JOURNAL OF COSMETIC SCIENCE 448 THE FORMATION OF THE CMC IN DEVELOPING HAIRS The following description of the formation of the CMC in the developing hair fi ber was taken from the work of Rogers (53), and also from the early work by Orwin and cowork- ers (54), with more recent work by Jones and coworkers (55). For more details of the formation of the CMC in developing hair fi bers, I recommend the review by Jones and Rivett (11) and the recent paper by Jones et al. (55). In the latter stages of development of the hair fi ber, desmosomes or intercellular bridges, gap junctions (where cells exchange molecules), and tight junctions (intercellular junc- tions where cell membranes fuse) are established between differentiating keratinocytes of the hair fi ber and the inner root sheath to varying extents as they move upward in the hair follicle (see Figure 8). Orwin et al. (54) observed that gap junctions and desmosomes cover about 10% of the plasma membrane of cortical cells in the bulb region and then gradually degenerate. Tight junctions are established between Henle’s outermost layer of the inner root sheath and Huxley’s layer of the inner root sheath and between Henle cells and the close com- panion layer of the outer root sheath (see Figure 8). These junctions are replaced with a new cell membrane complex that gradually develops as a continuous complex between the cells. Similar events should occur for cuticle–cuticle CMC, cuticle–cortex CMC, and cortex–cortex CMC, with appropriate distinctions to allow for the differences that arise between these three different cell-connecting units. LIPIDS OF THE CMC OF KERATIN FIBERS METHODS TO REMOVE LIPIDS FROM ANIMAL HAIRS FOR ANALYSIS Removal of external lipids. Wool fi bers are normally processed by scouring with a nonionic agent such as Lissapol TN 450 (a nonylphenol ethoxylate with an average of 8.5 ethoxy units CTFA = nonoxynol-8.5) and then in scientifi c studies treated with one or more solvents to remove any remaining external lipids. Non-swelling solvents and/or solvents of bulky molecules (like t-butyl alcohol) have been used to remove external lipids from keratin fi bers, that is, lipids that are believed to be soil and not part of the internal structure of animal hairs. Solvents such as hexane, t-butyl alcohol, or heptane, and sometimes Figure 8. Schematic of the different layers of an active hair follicle (not drawn to scale).

CELL MEMBRANE COMPLEX 449 t-butanol and heptane sequentially (36,56) have been used to extract “external lipids” such as wool wax or sebaceous matter from animal hairs. Such lipids are sometimes called ex- ternal, extrinsic, or even exogenous (57) and are primarily sebaceous in origin in humans. A large percentage of these lipids are not believed to be involved in the intercellular structure of animal hairs, but there is evidence (58) that some of this solvent-extractable lipid may be part of the structure of the surface lipids of hair fi bers. In the case of human hair, external lipids are normally removed by shampoo or sodium lauryl sulfate washing, by a combination of shampoo followed by incubation in hexane for only fi ve minutes (57), or in some cases by other solvents that most likely do not remove substantial amounts of internal hair lipids (e.g, ether or heptane). Removal of internal lipids not covalently bound to hair. Hair-swelling organic solvents alone or in combination with a second lipid solvent are used to remove internal lipids that are part of the internal structure of hair fi bers (of the CMC) but not covalently bonded to the hair protein structures. Solvents such as chloroform/methanol (44,56), methanol (52), ethanol (6), formic acid (50), n-propanol/water (50), or acetone (52) have been used to extract internal matter from animal hairs. The most frequently used solvent for removal of inter- nal lipids has been chloroform/methanol (70/30), although other mixtures have been used. Normally soxhlet extraction is employed however, multiple room temperature extractions have also been employed (57). Although formic acid and n-propanol/water (generally 1:1) do remove some internal lipids, these two solvents also remove some hair proteins of the CMC (most likely from the delta layers and possibly from other regions of the fi bers), as will be described in the section entitled “Proteins of the CMC.” Removal of covalently bound hair lipids plus salts insoluble in lipid solvents. Alkaline hydrolysis or methanolic alkali is used to remove covalently bound hair lipids. This technique can be used to remove total hair lipids, but it is generally used after extraction of external and internal lipids that are not covalently bound to the fi bers. Those lipids that are covalently bound at or near the fi ber surface in the cuticle are generally removed with potassium t- butoxide in t-butanol (bulky cleaving agent in a bulky solvent) (59), and total covalently bound lipids are generally removed with potassium hydroxide in methanol because alkali in a swelling solvent like methanol penetrates well into hair. In addition to covalently bound lipids, Wertz and Downing (34) have suggested that salts of cholesterol sulfate bind ionically to cationic groups on the hair proteins and will be insoluble in chloroform/methanol and therefore will remain in the fi brous residue after extraction with organic solvents. Korner et al. (48) used a solution of chloroform/metha- nol/aqueous potassium chloride to extract CMC lipids from wool and human hair and formed liposomes from the extracts. Since cholesterol sulfate is essential for liposome for- mation of keratin fi ber extracts, this solvent system must extract cholesterol sulfate too. TOTAL LIPIDS IN HAIR FIBERS The total amount of lipid extractable from hair is generally 1% to 9% of the weight of the hair (57,60). Masukawa et al. (57) have provided a relatively thorough analysis of hu- man hair lipids. In their study, the total hair lipid composition from 44 Japanese women, ages 1 to 81, was examined. The lipids were extracted/removed from hair in varying pro- cedures, to allow for analysis of several lipids and covalently bound 18-MEA. In this study, total fatty acids and 18-MEA were determined, but other important fatty acids,