9.1.2 Cutaneous Biochemistry

Keratin intermediate filament (KIF): Keratinocytes contain filaments (8-15 nm) of the KIF family that contribute to the cell cytoskeleton. KIF are constructed by proteins called keratins (K). The different layers of the epidermis contain varying K amounts; ranging from approximately 30% in the stratum basale to 80% in the stratum corneum. KIFs are elongated α-helix-enriched proteins that form dynamic coiled- coils, which are fashioned into filament collections called tonofilaments. The latter are only soluble in denaturants like urea and sodium dodecyl sulfate and reducing reagents like mercaptoethanol and dithiothreitol. Epithelial K are grouped as acidic (type I) and neutral-basic (type II) with sharing sequence homology in each group. Each epithelial cell/tissue type expresses at least one K pair, a type I K together with its preferred type II K partner. The K pair is regulated concomitantly. K5 and K14 are associated with normal cell proliferation and K6 and 16 with hyperproliferation in the epidermis (normally expressed in epithelial cells of the outer root sheath of hair follicles, nail beds and epithelium of oral mucosa), while terminal keratinocyte differentiation (maturation) is associated with expression of K1 and K10 as well as K2 and K11.

Biochemistry of keratinocyte differentiation: Major proteins involved in keratinocyte differentiation include involucrin, which can be extensively cross-linked to other proteins by transglutaminase (TGase). Integrins induce the synthesis of involucrin in keratinocytes, leading to their detachment from the basement membrane and interruption of proliferation. The transition of keratinocytes from the stratum spinosum to stratum granulosum is mediated by protein kinase C via suppression of K1 and K10 gene expression and the associated induction of the late differentiation markers loricrin and profilaggrin. Stratum granulosum keratinocytes contain protein- and lipid-rich granules. These structures (former known as keratohyalin granules) are irregularly shaped profilaggrin-containing granules that coalesce in the stratum granulosum and thereafter disperse in the stratum corneum upon dephosphorylation and subsequent maturation via proteolysis to filaggrin. These granules are lipid-rich lamellar structures (Odland granules), exist as lipid bilayers and fuse with the cell membranes to contribute lipids to the extracellular space of the stratum corneum. The lipids form the sheets of the lipid epidermal permeability barrier (EPB).

EPB function in human epidermis depends on TGase-mediated cross-linking of structural proteins and lipids during the terminal stages of keratinocyte differentiation. Several mutations in genes that encode for the EPB structural components and its enzymes and lipid processing can result in skin disease. Associated pathologies result from relevant mutations, such as in SPINK5 serine proteinase inhibitor (Netherton syndrome) and cathepsin C (Papillon–Lefevre syndrome).

EPB quality also depends on the presence of epidermal lipids, such as ceramides, cholesterol and free fatty acids. Ceramides account for approximately 50% of stratum corneum lipids followed by cholesterol and free fatty acids. Changes in the concentration of any of these can affect EPB quality. Aged and photo-aged skin exhibit a cholesterol-dominant barrier, atopic dermatitis with ceramide-dominance and psoriasis with a dominance of free fatty acids. EPB repair may be initiated by loss of Ca2+ and K+, which are important regulators of barrier function, during water influx in the damaged epidermis. These ions synergistically inhibit barrier repair via blocking the return of lipids to the stratum corneum. There is a 4-fold increase in extracellular Ca2+ from basal layer to the stratum corneum. An acid pH maintains EPB homeostasis by providing optimal pH for the enzymes involved in extracellular processing bilayer lipids.

Topical application of solvents to the skin removes lipids from the stratum corneum and increases trans- epidermal water loss (TEWL), followed by induction of a rapid barrier repair through epidermal metabolic changes. The lipid-rich cornified envelope (CE) that surrounds individual corneocytes is organized into lipid-rich lamellar bilayers. Aqueous pores interrupt the hydrophobic nature of this lipid-rich matrix representing potential routes of topical entry for water-soluble therapeutic compounds.

Skin fatty acids: The permeability of human skin is largely based on the quality and quantity of the lamellar lipids packed between corneocytes. Endogenously-produced fatty acids can be found in epidermal cell membranes and in the lipids located between corneocyte layers, which construct the hydro-lipid film at the skin surface. Fatty acids are involved in the end stages of epidermal cell life, participate in signaling events during keratinocyte proliferation and differentiation deep in the epidermis and even dermis, and are implicated in the skin immune response.

Modification of skin fatty acids can lead to disease. Atopic dermatitis is associated with abnormally reduced ω6 fatty acids, decreased ceramide content and increased TEWL. UV irradiation of skin can lead to increased casual skin surface lipid (CSL) levels leading to improved EPB performance. In contrast, photo-aged and intrinsically aged epidermis exhibit abnormal EPB homeostasis, CSL reduction and a dramatic fall of cholesterol synthesis.

Epidermal fatty acids can also derive from the diet. The skin is an active metabolizer of polyunsaturated fatty acids (PUFA). PUFA-deficient diets (e.g. linoleic acid) can result in both increased TEWL and scaly skin. Arachidonic acid, a 20-carbon PUFA, can be metabolized by the cyclooxygenase or lipoxygenase pathways in the skin producing prostaglandins and hydroxyeicosatetraenoic acids, which interact with signaling systems in the proliferating and differentiating epidermal cells.

Skin absorption of xenobiotics: Xenobiotics are foreign to the biological system chemical substances (natural compounds, drugs, environmental agents), which can be transported across the stratum corneum. The skin is constantly exposed to xenobiotics through occupational, environmental, therapeutic and systemic exposures. Human epidermis and sebaceous glands express cytochrome P450 (CYP) enzymes (capable of metabolizing drugs and chemicals), some of which are specific to skin epithelial cells. Especially relevant to skin is the role of CYPs in mediating photosensitivity, and reactions to drugs, such as sulfonamides, tetracyclines, retinoids. The observed pharmacogenetic polymorphisms in members of the CYP family explain inter-individual responses to drug therapy and determine individual absorption, disposition, metabolism, and excretion of xenobiotics.

Keratinocyte differentiation can be modulated by hormones and vitamins (retinoic acid, vitamin A/retinol from diet), vitamin D3, thyroid hormone and steroid hormones. These act as ligands of cell receptors localized in cell nuclei (nuclear receptors). Nuclear receptors influence the expression of multiple genes involved in cell differentiation. The skin has nuclear receptors for several steroid hormones, including glucocorticoids, estrogen, androgen and progesterone, and can produce active sex steroids through metabolism of adrenal C19 precursor steroids.

Defensins and other antimicrobial peptides are produced by keratinocytes of inflamed epidermis following proinflammatory cytokine release (IL-1) or bacterial infection.

Biochemistry of the dermis

The main cell type of the dermis is the fibroblast that produces and degrades extracellular matrix (ECM) components. Several other cell types are hosted in the dermis, including multi-functional cells of the immune system (macrophages and mast cells), whereas mast cells can trigger allergic reactions by secreting bioactive mediators such as histamine.

Dermal functionality is mediated through the ECM, which is a highly complex mixture of bioactive macromolecules produced by dermal cells and then either secreted intact or alternatively assembled later outside the cells. Collagens are the principal ECM component; other members include elastin, fibrillins, latent TGF-β-binding proteins, fibulins, laminins, proteoglycans, integrins, and the enzymes involved in their processing. ECM mutations can result in a broad range of human disorders.

Collagen: About 90% of total dermal protein consists of the collagen macromolecule, accounting for about 75% of skin total dry weight. At least 25 collagens exist, half are present in skin; consisting predominantly of collagen type I (85-90%), III (8-11%), and V (2-4%). Collagen type VII forms part of the so-called anchoring fibrils that attach the basement membrane to the ECM of the upper dermis. Collagen type XVII contributes to the so-called anchoring filaments that link the basal keratinocytes with the basement membrane. Collagen provides tissue integrity and facilitates tissue morphogenesis and platelet aggregation. Most skin collagens are dermal cell-derived but endothelial cells of dermal blood vessels also produce epidermal collagens.

Collagens share a 3 α-polypeptide (homotrimers or heterotrimers) chain format folded into a collagen triple helix. Every third amino acid in the chain is a glycine and the other two positions are over-represented by prolines or hyroxyprolines, with hydrogen bonding between hydroxyl groups on adjacent chains contributing to helix stability. Collagen biosynthesis involves the synthesis of procollagen polypeptides followed by both their intracellular and extracellular processing to generate a mature and functional collagen fibre. Procollagen molecules are aggregated into granules for secretion purposes from the cells. A battery of enzymes is involved in collagen biosynthesis and these require a diverse range of cofactors including O2, Fe2+ and ascorbate. Certain genetic syndromes are due to lacking cleavage at either or both procollagen polypeptide ends.

Elasticity of skin: The other major ECM fibrous protein is elastin, which provides the skin with its elasticity. Elastin is stretchable by more than its full resting length. There are three different classes of elastin fibers: Oxytalan fibers, elaunin fibers and elastic fibers. The elasticity of the fibers derive from the amorphous portion, which contains the highly cross-linked protein called elastin. Elastin molecules form cross-links that confer both elasticity and insolubility to them. The microfibrillar part of the elastic fiber is composed of fibrillin-1.

Extrafibrillar matrix: Extrafibrillar matrix, the dermal material lying outside the cells, not consisting of either collagen or elastic fibers, is an amorphous and hydroscopic material consisting of proteoglycans, glycoproteins, water and hyaluronic acid. Proteoglycans are formed by the binding of negatively charged, water/ion-binding and sulfated/ acetylated polysaccharides called glycosaminoglycans (GAGs) to a protein. Hyaluronic acid is protein-free GAG.

Biochemistry of Adnexal Structures

Eccrine sweat glands: They produce a watery perspiration that serves principally to cool and maintain the body core temperature at 37.5°C. At maximal output the eccrine glands of an adult human can excrete up to 3 litres sweat per hour. Osmotic force appears enough to move the sweat up to the skin surface. Eccrine gland activity is regulated via neural stimulation using sympathetic nerve fibres distributed around the gland. These use the neurotransmitter acetylcholine. Sweat is a clear, odour-free, colorless, slightly acidic fluid that is almost fully water (99.0-99.5%) with the remainder consisting of the electrolytes NaCl, K+ and HCO3−, as well as lactate, urea, ammonia, calcium, heavy metals. There are significant regional variations in the composition of sweat. The composition of sweat can be modulated by psycho-emotional and environmental factors. The sweat gland is also known to concentrate xenobiotics. Organophosphorus compounds (in insecticides and chemical warfare agents) can induce eccrine gland sweating. Raised NaCl levels in sweat are a reliable diagnostic feature of cystic fibrosis.

Sweat forms initially as an isotonic primary fluid in the secretory tubule that then passes through the duct for NaCl resorption, to yield a final hypotonic sweat. The steroid aldosterone is thought to regulate the reabsorption of Na+, as sweating is increased in Addison disease where aldosterone is reduced.

Apocrine sweat glands: They secrete their sterile, odour-free, weakly acidic product via exocytosis from secretory cells. Its viscous, milky consistency is due to its high content of lipids (fatty acids, cholesterol, squalene, triglycerides), androgens, ammonia, sugars, Fe2+. Some of these are odoriferous, especially so after their decomposition on the skin surface by bacteria. Human apocrine glands are not responsive to heat, although psycho-emotional stimuli are implicated in stimulating secretory activity.

Sebaceous glands: Their product, sebum, is released after sebocytes active DNA degradation burst and die (holocrine secretion) due to programmed cell death. There is a remarkable variation in the composition of sebum through human life and human sebum is unique, with no similarity to other species. Human sebaceous glands not only produce sebum but also regulate steroidogenesis, local androgen synthesis, skin barrier function, interaction with neuropeptides, potential production of both anti- and pro-inflammatory compounds and synthesis of anti-microbial lipids (sapienic acid, oleic acid).

The sebaceous gland contains all enzymes needed for transformation of cholesterol to steroids. Moreover, it expresses 5α-reductase isoenzymes, needed for the intracellular conversion of testosterone, or even the adrenal androgen dehydroepiandrosterone directly, to the more potent 5α-dihydrotestosterone. Sebaceous glands are part of the skin neuroendocrine system as they produce and release corticotropin- releasing hormone in response to systemic or local stress. Exogenous glutamine is required for sebocyte division and lipogenesis, though it can be replaced by spermidine.

Sebum is a yellowish viscous fluid containing lipids, such as triglycerides, free sterols and sterol esters, squalene, wax and free fatty acids, and cell debris. Sebaceous glands can synthesize considerable amounts of free fatty acids. Approximately half of the fatty acids in sebum are monosaturated with some unusually positioned double bonds (Δ6 unsaturation) unique to humans. Sebum is the only body secretion with a high squalene content, a lipid that cannot be crystalized. The composition of sebaceous gland lipids, including the relative proportions of different types of branched-chain fatty acids, is under both genetic and hormonal control and while significant inter-individual and inter-ethnic differences in sebum production exist, a common secretion rate of 0.3 mg sebum/10 cm3/h is detected.

Hair follicle: Hair fiber growth occurs in a highly time-resolved manner. Glucose (and glutamine) is the principal energy source. The hair follicle can react to most hormones of the human body and produces for itself a wide range of hormones, such as sex steroid hormones, proopiomelanocortin peptides, corticotropin-releasing hormone, prolactin. Neuropeptides, neurotransmitters and neurohormones are implicated in mediating hair follicle events particularly those related to stress. The observation that men castrated before puberty do not go bald nor grow beards, but they do so after treatment with testosterone, indicated a major role of androgens in hair growth. However, hair follicles in different regions of the body respond differently to different androgens. Drug-induced inhibition of the testosterone-metabolizing enzyme to 5α-dihydrotestosterone, 5α-reductase type II, induces hair regrowth in some balding men.

The hair fiber includes “hard” K, water, lipids, pigment, and trace elements (in order of decreasing amount). The biosynthesis of hair proteins begins in the bulb of the growing (anagen) hair follicle and it ceases approximately 500 µm above the zone of maximal keratinocyte proliferation in scalp terminal hair follicles. Neither the chemical composition of hair proteins nor its amino acid composition shows any difference across the ethnicities.

The “hard” K of the hair can be distinguished from epidermal “soft” K by their lack of extended glycine runs. “Hard” hair K contain many cysteine residues. Moreover, the dynamics of KIF assembly in epidermis and hair fibers are very different. Whereas these filaments disassemble during epidermal keratinocyte division, they are the product of non-viable K-producing cortical keratinocytes in the hair follicle. Abundant cysteine residues of the hair K are extensively cross-linked by disulfide bonds facilitated by more than 20 different K-associated proteins.

Hair fibers have a coating of long-chain fatty acids bonded covalently to the protein membrane of the epicuticle. Hair lipids contain a large amount (58%) of a hair-specific methyl-branched saturated 21-carbon fatty acid (18-methyl-eicosanoic acid) and provide a hydrophobic interface protecting the hair cortex from a hostile wet/dry environment. In humans the major fatty acids in hair fiber lipids include 16:0 (17%), 18:0 (10%), 18:1 (5%) 21:0 (48%). Hair fiber lipids are highly conserved, in marked contrast to the high inter-species variability in sebaceous gland lipids.

Nails: Corneocytes of the nail plate (onychocytes) are also filled with K filaments. Low-sulfur K form filaments running parallel to the nail surface and these are embedded in a non-K matrix material of high sulfur, high glycine, high tyrosine proteins. Up to 90% of nail K are of the hard “hair” K type. Water, lipids and trace elements including iron, zinc, and calcium are present in the nail plate. Overall, the nail contains much less lipid and water than the epidermis and becomes brittle at less than 7% water and soft at more than 30%. The nail low lipid content contributes to their 1.000-fold greater water permeability than the epidermis

Vitamin D production: Vitamin D3 (cholecalciferol) belongs to the group of fat-soluble secosteroids, which has an important impact in the intestinal absorption of calcium, magnesium, and phosphate. Besides oral ingestion it is photochemically in the epidermis through UVB radiation. Through radiation with UVB (290-315 nm) (sun exposure to face, arms and legs for 30 minutes twice per week, or approximately 25% Minimal Erythema Dose (MED)), 7-dehydrocholesterol is photochemically transformed into cholecalciferol (vitamin D3).

Steroidogenesis and neurohormones: Skin and its appendages respond to central hormone regulation but also act as an endocrine organ in a manner largely independent of body central control systems. The skin occupies a strategic location between the external and internal environments and so it can contribute to preserving body homeostasis. In addition, the cutaneous neuroendocrine system provides a capacity for stress sensing, been an equivalent of the central hypothalamic–pituitary–adrenal axis. The latter principal constituent activities include the production of corticotropin-releasing hormone and downstream proopiomelanocortin peptides, including endorphins and steroidogenesis (synthesis, metabolism and targeting of androgens and estrogens).

Pigmentation of skin and hair follicle: Despite the variations in skin and hair color, skin and hair pigmentation is derived from the pigment melanin, synthesized via a phylogenetically ancient biochemical process termed melanogenesis. Synthesis occurs within melanosomes, which are specialized organelles unique to highly dendritic, neural crest-derived, cells called melanocytes. Follicular melanocytes are derived from epidermal melanocytes during hair follicle development. Activity of the hair bulb melanocytes only occurs during the anagen phase of the hair growth cycle, that of epidermal melanogenesis is continuous.

Melanogenesis starts by the formation of the melanosome in which melanogenesis occurs and continues through the biochemical pathway that converts L-tyrosine into melanin. Both processes are under complex genetic control. Eumelanogenesis (brown-black melanin production) is critically dependent on the velocity of the tyrosinase reaction. For pheomelanogenesis (red/yellow melanin production), cysteinyl-DOPA is oxidized in multiple complex steps that may involve tyrosinase-dependent or independent reactions as well as glutathione reductase and peroxidase activities, in order to form pheomelanin. Mixed melanins (containing both eumelanin and pheomelanins) and neuromelanin are additional melanin types.

Both eu- and pheomelanin can be produced within the same melanocyte. Pheomelanins are photo- labile with photolysis products including superoxide, hydroxyl radicals and hydrogen peroxide.

The biosynthesis of melanins is based on the conversion of the amino acid L-tyrosine (additionally via L-phenylalanine) into a complex and heterogeneous group of compounds. This reaction follows three steps:

1. Hydroxylation of L-phenylalanine/L-tyrosine to L-dihydroxy-phenylalanine (L-DOPA);

2. The dehydrogenation/oxidation of L-DOPA to dopaquinone, which is the precursor for both eu/ pheomelanins and the limiting step in melanogenesis;

3. The dehydrogenation of dihydroxyindole to yield melanin pigment. Both eumelanogenesis and pheomelanogenesis require the oxidation of DOPA to dopaquinone. Thereafter, the conversion of dopaquinone to leuko-dopachrome signals eumelanin production, while the addition of cysteine to dopaquinone to yield cysteinyl-DOPA occurs in pheomelanin production. For eumelanogenesis (brown/black), L-DOPA needs to be oxidized by tyrosinase to L-dopaquinone and again by tyrosinase from dihydroxyindole to indole-5,6-quinone.

Despite the broad association of eumelanin with brown/black hair and pheomelanins with red/blonde hair, relatively minor differences in melanin content can have significant effects on visible hair color. Melanin in hair is tightly bound to the keratin and its isolation requires degradation of keratin by strong chemicals (NaOH, HCl).

Eumelanins are polymorphous nitrogenous biopolymers (mostly co-polymers of dihyroxyindole and dihyroxyindole carboxylic acid). They are insoluble in most solvents and are tightly associated with proteins through covalent bonds. Eumelanins exhibits unique stable paramagnetic states. Unlike eumelanin, pheomelanins are photo-labile with photolysis products including superoxide, hydroxyl radicals and hydrogen peroxide.