identified.

Indeed, the factors which control the activity of the desquamatory enzymes, and which therefore ultimately control desmosomal degradation, remain poorly understood. Protease–protease inhibitor complexation may play a physiologically relevant role in controlling desquamation, as suggested by the recent observation in vitro that the elastase inhibitor antileukoprotease, naturally present in the stratum corneum, can inhibit SCCE activity and corneocyte release [50]. Moreover, the loss of corneodesmosomes from the corneocyte surface appears to occur in a spatially controlled fashion during stratum corneum maturation ( Fig. 3). In superficial samples from normal skin residual corneodesmosomal staining is punctate and confined to regions of intercornoecyte overlap. The precise mechanism by which corneodesmosomes within these peripheral interdigitating regions are rendered less susceptible to hydrolysis is not known at present. The corneodesmosomes themselves may be heterogeneous in their composition, such that those present in these specific regions are relatively refractory to endogenous protease activity. Alternatively, proteolytic activity in these regions may be lower, either directly, through reduced levels of the enzyme, or indirectly, by changes in the surrounding lipid microenvironment having an impact upon enzyme activity/mobility (see below).

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Figure 3. .  Indirect immunofluorescence of superficial human corneocyte stained with an antidesmocollin1 antibody.

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Water activity and corneodesmosomal degradation

One known major factor which controls desquamation is water, and it is now established that corneodesmosomal degradation is inhibited at low environmental humidities [51]. The stratum corneum desquamatory proteases, similar to the enzymes involved in filaggrin hydrolysis (see below), are critically influenced by water activity within the tissue, and dsg1, dsc1 and corneodesmosin degradation are all reduced at low environmental humidities [37, 51[52]–53]. These observations emphasize that the maintenance of the water content of the stratum corneum is essential, not only for the mechanical properties of the tissue, but is also vital for specific enzyme reactions, which define optimal stratum corneum function and facilitate normal, orderly desquamation. Ultimately, therefore, these enzymic processes will depend upon the ability of the tissue to retain water against the desiccating action of the environment; a quality dependent on the integrity of intercellular barrier lipids and the effective and timely generation of the NMF within the stratum corneum. These two elements of the stratum corneum are discussed in detail below.

Stratum corneum barrier lipids

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References

The highly organized lipid lamellae surrounding the corneocytes are the major structural element designed to retain water within the stratum corneum. Although other lipids are present in the stratum corneum (low levels of glucosylceramides, cholesterol sulphate and phospholipids), the major lipids are ceramides, cholesterol and fatty acids. All these lipids are synthesized by the differentiating keratinocytes of the epidermis and stored either in the secretory vesicles of the lamellar bodies or in the plasma membrane of the cell, awaiting mobilization to form the lipid lamellae during cornification. Recent studies have shown that the major controlling element in barrier homeostasis is the epidermal calcium ion gradient [54]. Collapse of the ion gradient as a result of barrier damage results in up-regulation of the rate-controlling enzymes of lipid synthesis, namely fatty acid synthetase (fatty acid), hydroxymethylglutaryl (HMG) CoA reductase (sterol) and serine palmitoyl transferase (ceramides). Studies using selective inhibitors to these enzymes (for review see [55]) suggest that all major species of stratum corneum lipids are synthesized during barrier repair and are required for full barrier homeostasis. Unlike cholesterol and fatty acid biosynthesis, which increases almost immediately after barrier disruption, synthesis of the ceramide precursors, glucosylceramides, is delayed by approximately 7 h [56]. Possibly HMG CoA reductase and fatty acid synthetase, under acute metabolic control mechanisms, respond rapidly to barrier damage; whereas SPT is subjected to transcriptional and translational control only and takes longer to respond. Certainly, the recently discovered transcription factor, sterol regulatory element binding protein (SREBP), and in particular SREBP 2, has been shown to control epidermal sterol and fatty acid synthesis but does not regulate the ceramide synthetic pathway [57].

Although the correct ratio of these three classes of stratum corneum lipids is required for maintaining the lipid lamellae structures, the most important single component is the ceramides. These complex lipids are composed of a long chain sphingoid base (C16–C22), which is most commonly sphingosine, but can be dihydrosphingosine, phytosphingosine or 6-hydroxysphingosine, amide linked to the carboxylate head group of long chain non-hydroxy or alpha-hydroxy fatty acids [58].

Six major classes of ‘free’ ceramides, which differ mainly in the hydroxylation of the head group region, have been identified in the epidermis ( Fig. 4). Furthermore, in ceramide 1 and 6 there are also ω-hydroxylated amide-linked fatty acids that may be further esterified with other fatty acids. Although the precise contribution of each of these ceramide subspecies to the totality of barrier function remains to be elucidated, there is evidence to indicate that ceramide 1 plays a pivotal role in barrier function. This is the predominant ceramide species containing unsaturated fatty acids in the stratum corneum. It is remarkably enriched in linoleic acid, which comprises a minimum of 20–30% of the ω-esterified fatty acid. Indeed, the absolute requirement for linoleic acid in epidermal barrier function probably reflects its incorporation into ceramide 1.

The consequence of ω-esterification is that an unusual, long chain fatty acid species is produced, providing unique physical properties. Because of this extra long fatty acid extension, ceramide 1 linoleate has been proposed to undertake a membrane-organizing function in the stratum corneum by spanning adjacent lipid bilayers and acting as a ‘molecular rivet’[59]. However, more recent studies have indicated that this molecule influences stratum corneum extensibility behaviour by increasing flexibility at low environmental relative humidity [60]. Investigation into the behaviour of model lipid mixtures containing ceramide 1-linoleate led Oldroyd et al. [34] to conclude that ceramide 1-linoleate, unlike the oleate derivative, helped to maintain a fluid lipid phase, and prevented the crystallization of other stratum corneum lipids at physiological temperatures. Thus, ceramide 1-linoleate may be a naturally occurring ‘lipid crystallization inhibitor’ and this property may be vital, not only for the mechanical properties of the tissue, but also in facilitating the desquamatory process. Recent X-ray studies have further emphasized the importance of ceramide 1 to lipid organization in the stratum corneum [61]. Using defined lipid species to examine lipid lamellae structural organization in vitro, Bouwstra et al. have shown that difraction patterns similar to those obtained in vivo rely on the addition of ceramide 1.

In addition to the free ceramides species described above which form the lipid lamellae, the stratum corneum also contains two long chain ceramides, which are covalently bound to the corneocyte cornified envelopes [62]. These two species, named ceramide A and B, make up some 80% of the total bound lipid. Ceramide A, the major component (53%), consists of C30–34 ω-hydroxy acids amide linked to sphingosine. The more polar ceramide B contains the same hydroxy acids linked to the novel sphingoid base 6-hydroxy-4-sphingenine [63]. In addition to permitting cornified envelope interaction with the lipid lamellae, these lipids may be important for maintaining the correct orientation of the intercellular lipid bilayers.

Stratum corneum natural moisturising factor (NMF)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References

The role of NMF in the stratum corneum

The stratum corneum contains a high concentration of water-soluble, low molecular weight compounds, which are principally amino acids or their derivatives such as pyrrolidone carboxylic acid (PCA) and urocanic acid. Other major contributors are lactic acid and urea. This pool of compounds, known collectively as the NMF, is found exclusively in the corneocytes, where it can represent as much as 10% of their dry weight. The role of the NMF lies in the fact that its constituent chemicals, in particular its PCA salts (which account for some 30% of all the amino acids and derivatives in the stratum corneum), are intensely hygroscopic. These salts will therefore absorb atmospheric water (even at relative humidities as low as 50%) to the extent that they dissolve in their own absorbed water, thereby acting as very efficient humectants. Biologically, this allows the outermost layers of the stratum corneum to retain liquid water against the desiccating action of the environment.

The historical view of the importance of this liquid water was that it plasticised the stratum corneum [12], keeping it resilient and preventing cracking and flaking due to mechanical stresses. Keratin, the main stratum corneum structural protein, acquires its elastic properties with the help of hydrated NMF, specifically the neutral and basic free amino acids [64]. The specific ionic interaction between keratin and NMF, accompanied by a decreased mobility of water, leads to a reduction of intermolecular forces between the keratin fibres and increased elasticity.

In addition to its structural effects, by maintaining free water in the tissue the NMF also plays a critical role in facilitating key biochemical events. Within the stratum corneum the co-ordinated activity of specific proteases is essential for healthy stratum corneum function. Indeed, as we will discuss later, proteases are responsible for the very generation of the NMF itself. These hydrolytic processes can only function in an aqueous or semiaqueous environment; an environment effectively maintained by the water-retaining capacity of the NMF. Thus, this complex water-soluble pool of compounds fulfils a dual role in the stratum corneum.

The origin of the skin’s NMF

Studies conducted Scott and Harding in the early 1980s [65[66]–67] indicated that essentially all of the free amino acid components and their derivatives within the stratum corneum were derived from a short-lived, high molecular weight histidine-rich precursor protein. This unusual protein was shown to be the major component of the F-type keratohyalin granules [68]. Coincidental with the transition of the mature granular cell into the corneocyte, the protein is rapidly dephosphorylated and then undergoes a specific proteolytic processing to form the low molecular weight mature species [69]. These highly basic, so-called histidine-rich proteins proved to be identical to the protein filaggrin that was responsible for the correct formation and macrostructure of the keratin microfibrils in the stratum corneum–the so-called keratin matrix proteins [70]. The mechanism underlying the generation and subsequent catabolism of filaggrin within the stratum corneum is worthy of discussion at this juncture for, more than any other epidermal process, it emphasizes the subtlety and complexity of stratum corneum biochemistry and its dependence upon water activity.

Filaggrin is formed from a high molecular weight precursor, profilaggrin. This precursor consists of 10–12 repeating units of filaggrin flanked by truncated filaggrin sequences and N-and C-terminal peptides that share little homology with filaggrin [71, 72]. The presence of two calcium binding regions in the N-terminus portion implicates Ca2+ as a possible regulator of keratohyalin formation and/or processing of profilaggrin to filaggrin [73]. This processing of profilaggrin and the concomitant dissolution of the keratohyalin granules is likely to represent a pivotal control point in the final stages of terminal differentiation [74, 75], and the enzymes responsible for the dephosphorylation [76] and initial processing of profilaggrin [77[78]–79] are currently under intensive investigation.

Intact filaggrin, with the exception of a minor incorporation into the cornified cell envelope [80, 81], does not persist beyond the deepest two or three layers of the stratum corneum [67, 82]. Almost immediately after the filaggrin/keratin complex is formed within the deepest layers of the stratum compactum, extensive disulphide bonds form between the keratin fibres, catalysed by the filaggrin [83]. It has been proposed that the close-packed disulphide-bonded, keratin fibre network is sufficiently stable to make the continued presence of intact filaggrin unnecessary [69]. Following keratin stabilization filaggrin is extensively modified [69, 84] by the enzyme peptidyl deiminase to allow dissociation of the keratin/filaggrin complex and permit subsequent filaggrin proteolysis. The activity of peptidyl deiminase may therefore represent a pivotal phase of NMF generation, as it has been reported that the nascent filaggrin/keratin complex is proteolytically resistant [83], a paradoxical property in view of the desired fate of filaggrin. Recent evidence suggests that keratin may also by deiminated to facilitate dissociation of the filaggrin/keratin complex [85].

As emphasized in Fig. 5, the subsequent proteolysis of filaggrin is abrupt and complete. At the ultrastructural level the onset of filaggrin hydrolysis delineates the stratum compactum/dysjunctum boundary. Although filaggrin proteolysis is clearly a critical event for normal stratum corneum maturation and has been the subject of considerable research interest, the identity of the enzymes specifically responsible for ‘filaggrinolysis’ has proved elusive. Filaggrin, a protein specifically designed to be rapidly and completely degraded, is, perhaps not surprisingly, readily degraded by many proteases in vitro. Therefore, the in vivo significance of the ability of many proteases extracted from the stratum corneum, including cathepsin B [86] and L-like enzymes [87], to effectively degrade filaggrin in vitro, remains to be firmly established.

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Figure 5. nm diameter).

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Following filaggrin hydrolysis, some of its constituent amino acids are further metabolized, as mentioned above, to form specialized components of the NMF. Notably, glutamine is converted through non-enzymic cyclization into PCA [65], a potent humectant [88], while histidine is converted to urocanic acid, a natural UV absorber [89], by the enzyme histidase [90].

The control of filaggrin proteolysis in the stratum corneum

The conversion of filaggrin to NMF produces a large osmotic pressure within the corneocyte. Hence, the skin must delay activation of filaggrinolysis until the corneocyte (in particular its cornified cell envelope) has matured and strengthened and the cell has migrated up into the more desiccated regions of the stratum corneum. Ultimately, it is the control of this final enzymatic process which ensures that NMF is produced when and where it is needed to maintain the flexibility of the tissue and the activity of its desquamatory enzymes, without causing tissue damage [91]. The conversion of filaggrin to NMF only occurs within a narrow range of water activity (WA) between 0.7 and 0.95 [91]. Above a WA of 0.95 filaggrin is stable, whereas below 0.7 the whole tissue is essentially too dry to allow hydrolytic enzymes to operate and once again filaggrin cannot be degraded. This is illustrated by chronic skin occlusion [92], where filaggrin hydrolysis (despite hydration of the tissue) is blocked; the corneocytes remain filled with the intact protein and the stratum corneum NMF levels fall close to zero. However, the undegraded filaggrin recovered from occluded skin is nevertheless extensively deiminated, suggesting therefore that activation of peptidyl deiminase per se is not the critical step in NMF generation, which is controlled by changes in the WA gradient [93].

Conceptually, therefore, as filaggrin-containing cells move away from the deepest, most hydrated layers and begin to dry out, the WA within the cell decreases and at a specific point the ‘filaggrinases’ are activated and NMF is produced. The point at which this hydrolysis is initiated is independent of the age of the corneocyte [82] and is dictated ultimately by the water activity gradient present across the tissue. When the weather is humid the proteolysis occurs almost at the outer surface; in conditions of extreme low humidity the proteolysis is initiated deep within the tissue so that all but the deepest layers contain the NMF required to prevent desiccation. The stratum corneum has thus developed an elegant self-adjusting moisturization mechanism to respond to the different climatic conditions to which it is exposed. However, given the subtlety of the control mechanism, it is likely that when climatic conditions change suddenly and the environmental humidity and temperature fall rapidly, the associated changes in water activity within exposed stratum corneum may temporarily prevent effective filaggrin hydrolysis and predispose the skin to dryness.

Dry skin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References

The overall view that we have of ‘normal’ desquamation is that of a progressive and essentially compete destruction of corneodesmosomes intimately dependent upon the composition and organization of the intercellular lipids and effective tissue hydration. However, what are the underlying biochemical defects which result in the formation of the flaky scaling which we perceive as dry skin?

Aberrant corneodesmolysis

The most obvious consequence of this aberrant desquamation is the retention of corneodesmosomes in the superficial layers of the stratum corneum, as demonstrated by electron microscopy studies of winter-and soap-induced xerosis [21]. Biochemically, this increased retention is reflected in the increased levels of intact dsg1, dsc1 and corneodesmosin [51[52]–53, 94] in the superficial layers, indicating that hydrolysis of these molecules is inhibited. Indeed, similar findings of high levels of intact desmosomal cadherin are seen with the pathological scaling conditions such as psoriasis and ichthyosis [95] or following UV damage. The major consequence of this decreased hydrolysis and corneodesmosomal retention is that the predominant intercorneocyte linkages are not broken and the peripheral cells do not detach during desquamation. Hence, instead of the imperceptible loss of surface corneocytes, large clumps of cells accumulate. Cell loss eventually occurs by friction-induced fracture of visible corneoctye clusters or scales.

Yet what is the principal biochemical cause of these structural effects? A potential causative factor in reduced corneodesmosomal degradation is a reduction in SCCE and other proteolytic activity. Although several studies have been performed on SCCE and desquamation, precisely what changes in enzyme activity occur in perturbed desquamation is still poorly understood. Certainly, stratum corneum chymotryptic and tryptic activity levels are reduced in ichthyosis vulgaris [95] although whether this also specifically includes SCCE is unknown. This reduced activity may reflect intrinsic changes such as a reduced synthetic capacity for the enzyme or its aberrant delivery into the intercellular space [38, 96]. Alternatively, extrinsic factors may lead to reduced enzyme activity. In soap-induced xerosis we have shown a reduction in extractable SCCE activity from the peripheral layers of the stratum corneum. ( Fig. 6). This loss was attributed to the leaching out of SCCE by the action of the soap, because in deeper layers SCCE activity was unchanged. However, all studies reported to date invariably measure changes in putative desquamatory protease activities by measuring enzyme activity in aqueous solution using artificial substrates, e.g. casein zymography or fluorescent amidomethylcoumarin derivatives. Such approaches, at best, can only give a limited insight into potential alterations in enzyme activity likely to be occurring in situ within the hydrophobic intercellular space of deranged stratum corneum.

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Figure 6. .  SCCE activity levels in normal and soap-induced dry skin.

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Barrier lipids and dry skin condition

In addition to the proteolytic enzymes within the intercellular spaces, the stratum corneum contains a wide range of lipases, which are responsible for modification of the lipid matrix during the maturation process. Such enzymes include phospholipases, ceramidases, sphingomyelinases and glucosylcerebrosidases [97[98][99]–100]. However, although these lipases have the potential to modify the lipid lamellae (and thereby potentially influence desquamation) by their action on barrier lipids, their precise involvement in the defective cell shedding associated with general poor skin condition has not been reported.

The precise influence of the intercellular milieu on enzyme activity is poorly understood, but it is likely to be considerable: both the desquamatory proteases and the lipases mediate their action in the lipid-rich intercellular space and yet both have an absolute requirement for free water. As the stratum corneum lipid composition and phase behaviour will influence, and may indeed control, enzyme activity, lipid phase separation, induced by changes in lipid composition, will have profound effects on the hydrolases. Indeed, altered lipid levels and associated abnormalities in lipid lamellae structure are characteristic of several hereditary disorders of desquamation (reviewed in [101]).

The most closely studied of these scaling disorders is Recessive X-linked Ichthyosis (RXLI), in which there is a specific abnormality in sterol metabolism due to deficiency of cholesterol sulphatase [101]. The subsequent accumulation of cholesterol sulphate may influence protease activity indirectly by influencing lipid phase behaviour or directly, by inhibiting the desquamatory proteases as originally proposed by Williams [101] and demonstrated recently by Denda et al. [102]. SCCE has also been shown specifically to be sensitive to inhibition by this lipid [41]. Given that cholesterol sulphate is normally hydrolysed during stratum corneum maturation, its progressive hydrolysis and subsequent enhanced activity of SCCE may represent a subtle control mechanism regulating desmosomal hydrolysis and the desquamation process.

Abnormalities in lipid lamellae structure are also apparent in the outer layers of the stratum corneum in winter xerosis [21], where the normal ordered lipid bilayer structures ( Fig. 7) are replaced by a chaotic arrangement ( Fig. 8). This disorganized lipid structure is likely to influence both intercorneocyte cohesion and proteolytic activity and adversely effect the latter stages of desquamation. One reason for this loss of lipid structure may be an alteration in the lipid ratios. In skin suffering from soap-induced winter xerosis, the total levels of stratum corneum ceramides are decreased ( Fig. 9) [21, 103] and the levels of fatty acids are increased [20, 104]. In other studies examining ceramide levels in surfactant-induced xerosis, there was no change in total ceramide levels, although there was a change in the ratio of the ceramide subtypes [105]. The reported differences between these studies probably reflect differences in the protocols used to elicit the dryness and the precise methodologies used to sample the stratum corneum. In the more recent studies only the superficial layers of the stratum corneum have been sampled and these show a reduction in ceramide levels.

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Figure 7. .  Organization of stratum corneum lipids in tape-strippings of individuals with clinically normal skin. Transmission electron micrographs of tape-strippings. Ultrastructural changes in lipid organization towards the surface of the stratum corneum. (A) First strip; absence of bilayers and presence of amorphous lipidic material. (B) Second strip; disruption of lipid lamellae. (C) Third strip; normal lipid lamellae. (×200 000). Modified from J. Soc. Cosmet. Chem.45, 203–220 (1994).

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Figure 8. .  Organization of stratum corneum lipids in tape-stripping of subjects with winter xerosis. Transmission electron micrographs of tape-strippings of individuals with severe xerosis. Perturbation in lipid organization towards the surface of the stratum corneum. (A) First strip; disorganized lipid lamellae. (B) Second strip; disorganized lipid lamellae. (C) Third strip; normal lipid lamellae (×200 000). Modified from J. Soc. Cosmet. Chem.45, 203–220 (1994).

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Figure 9. .  Changes in ceramide content of stratum corneum with increasing skin dryness.

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Many factors influence the levels as well as types of stratum corneum lipids, and it is possible that their reduction leads directly to poor skin condition. Levels of lipids differ on different body sites, which may make those sites more or less prone to environmental damage [106]. Indeed, intrinsically reduced lipid levels or even surfactant-induced lipid extraction will leave the stratum corneum lamellae more prone to perturbation, resulting in abnormalities in function and culminating in visibly dry and flaky skin [107, 108]. Susceptibility to dry skin also shows seasonal variation and a tendency to increase with age. Many researchers have studied changes in stratum corneum lipids, in particular ceramides, associated with these events (reviewed in [109]). In general, lipid levels decrease in old age and show a decline in the winter months. Race- and gender-determined differences in intercellular lipid levels have been reported, but our understanding of the influence of these and other factors on the desquamatory process and the frequency and severity of dry skin episodes remains incomplete.

NMF levels and dry skin condition

Many studies have been conducted to examine, in detail, the consequences of altered NMF levels on skin condition. In atopic dermatitis there is evidence of decreased levels of NMF [109], whereas in more severe skin disorders, such as psoriasis [110] and ichthyosis [111], NMF is essentially absent. These disorders manifest themselves in abnormal desquamatory properties, leading to skin scale with severe cracking and fissuring of the tissue. Although clearly in all these disorders several aspects of keratinization are impaired, the inability to produce, or retain, NMF within the stratum corneum appears to be a significant factor contributing to the overall manifestation of the skin problem.

Reduced NMF levels are also implicated in the more common dry skin conditions. Denda et al. [112] reported that free amino acid levels decrease significantly in dry, scaly skin induced by repetitive tape-stripping, and also a significant correlation exists between hydration state of the stratum corneum and its amino acid content in elderly individuals with skin xerosis [113]. Furthermore, NMF levels on the leg are generally lower than on the forearm and this may relate to the increased incidence of dryness on the former [114].

We have studied factors influencing NMF levels by quantifying PCA levels present on sequential tape-stripping of stratum corneum. Figure 10 shows typical stratum corneum depth vs. PCA concentration profiles obtained from individuals aged between 20 and 70 years. The level of NMF declines markedly towards the surface of the skin, typical of normal skin exposed to routine soap washing, with subsequent NMF leaching from superficial stratum corneum [115]. By contrast, in healthy skin that has not been exposed to surfactant damage the NMF concentration is independent of depth, until the filaggrin-containing levels are reached. Secondly, the data illustrate that there is a significant age-related decline in NMF, which is particularly noticeable within the deeper layers of the stratum corneum. Hence, aged skin intrinsically has lower NMF levels than young skin, reflecting a general reduced synthetic capability and not solely a greater leaching of NMF during cleansing. These observations are confirmed by electron microscopical studies, which indicate decreased keratohyalin granules, and hence filaggrin, in senile xerosis [116]. Moreover, it is likely that in aged skin, loss of NMF may become more pronounced owing to a parallel age-related decline in water barrier repair. The decline in NMF production probably reflects the cumulative effects of actinic damage, being prominent in stratum corneum recovered from the back of the hand (photo-damaged), but not from the inner aspect of the biceps (photo-protected). In contrast, Tezuka has reported that although filaggrin (and hence NMF) levels show an age-related decline in leg stratum corneum, filaggrin levels on the face (a site particularly prone to photodamage) appear refractory to increasing age [114]. Whether these observations reflect ethnic differences or sensitivity of methodological approaches remains to be established.

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Figure 10. .  Profile of mean pyrrolidone carboxylic acid concentrations (PCA) vs. depth from the hands of young and old age groups.

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Suggestions that reduced functionality of the filaggrinases themselves, due, for example, to surfactant damage, may contribute to reduced NMF production must remain speculative until these enzymes can be specifically identified and measured in xerotic skin.

Clearly, many factors, both intrinsic and extrinsic, may reduce the generation of NMF within the stratum corneum and contribute to dry skin formation.

Moisturization technologies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References

Occlusive agents and humectants have been used widely in skin care products for the treatment of dry skin over many years. In fact the earliest use of oils for smoothing skin is reported to be as early as 2300 bc, but it was not until the 1950s that research focused on water-imbibing substances to help maintain stratum corneum hydration. However, until recently, due to a lack of understanding of both the mechanism of desquamation and the pathophysiology of dry skin, improvements in skin care treatments had remained empirical. Now, with the recent improvement in understanding of stratum corneum biology it is possible to begin to explain the mode of action of existing moisturizing technologies and to design novel approaches for improving the hydration and functionality of the cornified layer.

Petroleum jelly

The modern skin-care industry was born with the marketing of petroleum jelly, a by-product of the petroleum distillation, by Cheseborough under the Vaseline brand name. Petroleum jelly primarily acts as an occlusive agent, having been shown to reduce water loss by over 98%, whereas other oils only manage a 20–30% reduction. The efficacy of this product in trapping water within the stratum corneum means that it is the most effective moisturiser. Yet petroleum jelly does not act simply by forming an occlusive film over the surface of the skin. Recently, it has been shown to diffuse into the intercellular lipid domains, which may account for its efficacy. In addition, petroleum jelly has been shown to penetrate even down into the viable epidermis and may have physiological actions. Interestingly, in this respect petroleum jelly was shown to accelerate lipid biosynthesis, aiding in barrier repair [117].

Glycerol and other humectants

In the search for even more consumer acceptable moisturization formulations, several humectant agents have been tried. In general, the most effective humectants are polyols such as sorbitol, mannitol and glycerol. Moisturization is due to the high degree of hydroxyl groups on these molecules which bind and retain water. By far the most effective polyol for the treatment of low humidity-induced dry skin is the tri-hydroxylated molecule glycerol. Glycerol-containing emulsions produce a long-lasting decrease in trans epidermal water loss (TEWL) and generate a smoother skin surface. The particular effectiveness of glycerol in the treatment of dry skin is due to several mechanisms. As mentioned above, its polyol structure provides humectancy, binding and holding water. Glycerol is also an occlusive agent, preventing water loss, although this is a relatively weak property compared to petroleum jelly. More recent studies have highlighted additional functionalities of glycerol. Using optical microscopy, Froebe et al. [118] showed that glycerol prevented low humidity-induced crystal-phase transitions in model lipid mixtures; it was proposed that maintenance of a liquid crystalline state was important to reduce water loss. Finally, and most notably, glycerol is a corneodesmolytic agent ( Fig. 11), facilitating the perturbed desquamation associated with dry skin through enhanced activity of the desquamatory enzymes [51]. Although the precise mechanism is not known, increased corneodesmosomal hydrolysis probably occurs through a combination of humectancy, occlusivity and lipid phase modulation.

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Figure 11. .  Transmission electron micrographs showing the distribution of desmosomes in stratum corneum. (A) Control tissue, no glycerol treatment, incubated at 44% relative humidity (RH) for 7 days. (B) Tissue incubated at 80% RH for 7 days (no glycerol treatment). (C) Tissue incubated at 80% RH for 7 days following 5% glycerol treatment. Modified from Arch. Derm. Res.287, 457–466 (1995).

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Despite its multiple beneficial effects for dry skin, simply delivering glycerol to the skin is, in itself, not enough. The composition of the formulation has been shown to be critical for delivering the maximal glycerol benefit. In moisturization efficacy tests examining the effects of glycerol on dry skin relief, low levels of glycerol (1%) in a simple stearate emulsion had a minimal effect on the alleviation of dry skin [119], ostensibly through the absence of an effective occlusive agent in the formulation. When a clinically ineffective mixture of lipids (lecithin: cholesterol: stearic acid 1:2:1 w/w/w) was combined with glycerol a synergistic alleviation of dry skin was apparent.

Surfactants

An alternative approach to modifying the barrier lipids, other than topical application of the lipids themselves, is to add an agent which modifies the phase behaviour of the lamellae. Glycerol has already been described as such an agent (see above). In addition, the emulsifiers included in the cosmetic base product can have a similar effect. In many cases these emulsifiers are surfactants, which will increase the fluidity of the lipid lamellae and subsequently facilitate the desquamatory process. The zwitterionic surfactant (octadecyldimethyl-ammoniohexanoate) amine oxide (DMDAO), alone or in synergy with SDS (8:2), has been shown to lead to enzyme-mediated dissolution of the stratum corneum [120[121]–122]. The addition of a protease inhibitor to this mixture inhibited the surfactant effect. Theoretically, one can envisage at least two potential mechanisms to explain such observations; the enzymes may have increased mobility to reach their substrates and/or the water associated with the more fluid lipids is increased.

Urea

Urea, a natural component of the stratum corneum is commonly used in skin-care and dermatological formulations. This unique physiological substance has been demonstrated to be a potent skin humidifier and descaling agent, and as long ago as 1943 Rattner used urea in hand creams as a humidity-promoting additive [123]. Ten percent urea has been shown to be effective for the management of dry skin, being more efficacious than salicylic acid and petroleum jelly [124]. Urea-containing moisturisers influence the barrier properties of the skin by reducing TEWL [125, 126], increasing skin capacitance and reducing irritant reactions [127]. Recent data suggest that lipid biosynthesis may also be increased by topical application of high concentrations of urea [128]. In combination with lactic acid, urea has been shown to be an effective treatment of ichthyosis [129] and in combination with polidocanol urea is an effective treatment of juvenile atopic dermatitis [130].

α-Hydroxyacids

α-Hydroxyacids (AHAs) constitute a class of compounds which exert specific and unique benefits on skin structure and function. These compounds were originally used to alleviate the symptoms of ichthyosis, with lactic acid (3%) being proposed as a treatment in 1946 [131], and similar benefits were subsequently obtained with the β-hydroxyacid, salicylic acid [132]. For the more mundane problem of dry skin, Middelton [133] and Van Scott [134] demonstrated the efficacy of short-chain α-hydroxyacids in the early 1970s. In classical moisturization efficacy tests, racemic mixtures of lactic acid were shown to ameliorate the symptoms of dry skin [135], and in those same studies lactic acid-containing products were also more effective in preventing the reappearance of dry skin compared with lactic acid-free products [135, 136]. α-Hydroxyacids were originally described as keratolytics, although this is a misnomer, as they do not degrade keratin; rather their mode of action in alleviating dry-skin stems largely from their ability to promote desquamation. Ultrastructural studies on glycolic acid-treated stratum corneum suggest a targeted action towards corneodesmosomes within the stratum dysjunctum, the outer region of the cornified layer [137]. This mode of action would suggest that a more correct name for these compounds would be corneodesmolytics; however, the precise mechanism by which α-hydroxyacids have an impact upon desquamation is not fully understood. One possiblity is that the molecules act like NMFs and aid in tissue moisturization. Alternatively, for the long-chain AHAs the functionality may reside in the intrinsic surfactant properties of the molecule.

α-Hydroxyacids also provide skin condition benefit by enhancing barrier function. In clinical studies the l-chiral isomer improved stratum corneum barrier function, as measured by both reduction in TEWL values, following a challenge with SLS, and by improved resistance to the appearance of dry skin in moisturization efficacy studies [138]. These improvements were related to the overall increase in stratum corneum ceramide levels and especially ceramide 1-linoleate levels.

Although α-hydroxyacids are beneficial for the treatment of dry skin, these compounds are primarily used as anti-ageing actives. Here, they work by stimulating cell renewal, an effect which is related to the pH of the preparation [139, 140].

Thus, the actions of α-hydroxyacids on skin are multiple. Their effects are mediated by improving moisturization, facilitating desquamation, reducing corneocyte cohesion, increas-ing stratum corneum ceramide levels and strengthening the barrier. They also increase keratinocyte proliferation, which will lead to smaller corneocytes and a smoother skin surface. Clearly, therefore, although these molecules ultimately mediate their actions in the stratum corneum by facilitating corneodesmolysis, their skin benefits are more far-reaching.

Pyrrolidone carboxylic acid (PCA)

As the principal component of the NMF, considerable interest has been paid to the ability of PCA and its derivatives (including simple [141] and novel [115] esters and sugar complexes [142]) to moisturize the stratum corneum following topical application. Creams and lotions containing the sodium salt of PCA are widely reported to help hydrate the skin and improve dry flaky skin conditions [143, 144].

Barrier lipids

The critical importance of the intercellular lipid lamellae to stratum corneum barrier function has been utilized in developing novel approaches to moisturization. Imokawa and coworkers [107, 108] topically applied human stratum corneum ceramides to solvent-and surfactant-induced scaly skin, producing reductions in scaling and increased skin moisturization. This amelioration of skin condition was superior to placebo and lotions containing sebaceous lipids. Elias and coworkers have also researched extensively the use of exogenously supplied lipid species to repair water barrier function [145, 146]. Equimolar mixtures of ceramides, cholesterol and fatty acids allow the barrier to repair at normal rates, whereas an optimized mixture (cholesterol, ceramide, palmitate and linoleate: 4.3 : 2.3: 1: 1.08) was seen to accelerate barrier repair following acetone-induced barrier disruption. Moreover, this optimized mixture accelerated barrier repair following a range of barrier insults (tape-stripping, treatment with N-laurosarcosine or dodecylbenzene sulphonic acid). By contrast, the mixture was not effective following barrier damage with SLS or ammonium laurylsulphosuccinate [147]. These studies suggest that customised mixtures of the critical lipid species are required to repair barrier damage in different conditions. Further studies are required to relate the significance of these observations to human skin.

Recent studies have shown an inverse correlation between stratum corneum ceramide species and the erythema and TEWL following barrier insult by SLS [148]. Similarily, exogenously supplied ceramides 1 and 2 have been shown to reduce the detrimental effects of SLS on disturbing skin barrier function [149] Hence, low levels of particular ceramides may determine the likelihood of SLS irritant contact dermatitis [149] and improved barrier function may be obtained by the application of specific ceramide species. Similar approaches may also improve the quality of atopic skin, where low ceramide to cholesterol levels and, in particular, decreased ceramide 3 levels have been reported [150].

Enhanced lipid synthesis through delivery of lipid precursors

An alternative to improving barrier function by topical application of the various mature lipid species is to enhance the natural lipid-synthetic capability of the epidermis through the topical delivery of lipid precursors. The earliest work in this field relates to the alleviation of essential fatty acid deficiency (EFAD) in animal models. The absence of essential fatty acids, such as linoleic and linolenic acid, from the diet leads to the formation of a stratum corneum characterized by defective barrier properties and aberrant desquamation. Topical application of linoleic and linolenic acid reverses the condition and restores normal barrier functionality [151]. As already described, a significant proportion of the linoleic acid in the skin is destined for ω-hydroxylation and ceramide 1 linoleate synthesis, and it is the deficiency of this molecule in the EFAD stratum corneum (where it is replaced by ceramide 1 oleate) which appears to be directly responsible for the aberrant barrier. However, enhanced availability of linoleic acid to the skin through topical supplemenation is also likely to improve skin quality in more cosmetically relevant conditions. In healthy skin the ceramide 1 linoleate: ceramide 1-oleate ratio is typically 2:1. In winter, the ratio shows a characteristic decline and may predispose such skin to xerosis (either directly through reduced barrier functionality or indirectly through reduced desquamatory enzyme activity). Indeed, topical application of formulations containing linoleic acid in the form of natural oils [152] leads to enhanced synthesis of ceramide 1 linoleate and a subsequent normalization of the linoleate/oleate ratio. Direct topical ap-plication of linoleic acid has also been shown to alleviate the symptoms of dry skin [153].

In essence, because the skin relies on the systemic supply of essential fatty acids, under certain circumstances such as low temperature and humidity, and with increasing age, this may be barely adequate for optimal barrier function. Hence, the skin could be regarded as undergoing frequent episodes when it can be considered essential fatty acid-deficient. Therefore, amelioration of this deficiency by topical application of linoleic acid will have considerable benefits for skin condition, by increasing ceramide 1-linoleate levels.

Approaches to improving barrier function are not restricted to supplying complex lipid components to the stratum corneum. Improvements can also be achieved by simply providing the precursor molecules required for the synthesis of a particular lipid species. As already described, topically applied lactic acid, especially the l-isomer, can function as a general precursor to ceramides [138]. Other precursors such as serine, the primary substrate for serine palmitoyl transferase (SPT), the rate-limiting enzyme in the ceramide biosynthesis pathway, are utilized by keratinocytes in the presence of thiols (lipoic acid and N-acetylcysteine) to stimulate ceramide biosynthesis [154]. These thiols presumably activate SPT by thiol disulphide exchange mechanisms, and might be expected to enhance barrier performance in damaged skin.

The flaw in the approach of supplying precursor molecules to boost the synthesis of barrier components is that it is reliant on the synthetic capacity of each individual pathway and on the relevant control mechanisms. An alternative approach is to provide substrates which can feed into the biosynthetic pathways beyond the rate-limiting step. A good example is tetra-acetylphytosphingosine (TAPS), a modified sphingoid base, which has been shown to stimulate ceramide biosynthesis in cultured keratinocytes [155]. In subsequent clinical evaluation, topically applied TAPS increased stratum corneum ceramide levels in the majority of subjects treated [156]. Moreover, in these individuals a corresponding increase in resistance to surfactant damage was also observed. In further studies, a synergistic improvement in stratum corneum ceramide levels, and a corresponding increase in barrier function, was achieved when TAPS was combined with ω-hydroxy acids and linoleic acid. This triple lipid combination preferentially increased ceramide 1 levels [156].

Epidermal differentiation modulators

The last decade has not only increased our understanding of ceramides as barrier lipids, but has also shown that these complex lipid structures function as important cellular regulatory molecules. In the cell it has been shown that ceramides are produced in response to a range of signalling mechanisms by the action of sphingomyelinase enzymes [157]. These enzymes remove the phosphorylcholine from sphingomyelin to release the bioactive ceramide, which is then capable of activating a range of ceramide-inducible kinases and phosphatases to modify cellular function. Inactivation of the ceramide is either by ceramidase-induced degradation or by conversion back to sphingomyelin, to complete the sphingomyelin cycle. In the epidermis specifically, ceramides and other sphingolipids are known to be involved in cellular decisions on differentiation and apoptosis [158[159]–160]. Furthermore, recent studies have shown that barrier disruption increases both neutral and acidic sphingomyelinase activity [161]. It is hypothesized that the neutral enzyme may function in a signalling capacity, whereas the acidic enzyme acts to supply barrier ceramides. Hence, exogenously added ceramides may play an important pro-differentiation role, aiding barrier recovery. For example, exogenously supplied short-chain ceramides induce keratinocyte differentiation in vitro[162] and short-chain ceramides and pseudoceramides also potentiate the pro-differentiation effects of vitamin D [162]. Indeed, vitamin D is known to activate keratinocyte sphingomyelinase, leading to increased intracellular levels of ceramide [163]. Although the precise structure/function requirements for ceramide-induced keratinocyte differentiation remain to be elucidated, hydroxy acid-containing ceramides have been shown to be superior to non-hydroxy acid-containing ceramides in inducing keratinocyte differentiation, and there is a requirement for unsaturation of the sphingoid base [164].

But what of the intrinsic stratum corneum ceramides? The glycosylated ceramides have been reported to have both proliferative [165] and differentiation enhancing [166] properties, and long-chain barrier ceramides have pro-differentiation activity in cultured keratinocytes. Indeed, ceramide 1 ω-esterified fatty acid variants have been shown to have differing potencies in enhancing keratinocyte differentiation, with ceramide 1-linoleate being the most potent form [164]. Such observations raise the question as to whether topical application of long-chain ceramides can, in addition to a direct, physical effect on barrier function, also have an impact on the underlying proliferation/differentiation responses of the epidermis. Indeed, it raises the issue of whether the intrinsic ceramide molecules have such functionality. However, due to their extremely hydrophobic nature it is considered unlikely that topically applied long-chain ceramides can penetrate through intact stratum corneum to influence the underlying epidermis.

Desquamation enhancers

As an adjuvant to moisturization (as, in itself, it the treatment does not influence the water content of the stratum corneum) is the approach of ameliorating dry skin by enzymatic scale removal. The use of topically applied proteolytic enzymes has been shown to be beneficial in removing surface dry skin and ameliorating the symptoms of xerosis, if not the underlying cause [167]. These enzymes have also been chemically modified by linkage to glycans to increase stability and reduce potential irritancy and allergy responses. Currently, several cosmetic products containing such enzymes are commercially available, although these preparations have not been used in a mass-market product. Ultimately, the topical application of a formulation containing the skin’s own specific desquamatory proteases (e.g. human recombinant SCCE), rather than bacterial proteases, may prove more efficaceous.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References

Research into the structure and function of the stratum corneum has increased substantially over the past two decades. With it has come an increased understanding of the role of desquamation in the normal functioning of the skin. Perturbation of the desquamatory process leads to dry, flaky skin, whatever the cause. The dry skin condition has been shown to be largely due to the inhibition of the corneodesmosomal degradative process, with the resultant retention of these binding complexes in the superficial layers of the stratum corneum. The underlying cause appears to be the loss of water from the tissue owing to perturbations of one or both of the systems which are primarily responsible for maintaining stratum corneum water content; namely, the NMF and barrier lipids. As a result of this water loss, the most efficacious treatment for dry skin is moisturization. Traditionally, moisturization agents have either been occlusive agents or humectants. With new understanding of stratum corneum biology comes novel methodologies for improving the treatment of dry skin. Moisturizing agents, which supplement the barrier lipids, either by direct application or by increasing intrinsic lipogenesis, are becoming available. In addition, the realization that specific moisturizing agents, such as glycerol, can enhance corneodesmosomal degradation provides new avenues for novel moisturization products.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stratum corneum and desquamation
  5. Stratum corneum barrier lipids
  6. Stratum corneum natural moisturising factor (NMF)
  7. Dry skin
  8. Moisturization technologies
  9. Summary
  10. References
  • 1
    Odland, G.F. Structure of the skin. In Biochemistry and physiology of the skin Vol. 1 (L. A. Goldsmith, ed), pp. 3 63. Oxford University Press, Oxford (1991).
  • 2
    Winsor, T. & Burch, G.E. Differential roles of layers of human epigastric skin on diffusion of water. Arch. Intern. Med. 74, 428 444 (1944).
  • 3
    Matolsky, A.G, Downes, A.M., Sweeney, T.M. A study of the cornified epithelium of human skin. J. Invest Dermatol. 50, 19 26 (1968).
  • 4
    Elias, P.M. Epidermal lipids, barrier function and desquamation. J. Invest. Dermatol. 80(Suppl.), 44 49 (1983).
  • 5
    Rice, R.H. & Green, H. Cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked proteins. Cell 11, 417 422 (1977).
  • 6
    Tabachnick, J. & Labadie, J.H. Studies on the biochemistry of epidermis, IV – The free amino acids, ammonia, urea and pyrrolidone carboxylic acid content of conventional and germ free albino guinea pig epidermis. J. Invest. Dermatol. 54, 24 31 (1970).
  • 7
    Swartzendruber, D.C, Wertz, P.W, Kitko, D.J, Madison, K.C., Downing, D.T. Evidence that the corneocyte has a chemically-bound lipid envelope. J. Invest. Dermatol. 88, 181 193 (1987).
  • 8
    Wertz, P.W., Madison, K.C., Downing, D.T. Covalently bound lipids of human stratum corneum. J. Invest. Dermatol. 92, 109 111 (1989).
  • 9
    Chapman, S. & Walsh, A. Desmosomes, corneosomes and desquamation. An ultrastructural study of adult pig epidermis. Arch. Dermatol. Res. 282, 304 310 (1990).
  • 10
    Skerrow, C.J., Clelland, D.G., Skerow, D. Changes to desmosomal antigens and lectin-binding sires during differentiation in normal epidermis: a quantitative ultrastructural study. J. Cell Sci. 92, 667 677 (1989).
  • 11
    Scott, I.R., Harding, C.R., Barrett, J.G. Histidine-rich proteins of the keratohyalin granules: source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum. Biochim. Biophys. Acta. 719, 110 117 (1982).
  • 12
    Blank, I.H. Factors which influence the water content of the stratum corneum. J. Invest. Dermatol. 18, 433 440 (1952).
  • 13
    King, I.A., O’Brien, T.J, Buxton, R.S. Expression of the skin type desmosomal cadherin Dsc1 is closely linked to the keratinisation of epithelial tissues during mouse development. J. Invest. Dermatol. 107, 531 538 (1996).
  • 14
    King, I.A., Angst, B.D., Hunt, D.M., Kruger, M., Arnemann, J., Buxton, R.S. Hierarchical expression of desmosomal cadherins during stratified epithelial morphogenesis in the mouse. Differentiation 62, 83 96 (1997).
  • 15
    Serre, G., Mils, V., Haftek, M., et al.Identification of late differentiation antigens of human cornified epithelia, expressed in re-organised desmosomes and bound to cross-linked envelopes. J. Invest. Dermatol. 97, 1061 1072 (1991).
  • 16
    Montezin, M., Simon, M., Guerrin, M, Serre, G. Corneodesmosin, a corneodesmosome-specific basic protein, is expressed in the cornified epithelia of the pig, guinea pig rat and mouse. Exp. Cell Res. 231, 132 140 (1997).
  • 17
    Zhou, Y. & Chaplin, D.D. Identification in the HLA class 1 region of a gene expressed late in keratinocyte differentiation. Proc. Natl. Acad. Sci. 90, 9470 9474 (1993).
  • 18
    Lundstrom, A., Serre, G., Haktek, M., Egelrud, T. Evidence for a role of corneodesmosin, a protein which may serve to modify desmosomes during cornification, in stratum corneum cell cohesion and desquamation. Arch. Dermatol. Res. 286, 369 375 (1994).
  • 19
    Simon, M., Montezin, M., Guerrin, M., Durieux, J.J., Serre, G. Characterisation and purification of human corneodesmosin, an epidermal basic glycoprotein associated with corneocyte-specific modified desmosomes. J. Biol. Chem. 272, 31770 31776 (1997).
  • 20
    Fartasch, M., Bassukas, I.D., Diepgen, T.L. Structural relationship between epidermal lipid lamellae, lamellar bodies and desmosomes in human epidermis, an ultrastructural study. Br. J. Dermatol. 128, 1 9 (1993).
  • 21
    Rawlings, A.V., Watkinson, A.W, Rogers, J., Mayo, A.M., Hope, J., Scott, I.R. Abnormalities in stratum corneum structure, lipid composition and desmosomal degradation in soap-induced winter xerosis. J. Soc. Cosmet. Chem. 45, 203 220 (1994).
  • 22
    Brysk, M.M. & Miller, J. Concanavalin-A binding glycoprotein in human stratum corneum. J. Invest. Dermatol. 82, 280 284 (1984).
  • 23
    Brysk, M.M., Bell, T., Rajaraman, S. Sensitivity of desquamin to proteolytic degradation. Pathobiology 59, 109 112 (1991).
  • 24
    Brysk, M.M., Bell, T., Brysk, H., Selvanayagam, P., Rajaraman, S. Enzymatic-activity of desquamin. Exp. Cell Res. 214, 22 26 (1994).
  • 25
    Selvanayagam, P., Lei, G., Ram, S., et al.Desquamin is an epidermal ribonuclease. J. Cell Biol. 68, 74 82 (1998).
  • 26
    Smith, W.P., Christensen, M.S., Nacht, S., Gans, E.H. Effect of lipids on the aggregation and permeability of human stratum corneum. J. Invest. Dermatol. 78, 7 11 (1982).
  • 27
    Chapman, S.J., Walsh, A., Jackson, S.M., Friedmann, P.M. Lipids, proteins and corneocyte adhesion. Arch. Dermatol. Res. 283, 1729 1732 (1991).
  • 28
    Abraham, W. & Downing, D.T. Interaction between corneocytes and stratum corneum lipid liposomes in vitro. Biochim. Biophys. Acta 1021, 119 125 (1990).
  • 29
    Ranasinghe, A.W., Wertz, P.W., Downing, D.T., McKenzie, I. Lipid composition of cohesive and desquamated corneocytes from mouse ear. J. Invest. Dermatol. 94, 216 220 (1985).
  • 30
    Bointe, F., Saunois, A., Pinguet, P., Meybeck, A. Existence of a lipid gradient in the upper stratum corneum and its possible significance. Arch. Dermatol. Res. 289, 78 82 (1997).
  • 31
    Elias, P.M. & Menon, G.K. Structural and lipid biochemical correlates of the epidermal permeability barrier. In Advances in lipid research Vol. 24 (P. M. Elias, ed), pp. 1 26. Academic Press, London (1991).
  • 32
    Azimi, N.A., Spencer, I.S., Potts, R.O, Lyttle, F.E., Chen, D.A. Fluorescence spectroscopic evaluation of the fluidity gradient in stratum corneum lipids. J. Invest. Dermatol. 98, 641 (1992).
  • 33
    Wertz, P.M. & Downing, D.T. Ceramidase activity in porcine epidermis. Febs Lett. 268, 110 112 (1990).
  • 34
    Oldroyd, J., Critchley, P., Tiddy, G, Rawlings, A.V. Specialised role for ceramide one in the stratum corneum water barrier. J. Invest. Dermatol. 102, 525 (1994).
  • 35
    Bointe, F., Saunois, A., Pinguet, P., et al.Thermotrophic phase behaviour of in vivo extracted human stratum corneum lipids. Lipids 32, 653 660 (1997).
  • 36
    Barton, S.P, King, C.S., Marks, R., Nicholls, S. Technique for studying the structural detail of isolated human corneocytes. Br. J. Dermatol. 102, 63 73 (1980).
  • 37
    Long, S., Banks, J., Watkinson, A., Harding, C.R., Rawlings, A.V. Desmocollin 1: a key marker for desmosome processing in the stratum corneum. J. Invest. Dermatol. 106, 397 (1996).
  • 38
    Menon, G.K., Ghadially, R., Williams, M.L., Elias, P.M. Lamellar bodies as delivery systems of hydrolytic enzymes: implications for normal and abnormal desquamation. Br. J. Dermatol. 126, 337 345 (1992).
  • 39
    Egelrud, T. Purification and preliminary characterisation of stratum corneum chymotryptic enzyme – a proteinase that may be involved in desquamation. J. Invest. Dermatol. 101, 200 204 (1993).
  • 40
    Sondell, B., Thornell, L.E., Stigbrand, T., Egelrud, T. Immunolocalisation of stratum-corneum chymotryptic enzyme in human skin and oral epithelium with monoclonal antibodies – Evidence of a proteinase specifically expressed in keratinising squamous epithelia. J. Histochem. Cytochem. 42, 459 (1994).
  • 41
    Rogers, J.S., Watkinson, A., Harding, C.R. Characterisation of the effects of protease inhibitors and lipids on human stratum corneum chymotrytic-like enzyme supports a role in desquamation. J. Invest Dermatol. 110, 672 (1998).
  • 42
    Lundstrom, A. & Egelrud, T. Evidence that cell shedding from plantar stratum corneum in vitro involves endogenous proteolysis of the desmosomal protein desmoglein-1. J. Invest. Dermatol. 94, 216 220 (1990).
  • 43
    Sondell, B., Thornell, L.E., Egelrud, T. Evidence that stratum corneum chymotryptic enzyme is transported to the stratum corneum extracellular space via lamellar bodies. J. Invest. Dermatol. 104, 891 823 (1995).
  • 44
    Nylander Lundqvist, E. & Egelrud, T. Formation of active IL-1 beta from pro-IL-1beta catalysed by stratum corneum chymotryptic enzyme in vitro. Acta-Dermato-Venereo. 77, 203 206 (1997).
  • 45
    Suzuki, Y., Nonura, J., Hori, J, Koyama, J., Takahashi, M., Horii, I. Detection and characterisation of endogenous proteases associated with desquamation of stratum corneum. Arch. Dermatol. Res. 285, 327 337 (1993).
  • 46
    Sato, J., Nakanishi, J., Denda, M., Nomura, J., Koyama, J. Cholesterol sulphate inhibits proteases which are involved in desquamation of stratum corneum. J. Invest. Dermatol. 108, 396 (1997).
  • 47
    Franzke, C.W., Wiedow, O, Christophers, E. Detection of trypsin-like activity in human stratum corneum. J. Invest. Dermatol. 109, 431 (1997).
  • 48
    Hansson, L., Stromqvist, M., Backman, A., Egelrud, T. Cloning, expression and characterisation of stratum corneum chymotrypic enzyme-a skin-specific human serine protease. J. Biol. Chem. 269, 19420 19426 (1994).
  • 49
    Walsh, A. & Chapman, S. Sugars protect desmosomal proteins from proteolysis. Br. J. Dermatol. 122, 289 (1990).
  • 50
    Franzke, C.W., Baici, A., Bartels, J, Christophers, E., Wiedow, O. Antileukoprotease inhibits stratum corneum chymotryptic enzyme – Evidence for a regulatory function in desquamation. J. Biol. Chem. 271, 21886 21890 (1996).
  • 51
    Rawlings, A.V, Harding, C.R, Watkinson, A., Banks, J., Ackerman, C, Sabin, R. The effect of glycerol and humidity on desmosome degradation in stratum corneum. Arch. Dermatol. Res. 287, 457 464 (1995).
  • 52
    Bartolone, J., Doughty, D., Egelrud, T. A non-invasive approach for assessing corneocyte cohesion: immunocytochemical detection of Dsg1. J. Invest. Dermatol. 96, 596 (1991).
  • 53
    Bernard, D., Camus, C, Nguyen, Q.L., Serre, G. Proteolysis of corneodesmosomal proteins in winter xerosis. J. Invest. Dermatol. 105, 176 (1995).
  • 54
    Lee, S.H., Elias, P.M., Proksch, E., Feingold, K.M. Calcium and potassium are important regulators of barrier homeostasis. J. Clin. Invest. 89, 530 538 (1992).
  • 55
    Feingold, K.R. The regulation and role of epidermal lipid synthesis. In: Advances in lipid research Vol. 24 (P. M. Elias, ed), pp. 57 82. Academic Press, London (1991).
  • 56
    Holleran, W.M., Man, M.Q, Wen, N.G., Menon, G.K., Elias, P.M., Feingold, K.R. Sphingolipids are required for mammalian epidermal barrier function-inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation. J. Clin. Invest. 88, 1338 1345 (1991).
  • 57
    Harris, I.R., Farrell, A.M., Holleran, W.M. et al. Parallel regulation of sterol regulatory element binding protein-2 and the enzymes of cholesterol and fatty acid synthesis in cultured human keratinocytes and murine epidermis. J. Lipid Res. 39, 412 422 (1997).
  • 58
    Wertz, P.W., Miethke, M.C., Long, S.A., Strauss, J.S., Downing, D.T. Composition of ceramides from human stratum corneum and comedones. J. Invest. Dermatol. 84, 410 412 (1985).
  • 59
    Swartzendruber, D.C, Wertz, P.W., Kitco, D.J., Madison, K.C., Downing, D.T. Molecular models of intercellular lamellae in mammalian stratum corneum. J. Invest. Dermatol. 92, 215 257 (1991).
  • 60
    Rawlings, A., Critchley, P., Ackerman, C., Scott, I.R. The functional roles of ceramide one. Proceedings of the 17th IFSCC, International Federation Society Cosmetic Chemists. Int. Cong. 1, 14 19 (1992).
  • 61
    Bouwstra, J.A., Gooris, G.S., Dubbelaar, F.E.R., Weerheim, A.M., Iljerman, A.P., Ponec, M. Article title. J. Lipid Res. 39, 186 196 (1998).
  • 62
    Wertz, P.W., Madison, K.C., Downing, D.T. Covalently bound lipids of human stratum corneum. J. Invest. Dermatol. 92, 109 111 (1989).
  • 63
    Robson, K.J., Stewart, M.E., Michelsen, S., Lazo, N.D., Downing, D.T. 6-Hydroxy-4-sphingenine in human epidermal ceramides. J. Lipid Res. 35, 2060 2068 (1994).
  • 64
    Jokura, Y., Ishikawa, S., Tokuda, H, Imokawa, G. Molecular analysis of elastic properties of the stratum corneum by solid-state C-13 – nuclear magnetic resonance spectroscopy. J. Invest. Dermatol. 104, 806 812 (1995).
  • 65
    Barrett, J.G. & Scott, I.R. Pyrrolidone carboxylic acid synthesis in guinea pig epidermis. J. Invest. Dermatol. 81, 122 124 (1983).
  • 66
    Scott, I.R. & Harding, C.R. Studies on the synthesis and degradation of a histidine-rich phosphoprotein from mammalian epidermis. Biochim. Biophys. Acta. 669, 65 78 (1981).
  • 67
    Scott, I.R., Harding, C.R., Barrett, J.G. Histidine rich proteins of the keratohyalin granules: Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum. Biochim. Biophys. Acta. 719, 110 117 (1982).
  • 68
    Ishidayamamoto, A., Hohl, D., Roop, D.R., Iizuka, H., Eady, R.A.J. Loricrin immunoreactivity in human skin – Localisation of specific granules (L-granules) in acrosyringia. Arch. Dermatol. Res. 285, 491 498 (1993).
  • 69
    Harding, C.R. & Scott, I.R. Histidine-rich proteins (filaggrins). Structural and functional heterogeneity during epidermal differentiation. J. Mol. Biol. 170, 651 673 (1983).
  • 70
    Steinert, P.M., Cantieri, J.S., Teller, J.D., Lonsdale-Eccles, J.D., Dale, B.A. Characterisation of a class of cationic proteins that specifically interact with intermediate filaments. Proc. Natl. Acad. Sci. USA 78, 4097 4101 (1981).
  • 71
    Gan, S.-Q., McBride, O.W., Idler, W.W., Marakova, N, Steinert, P.M. Organisation, structure and polymorphism of the human profilaggrin gene. Biochemistry 29, 9432 9440 (1990).
  • 72
    Presland, R.B., Haydock, P.V., Fleckman, P., Nirunsuksiri, W, Dale, B.A. Characterisation of the human profilaggrin gene. Genomic organisation and identification of an S-100 calcium binding domain at the amino terminus. J. Biol. Chem. 267, 23772 23781 (1992).
  • 73
    Presland, R.B., Bassuk, J.A, Kimball, J.R., Dale, B.A. Characterisation of two distinct calcium-binding sites in the amino terminus of of human profilaggrin. J. Invest. Dermatol. 104, 218 223 (1995).
  • 74
    Dale, B.A., Preland, R.B., Lewis, S.P., Underwood, R.A., Fleckman, P. Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity. J. Invest. Dermatol. 108, 179 187 (1997).
  • 75
    Scott, I.R. & Harding, C.R. Profilaggrin phosphatase activity – A key control step in the pathway of epithelial differentiation. J. Invest. Dermatol. 96, 1006 (1993).
  • 76
    Kam, E, Resing, K.A., Lim, S.K., Dale, B.A. Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase-2A family. J. Cell Sci. 106, 219 226 (1993).
  • 77
    Presland, R.B., Kimball, J.R., Kautsky, M.B., Lewis, S.P., Lo, C.Y., Dale, B.A. Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation. J. Invest. Dermatol. 108, 170 178 (1997).
  • 78
    Yamazaki, M., Ishidoh, K., Suga, Y. et al. Cytoplasmic processing of human profilaggrin by active mu-calpain. Biochim. Biophys. Res. Comm. 235, 652 656 (1997).
  • 79
    Resing, K.A., Thulin, C., Whiting, K., Alalawi, M., Mostad, S. Characterisation of profilaggrin endoproteinase-1: a regulated cytoplasmic endoproteinase of epidermis. J. Biol. Chem. 270, 28193 28198 (1997).
  • 80
    Richards, S., Scott, I.R., Harding, C.R., Liddell, E., Curtis, G.C. Evidence for filaggrin as a component of the cell envelope of the newborn rat. Biochem. J. 253, 153 160 (1988).
  • 81
    Steinert, P.M. & Marekov, L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins are isodipeptide cross-linked components of the human cornified cell-envelope. J. Biol. Chem. 270, 17702 17711 (1995).
  • 82
    Scott, I.R. Alterations in the metabolism of filaggrin in the skin after chemical and ultraviolet induced erythema. J. Invest. Dermatol. 87, 460 465 (1986).
  • 83
    Steinert, P.M. Epidermal keratin: filaments and matrix. In: Stratum corneum (R. Marks and G. Plewig, eds), pp. 25 38. Springer-Verlag, Berlin (1983).
  • 84
    Kan, S.H., Asaga, H., Senshu, T. Detection of several families of deiminated proteins derived from filaggrin and keratins in guinea pig skin. Zool. Sci. 13, 673 678 (1996).
  • 85
    Senshu, T., Kan, S.H, Ogawa, H., Manabe, M., Asaga, H. Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis. Biochem. Biophys. Res. Comm. 225, 712 719 (1996).
  • 86
    Kawada, A., Hara, K., Morimoto, K. , Hiruma, M., Ishibasha, A. Rat epidermal cathepsin-B – Purification and characterisation of proteolytic properties towards filaggrin and synthetic substrates. Int. J. Biochem. Cell Biol. 27, 175 183 (1995).
  • 87
    Kawada, A., Hara, K., Hiruma, M., Noguchi, H., Ishibashi, A. Rat epidermal cathepsin L-like proteinase – Purifiction and some hydrolytic properties towards filaggrin and synthetic substrates. J. Biol. Chem. 118, 332 337 (1995).
  • 88
    Laden, K. & Spitzer, R. Identification of a natural moisturising agent in skin. J. Soc. Cosmet. Chem. 18, 351 360 (1976).
  • 89
    Angelin, J.H. Urocanic acid a natural sunscreen. Cosmetics Toiletries 91, 47 49 (1976).
  • 90
    Scott, I.R. Factors controlling the expressed activity of histidine ammonia lyase in the epidermis and the resulting accumulation of urocanic acid. Biochem. J. 194, 829 838 (1981).
  • 91
    Scott, I.R. & Harding, C.R. Filaggrin breakdown to water binding components during development of the rat stratum corneum is controlled by the water activity of the environment. Dev. Biol. 115, 84 92 (1986).
  • 92
    Scott, I.R. & Harding, C.R. Physiological effects of occlusion-filaggrin retention. Proc. Dermatol. 2000, 773 (1993).
  • 93
    Harding, C.R., Ellis, K., Scott, I.R. Alterations in the processsing of human filaggrin following skin occlusion in vitro and in vivo. J. Invest. Dermatol. 100, 579 000 (1993).
  • 94
    Haftek, M., Simon, M., Kanitakis, J, et al.Expression of corneodesmosin in the granular layer and stratum corneum of normal and diseased skin. Br. J. Dermatol. 137, 864 873 (1998).
  • 95
    Suzuki, Y., Koyama, J, Moro, O., Kikuchi, K., Tanida, M., Tagami, H. The role of two endogenous proteases of the stratum corneum in degradation of desmoglein-1 and their reduced activity in the skin of ichthyotic patients. Br. J. Dermatol. 134, 460 464 (1996).
  • 96
    Fartasch, M., Bassukas, I.D., Diepgen, T.L Disturbed extruding mechanism of lamellar bodies in dry non-eczematous skin of atopics. Br. J. Dermatol. 127, 221 227 (1992).
  • 97
    Mao-Qiang, M., Jain, M., Feingold, K.R., Elias, P.M. Secretory phospholipase A2 activity is required for permeability barrier homeostasis. J. Invest. Dermatol. 106, 57 63 (1996).
  • 98
    Menon, G.K., Grayson, S., Elias, P.M. Cytochemical and biochemical localisation of lipase and sphingomyelinase activity in mammalian epidermis. J. Invest. Dermatol. 86, 591 597 (1986).
  • 99
    Holleran, W.M., Takagi, Y., Menon, G.K., Legler, G., Feingold, K.R., Elias, P.M. Processing of epidermal glucosylceramides is required for optimal mammalian permeability barrier function. J. Clin. Invest. 91, 1656 1664 (1993).
  • 100
    Jin, K., Higaki, Y., Higuchi, K., Yada, Y., Kawashima, M., Imokawa, G. Analysis of beta glucocerebrosidase and ceramidase activities in atopic and aged dry skin. Acta Derm. Venereo 74, 337 340 (1994).
  • 101
    Williams, M.L. Lipids in normal and pathological desquamation. In Advances in lipid research Vol. 24 (P. M. Elias, ed), pp. 211 262. Academic Press, London (1991).
  • 102
    Sato, J, Nakanishi, J., Denda, M., Nomura, J., Koyama, J. Cholesterol sulphate inhibits proteases which are involved in desquamation of stratum corneum. J. Invest. Dermatol. 108, 396 (1997).
  • 103
    Saint-Leger, D., Francois, A.M., Leveque, J.L., Stoudemayer, T., Kligman, A.M., Grove, G.L. Stratum corneum lipids in winter xerosis. Dermatologica 178, 151 155 (1989).
  • 104
    Nappe, C., Delesalle, G., Jansen, A, De Rigal, J., Camus, C. Decrease in ceramide II in skin xerosis. J. Invest. Dermatol. 100, 530 (1993).
  • 105
    Fulmer, A.W. & Kramer, G.J. Stratum corneum abnormalities in surfactant induced dry scaly skin. J. Invest. Dermatol. 80, 598 602 (1989).
  • 106
    Lampe, M.A., Burlingame, A.L, Whitney, J, et al.Human stratum corneum lipids: characterisation and regional variation. J. Lipid Res. 24, 120 130 (1983).
  • 107
    Imokawa, G, Akasaki, S., Hattori, M., Yoshizuka, N. Selective recovery of deranged water-holding properties by stratum corneum lipids. J. Invest. Dermatol. 87, 758 761 (1986).
  • 108
    Imokawa, G., Akasaki, S., Minematsu, Y, Kawai, M. Importance of intercellular lipids in water retention properties of the stratum corneum: induction and recovery study of surfactant dry skin. Arch. Dermatol. Res. 281, 45 51 (1989).
  • 109
    Rawlings, A.V., Harding, C.R., Schilling, K.S. Ceramides and the skin. In: Textbook of cosmetic dermatology (R. Baran and H. Maibach, eds), pp. 99 112. Martin Dunitz Ltd, London (1998).
  • 110
    Seguchi, T., Chang, Y.C., Kusuda, S., Takahashi, M., Aisuy, K., Tezuka, T. Decreased expression of filaggrin in atopic skin. Arch. Derm Res. 288, 442 446 (1996).
  • 111
    Marstein, S., Jellum, E., Eldjarn, L. The concentration of pyrroglutamic acid (2-pyrrolidone-5-carboxylic acid) in normal and psoriatic epidermis determined on a microgram scale by gas chromatography. Clin. Chim. Acta. 49, 389 395 (1973).
  • 112
    Sybert, V.P., Dale, B.A., Holbrook, K.A. Ichthyosis vulgaris: identification of a defect in filaggrin synthesis correlated with an absence of keratohyalin granules. J. Invest. Dermatol. 84, 191 194 (1985).
  • 113
    Denda, M., Hori, J., Koyama, J, et al.Stratum corneum sphingolipids and free amino acids in experimentally-induced scaly skin. Arch. Derm. Res. 284, 363 367 (1992).
  • 114
    Horii, I., Nakayama, Y., Obata, M., Tagami, H. Stratum corneum hydration and amino acid content in xerotic skin. Br. J. Dermatol. 121, 587 592 (1989).
  • 115
    Tezuka, T., Qing, J., Saheki, M., Kusuda, S., Takahashi, M. Terminal differentiation of facial epidermis of the aged – immunohistochemical studies. Dermatology 188, 21 24 (1994).
  • 116
    Scott, I.R. & Harding, C.R. A filaggrin analogue to increase natural moisturising factor synthesis in skin. Dermatology 2000, 773 (1993).
  • 117
    Tezuka, T. Electron microscopal changes in xerotic senilis epidermis. Its abnormal membrane coating granule formation. Dermatologica 166, 57 61 (1983).
  • 118
    Ghadially, R, Halkiersorenson, L., Elias, P.M. Effects of petrolatum on stratum corneum structure and function. J. Am. Acad. Dermatol. 26, 387 396 (1992).
  • 119
    Froebe, C.L., Simion, F.A., Ohlemeyer, H et al. Prevention of stratum corneum lipid phase transition by glycerol – an alternative mechanism for skin moisturisation. J. Soc. Cosmet. Chem. 41, 51 65 (1990).
  • 120
    Summers, R.S., Summers, B, Chandar, P., Feinberg, C., Gursky, R., Rawlings, A.V. The effects of lipids with and without humectant on skin xerosis. J. Soc. Cosmet. Chem. 47, 27 39 (1996).
  • 121
    Bisset, D.L., McBride, J.F., Patrick, L.F. Role of protein and calcium in stratum corneum cell cohesion. Arch. Derm Res. 279, 184 189 (1987).
  • 122
    Takahashi, M., Aizawa, M., Miyazawa, K., Machida, Y. Effects of surface active agents on stratum corneum cell cohesion. J. Soc. Cosmet. Chem. 38, 21 28 (1987).
  • 123
    Lundstrom, A. & Egelrud, T. Cell shedding from human plantar skin in vitro: evidence that two different types of protein structures are degraded by a chymotryptic-like enzyme. Arch. Derm Res. 282, 234 237 (1990).
  • 124
    Rattner, H. Dermatologic uses of urea. Acta Derm. Venerol. 37, 155 165 (1943).
  • 125
    Fredrikkson, T. & Gip, L. Urea creams in the treatment of dry skin and hand dermatitis. Int. J. Dermatol. 14, 442 444 (1975).
  • 126
    Serup, J. A double blind comparison of 2 creams containing urea as the active agent – assessment of efficacy and side-effects by non-invasive techniques and a clinical scoring scheme. Acta Dermato. Venereo. Supplement 177, 34 38 (1992).
  • 127
    McCallion, R. & Po, A.L. Modelling transepidermal water-loss under steady state and non-steady state relative humidities. Int. J. Pharmaceutics 105, 103 112 (1994).
  • 128
    Lodon, M. Urea-containing moisturisers influence barrier properties of normal skin. Arch. Dermatol. Res. 288, 103 107 (1996).
  • 129
    Pigatto, P.D., Bigardi, A.S, Cannistraci, C., Picardo, M. 10% urea cream (Laceran) for atopic dermatitis: a clinical and laboratory evaluation. J. Dermatol. Treat. 7, 171 175 (1996).
  • 130
    Swanbeck, G. Treatment of dry hyperkeratotic, itchy skin with urea containing preparations. Dermatol. Digest. 11, 39 43 (1972).
  • 131
    Hauss, H., Proppe, A, Matthies, C.A. Formulation for the treatment of dry, itching skin in comparison –results from therapeutic use. Dermatosen Beruf Umwelt 41, 184 188 (1993).
  • 132
    Stern, E.C. Topical application of lactic acid in the treatment and prevention of certain disorders of the skin. Urologic Cutaneous Review 50, 106 107 (1943).
  • 133
    Baden, H.P. & Alper, J.C. Keratolytic gel containing salicylic acid in propylene glycol. J. Invest. Dermatol. 61, 330 333 (1973).
  • 134
    Middleton, J.D. Development of a skin cream designed to reduce dry and flaky skin. J. Soc. Cosmet. Chem. 25, 519 534 (1974).
  • 135
    Van Scott, E. & Yu, R.J. Control of keratinisation with α-hydroxy acids and related compounds. Arch. Dermatol. 110, 586 590 (1974).
  • 136
    Wehr, P.W., Krochmal, L., Bagatell, F., Ragsdale, W. A controlled two center study of lactate 12 percent lotion and a petrolatum-based creme in patients with xerosis. Cutis 23, 205 209 (1986).
  • 137
    Dahl, M.C. & Dahl, A.C. 12% lactate lotion for the treatment of xerosis. Arch. Dermatol. 119, 27 30 (1983).
  • 138
    Fartasch, M., Teal, J., Menon, G.K. Mode of action of glycolic acid on human stratum corneum; Ultrastructural and functional evaluation of the epidermal barrier. Arch. Dermatol. Res. 289, 404 409 (1997).
  • 139
    Rawlings, A.V., Davies, A., Carlomusto, M., Pillai, S, Zhang, K., Verdejo, P., Feinberg, C, Nguyen, L., Chandar, P. Effect of lactic acid isomers on keratinocyte ceramide synthesis, stratum corneum lipid levels and stratum corneum barrier function. Arch. Dermatol. Res. 288, 383 390 (1996).
  • 140
    Smith, W.P. Epidermal and dermal effects of topical lactic acid. J. Am. Acad. Dermatol. 35, 388 391 (1996).
  • 141
    Hall, K.J. & Hill, J.C. The skin plasticisation effect of 2-hydroxyoctanoic acid. 1. The use of potentiators. J. Soc. Cosmet. Chem. 37, 397 407 (1986).
  • 142
    Kwoyo Hakko Kogyo Co. Pyrrolidone carboxylic acid esters containing composition used to prevent loss of moisture from the skin. Patent JA 48 82 046 (1982).
  • 143
    Org Santerre. l-pyrrolidone carboxylic acid-sugar compounds as rehydrating ingredients in cosmetics Patent Fr 2 277 823 (1977).
  • 144
    Clar, E.J. & Fourtanier, A. L’acide pyrrolidone carboxylique (PCA) et la peau. Int. J. Cosmet Sci. 3, 101 113 (1981).
  • 145
    Middleton, J.D. & Roberts, M.E. Effects of a skin cream containing the sodium salts of pyrrolidone carboxylic acid on dry and flaky skin. J. Soc. Cosmet. Chem. 29, 201 205 (1978).
  • 146
    Mao-Qiuang, M., Feingold, K.R., Elias, P.M. Exogenous lipids influence permeability barrier recovery in acetone-treated murine skin. Arch. Dermatol. 129, 728 738 (1993).
  • 147
    Zettersten, E.M., Ghadially, R., Feingold, K.R., Crumrine, D, Elias, P.M. Optimal ratios of topical stratum corneum lipids improve barrier recovery in chronologically aged skin. J. Am. Acad. Dermatol. 37, 403 408 (1997).
  • 148
    Yang, L., Mao-Qiang, M., Taljbeni, M., Elias, P.M., Feingold, K.R. Topical stratum corneum lipids accelerate barrier repair after tape-stripping, solvent treatment and some but not all types of detergent treatment. Br. J. Dermatol. 133, 679 685 (1995).
  • 149
    Di Nardo, A., Sugino, K., Wertz, P.W., Ademola, J., Maibach, H.J. Sodium lauryl sulphate (SLS) induced irritant contact dermatitis: a correlation between ceramides and in vivo parameters of irritation. Contact Dermatitis 35, 86 91 (1996).
  • 150
    Linter, K., Mondon, P., Girard, F. The effect of a synthetic ceramide 2 on transepidermal water loss after stripping or SLS treatment: an in vivo study. Int. J. Cosmet. Chem. 19, 15 25 (1997).
  • 151
    Di Nardo, A., Wertz, P., Giannetti, A., Seidenari, S. Ceramide and cholesterol composition of the skin of patients with atopic dermatitis. Acta Dermato. Venereo. 78, 27 30 (1998).
  • 152
    Prottey, C., Hartop, P.J., Press, M. Correction of the cutaneous manifestations of essential fatty acid deficiency in man by application of sunflower seed oil to the skin. J. Invest. Dermato. 64, 228 234 (1975).
  • 153
    Conti, A., Rogers, J., Verdejo, P., Rawlings, A.V. Seasonal influences on stratum corneum ceramide 1 fatty acids and the influence of topical essential fatty acids. Int J. Cosmet. Sci. 18, 1 12 (1996).
  • 154
    Brod, J., Traitler, H., De Studer, A., LaCharriere, M. Evolution of lipid composition in skin treated with blackcurrant seed oil. Int. J. Cosmet. Sci. 10, 149 159 (1993).
  • 155
    Zhang, K., Kosturko, R., Rawlings, A.V. The effect of thiols on epidermal lipid biosynthesis. J. Invest. Dermatol. 104, 687 (1995).
  • 156
    Carlomusto, M., Pillai, K., Rawlings, A.V. Human keratinocytes in vitro can utilise exogenously supplied sphingolipid analogues for keratinocyte ceramide biosynthesis. J. Invest. Dermatol. 106, 919 (1996).
  • 157
    Davies, A., Verdejo, P., Feinberg, C., Rawlings, A.V. Increased stratum corneum ceramide levels and improved barrier function following treatment with tetraacetylphytosphingosine. J. Invest. Dermatol. 106, 918 000 (1996).
  • 158
    Liu, B., Obeid, L.M., Hannun, Y. Sphingomyelinases in cell regulation. Seminars Cell. Dev. Biol. 8, 311 322 (1997).
  • 159
    Spiegel, S. & Merrill, A.H. Sphingolipid metabolism and cell growth regulation. FASEB J. 10, 1388 1397 (1996).
  • 160
    Hunnan, Y.A. Functions of ceramides in cordinating cellular responses to stress. Science 274, 1855 1859 (1996).
  • 161
    Merrill, A.H., Schmelz, E.-M., Dillehay, D.L, et al.Sphingolipids – The enigmatic lipid class: biochemistry, physiology and pathophysiology. Toxic Appl. Pharmacol. 142, 208 225 (1997).
  • 162
    Fori, M., Jensen, J.M., Schutze, S., Kronke, M., Proksch, E. Acidic and neutral sphingomyelinases generating ceramides for the skin barrier in outer and inner epidermal layers of aged mice. J. Invest. Dermatol. 110, 672 (1998).
  • 163
    Pillai, K., Frew, L., Cho, S., Rawlings, A.V. Synergy between the vitamin D precursor, 25 hydroxyvitamin D and short chain ceramides on human keratinocyte growth and differentiation. J. Invest. Dermatol. Suppl. 1, 39 45 (1996).
  • 164
    Carlomusto, M., Mahajan, M., Pillai, S. Vitamin D-mediated keratinocyte differentiation does not involve sphingomyelin hydrolysis. J. Invest. Dermatol. 108, 660 (1997).
  • 165
    Bosko, C., Samares, S., Santanastasio, H., Rawlings, A.V. Influence of fatty acid composition of acylceramides on keratinocyte differentiation. J. Invest. Dermatol. 106, 871 (1996).
  • 166
    Marsh, N.N., Elias, P.M., Holleram, W.M. Glucosylceramides stimulate murine epidermal hyperproliferation. J. Clin. Invest. 95, 2903 2909 (1995).
  • 167
    Uchida, Y., Iwamori, M, Nagai, Y. Activation of keratinisation of keratinocytes from fetal rat skin with N (linoleoyl) ω-hydroxy fatty acyl sphingosyl glucose as a marker of epidermis. Biochim. Biophys. Res. Commun. 179, 162 168 (1990).
  • 168
    Takuji, M., Yasukohchi, T., Hirobe, M., Arakane, K., Adachi, K. The protease as a cleansing agent and its stabilisation by chemical modification. J. Soc. Cosmet. Chem. Jpn 27, 276 288 (1993).