CELL MEMBRANE COMPLEX 441 stratum corneum. As early as 1945, Weitkamp (20) reported 18-MEA in wool wax (de- gras). In 1985, Evans et al. (21) demonstrated that 18-MEA is covalently bonded to the keratin fi ber surface by reacting wool fi ber with anhydrous alkali after solvent-extractable lipids have been removed. The cleavage of 18-MEA with chlorine water by Negri et al. (12) and by hydroxyl amine at neutral pH by Evans and Lanczki (13) support the attach- ment by a thioester linkage rather than an ester or amide linkage, although there is evi- dence for ester and/or amide attachment of some fatty acids (primarily palmitic, stearic, oleic, and others), mainly in the lower beta layer in cuticle–cuticle CMC by Evans and Lanczki (13) and by Korner and Wortmann (22). Essentially all of the MEA is in the upper beta layer of the cuticle–cuticle CMC, as dem- onstrated in a paper by Jones et al. (23). Maple syrup urine disease (MSUD) is a genetic defect in humans and Poll Herford cattle (24) involving 18-MEA. MSUD is caused by a defi ciency in an enzyme involved in the complicated synthesis of 18-MEA. Isoleucine serves as a precursor in the biosynthesis of 18-MEA via the branched chain 2-oxo acid dehydrogenase, which is the enzyme that is defi cient in this genetic defect (10). Jones and Rivett in their TEM studies of MSUD (10,23) found that the structural defect of MSUD in human hair occurs only on the upper surface of cuticle cells (upper beta layer), where 18-MEA is replaced by straight chain C18 and C20 fatty acids, and that the undersides of cuticle cells (lower beta layer) are not affected. These facts confi rm that 18-MEA is at- tached to the top surface of cuticle cells (upper beta layer) and not to the underside. Only a few years after the discovery by Evans et al. (21) that 18-MEA is covalently bound to the keratin fi ber surface, Negri et al. (12) proposed a model for the keratin fi ber surface consisting of a monolayer of 18-MEA covalently bonded to an ultra-high sulfur protein through a thioester linkage at approximately 1-nm spacings, and they suggested that the protein support was in the beta confi guration and that it might be attached to the All- worden membrane. Although widely varying estimates of the thickness of the epicuticle have been made from 5 to 14 nm, one of the more recent and perhaps reliable estimates is by Swift and Smith (25), who examined wool fi ber, human hair, and several other mam- malian hairs using high-resolution TEM and identifi ed that the epicuticle is approxi- mately 13-nm thick and is rich in cystine, and that thioester-bound lipids might be present within its bulk. Swift’s estimate of the epicuticle thickness is consistent with the maximum thickness reported by several other workers (26–28). Leeder and Bradbury (18,29) discovered that the Allworden reaction takes place with isolated cuticle cells from several different animal hairs including wool and human hair fi ber, proving that this proteinaceous membrane material completely surrounds each cu- ticle cell and is not a continuous external membrane on hair fi bers. In this important scientifi c effort, cuticle cells were isolated by shaking fi bers in formic acid and then ex- posing the isolated cells to chlorine water. Formic acid is known to attack and to solubi- lize some proteins believed to be largely from the delta layer of the cell membrane complex, and its effects will be discussed later in the section entitled “Proteins of the CMC.” In the intact fi ber Allworden sacs form over the top of cuticle cells (the exposed surface). Leeder and Bradbury suggested that “the sac always occurs on only one side of the cuticle cell,” i.e., the top of cuticle cells and not the bottom (15,18,29). They ex- plained that this effect occurs because the connecting bonds on the top of cuticle cells are between the epicuticle and the A-layer and therefore are most likely through disul- fi de crosslinks that are vulnerable to chlorine water oxidation (15). Furthermore, they suggested that the connecting bonds on the underside of cuticle cells are between the

JOURNAL OF COSMETIC SCIENCE 442 membrane and the endocuticle (actually the inner layer, a layer about 10- to 40-nm thick (7) between the endocuticle and the cell membrane and similar in composition to the exocuticle (see Figure 6), through bonding that is resistant to chlorine water oxidation (15) (possibly isopeptide linkages). Negri et al. (12) have shown that the Allworden reaction is an effect of the membranous proteins around cuticle cells and that 18-MEA is not required for the formation of All- worden sacs because the sacs can be produced from cuticle in which 18-MEA has been removed by prior treatment with either methanolic KOH or potassium t-butoxide in t- butanol. Because of the bulky nature of the t-butoxide anion, it removes only covalently bound fatty acid at or near the fi ber surface. Furthermore, Negri et al. (12) have shown that removal of the covalently bound fatty acid facilitates the formation of Allworden sacs because the rate of formation of the sacs increases with prior removal of the covalently bound 18-MEA. The separation and analysis of the Allworden membrane by Allen et al. (30), together with the work of Zahn et al. (31), provided indirect evidence for the cuticle cell mem- brane containing an ultra-high sulfur protein, loricrin, and involucrin. This important contribution will be described in more detail later in this paper in the section entitled “Proteins of the CMC.” Bilayers versus monolayers in the cuticle–cuticle CMC. Whether or not the covalently bound lipids of the cuticle–cuticle CMC are bonded to another lipid layer on their hydrophobic end, forming a bilayer, or they are bonded to a hydrophobic protein in the delta layer is still debated, but this author believes the evidence clearly favors the monolayer model (4,7) for the following reasons: O If the beta layers are monolayers, then 18-MEA is linked to the delta layer through short hydrophobic bonds, making the upper beta layer susceptible to failure at the delta layer where it has been shown to occur (4,7,32,33). O Swift (7) has pointed out that a monolayer model fi ts better from the point of view of CMC measurements. O If bilayers exist (a schematic of the bilayer model is shown in reference 4), then there are two options for bonding of the second fatty acid layer to the delta layer. One op- tion is for fatty acids to be covalently bonded to the delta layer, but this option is not plausible because in human hair and wool fi ber 40% to 50% of the covalently bonded fatty acids are 18-MEA (34–36) therefore, there are insuffi cient covalently bound fatty acids in human hair and wool fi ber to account for this type of bonding. The other option is bonding of the second layer of fatty acids through hydrophobic link- ages to the covalently bound fatty acids and bonding to the delta layer through polar attachments and ionic bonding. However, this type of bonding would provide beta– beta failure, not beta–delta failure, and it would allow for solvent removal of the non-covalently bound lipid layer by chloroform/methanol extraction, which has been shown to occur in cortex–cortex CMC but not in cuticle–cuticle CMC, which is more resistant to this type of solvent system (37,38). To provide beta–delta failure from this bilayer model, the new hair surface would form a bilayer consisting pri- marily of hydrophilic acid groups at the very surface, and so this bilayer model is also not plausible. O Negri et al. (39) noted that formic acid removes proteins more readily from the cortex– cortex CMC and that it modifi es CMC junctions of the cortex more than those of the