The role of 18-methyleicosanoic acid in the structure and formation of mammalian hair fibres

Micron Vol. 28. No. 6, pp. 469 485, 1997

Pergamon PII: S0968-4328(97)00039-5

1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0968 4328/97 SI 7.00+0.00

REVIEW PAPERS

The Role of 18-Methyleicosanoic Acid in the Structure and Formation of Mammalian Hair Fibres LESLIE N. JONES*~ and D O N A L D E. R1VETTq" *CSIRO Division of Wool Technology, P.O. Box 21, Belmont 3216, Australia t C S I R O Division of Biomolecular Engineering, 343 Royal Parade, Parkville 3052, Australia (Received 15 March 1997; accepted 10 June 1997)

Abstract- Although branched chain fatty acids perform many functions in biological systems, the importance of the anteiso 18 methyleicosanoic acid (MEA) has only recently been recognized. In this first review on MEA its role and distribution is ~xplored. MEA has been found in minor amounts in the fatty acid components of a wide range of biological materials, but th~ current interest results from it being the major covalently bound fatty acid in mammalian hair fibres, a finding which is unusual I because protein-bound fatty acids are typically straight-chain, even-numbered acids (C14 C18). MEA is released by surface ~estricted reagents indicating that it is located exclusively in or on the surface of the cuticle cells, a conclusion that has been ve)-ified by analysis of isolated cuticle cells. X-ray photoelectron spectroscopy. (XPS) and ,secondary-ion. mass spectroscopy (SIMS).[ studies support these results in that they show the surface of the cuticle to be predominantly hydrocarbon. When elthe~ neutral hydroxylamine or acidic chlorine solutions are applied to hair and wool fibres fatty acids are liberated, indicating the pr#sence of thioester bonds. Calculations, based on fatty acid and amino acid analysis, indicate that approximately one residue in i0 of the cuticular membrane protein is a fatty acid thioester of cysteine. Removal of this covatently linked fatty acid renders the fibre hydrophilic, thus offering a chemical explanation for many technological and cosmetic treatments of mammalia~ fibres. Examination of the fibre surface and that of isolated cuticle cells by transmission electron microscopy (TEM) confirms thepresence of a thin non-staining continuous layer surrounding the cuticle cells. Alkaline treatments which remove the bound fatty a~ids were found to disrupt this layer. TEM examination of developing hair fibres has indicated that the fatty acid layer on the uppe~ surface and scale edges of the cuticle cell differs from that o f the underside of the cell. Similar structural studies of hair from pationts with maple syrup urine disease (MSUD) support the findings that thioester-bound MEA is limited to the upper surface of fibre cuticle cells. The current model proposed for the boundary layer consists of crosslinked protein with surface thioester-linked fatly acids, forming a continuous hydrophobic layer on the upper surface and scale edges of the cells. ~: 1997 Elsevier Science Ltd. All rights reserved

Key words: branched chain fatty acid, fibre cuticle surface membrane, hydrophobic monolayer, 18 methyleicosanoic acid.

CONTENTS I.

ll.

III.

Distribution and chemistr). ..................................................................................................................................................;............... 469 A. Early observations .......................................................................................................................................................................... 469 B. MEA in animal fibres ..................................................................................................................................................................... 470 C. Mode of attachment of the bound fatty acids ................................................................................................................................ 471 D. Biosynthesis of anteiso fatty acids .................................................................................................................................................. 473 E. Physical measurements of bound lipids on keratin fibres ............................................................................................................... 473 Involvement in fine structure ................................................................................................................................................................ 474 A. Structure of the mammalian hair fibre ........................................................................................................................................... 474 B. Fibre cuticle .................................................................................................................................................................................... 474 C. Resistant membranes ....................................................................................................................................................................... 474 D. Proteinaceous resistant barriers ....................................................................................................................................................... 476 E. Fibre cortex ....................................................................................................................................................................................... 476 F. Intercellular regions (cell membrane complex) ............................................................................................................................... 477 G. The mammalian hair follicle ............................................................................................................................................................ 478 H. Formation of fibre-cuticle surface membranes ................................................................................................................................ 478 I. Mutation involving MEA ................................................................................................................................................................ 479 Conclusions ........................................................................................................................................................................................... 482 Acknowledgements ................................................................................................................................................................................ 482 References ............................................................................................................................................................................................. 482

Weitkamp (1945). MEA was found to be a significant component of the fatty acids found in an hydrolysate of wool wax, representing 5.6% of the total welght of fatty acids or approximately 2.6% of the original wax. The racemic form of MEA was obtained synthetically by Nunn (1951) and was reported as colouriess needles having a melting point of 57.2-57.6°C. In ai later study

I. DISTRIBUTION AND CHEMISTRY

A. Earl)' observations The anteiso fatty acid (+)-18-methyleicosanoic acid (MEA) was first reported as a component of wool wax, ++Corresponding

author, 469

L. N. Jones and D. E. Rivett

470

Table 1. Major fatty acids in the bound lipids of wool

Fatty acid 16:0 18:0 18:1 C21a(MEA) Total fatty acid:

Logan et al. (1990) b

Amount of fatty acid (mg/g fibre)" Negri et al. (1991) c Wertz and Downing (1989) b

0.11 (11%) ~ 0.12 (12%) 0.08 (8%) 0.43 (43%) 1.0

0.10 (8%) 0.08 (6%) 0.06 (5%) 0.93 (72%) 1.3

0.62 (17%) 0.37 (10%) 0.19 (5%) 1.71 (48%) 3.6

aBound fatty acids liberated from solvent extracted wool with alcoholic alkali treatment. bDetails of wool used not given. CFrom 16pm Australian Merino wool. UValues in parenthesis refer to percentage composition of fatty acids by weight.

Downing et al. (1960) confirmed the presence of MEA in wool wax using gas chromatography and reported a yield of 5.8-6.1% (weight) of the fatty acid component. The analysis of wool wax acids was reviewed by Motiuk (1979), where the presence of MEA was again reported. Crabtree and Truter (1974) showed that the bulk of the MEA was present in the wool wax as cholesterol and lanosterol esters. MEA has also been found as a significant component in the extractable lipids of wool (see Table 1). It is further present in trace amounts in lipids from other biological sources, including vernix caseosa (Downing, 1963; Karkkainen et al., 1965; Nicolaides et al., 1976), mouse epidermis (Wilkinson and Karasek, 1966), meibomian glands (Harvey and Tiffany, 1984), mole skin surface (Downing and Stewart, 1987), and bacteria (O'Donnell et al., 1985). It is possible that the significance of MEA would have been ignored but for a paper presented at the 7th International Wool Conference held in Tokyo, 1985. Evans et al. (1985) demonstrated that MEA was the major component of a group of bound lipids released from the surface of wool fibres by an anhydrous solution of potassium t-butoxide.

B. M E A in animal fibres

The concept of bound lipids in mammalian hair fibres was put forward as early as 1952, when Elliot and Manogue (1952) proposed that sterols were linked through thiol groups to wool fibres. K6pke and Nilssen (1960) first postulated the presence of bound fatty acids on the surface of wool fibres, and the later work of Leeder and Rippon (1985) supported their hypothesis. Currey and Golding (1971) also suggested the possibility that bound fatty acids were present on the surface of human hair fibres. The partial liberation, detection and analysis of bound lipids was first performed by Evans et al. (1985), using anhydrous alkali (potassium t-butoxide in t-butanol) as a surface treatment for wool fibres. Analysis of the extract revealed that it was composed predominantly of free fatty acids, with an unusual methyl-branched 21carbon fatty acid (MEA) being the major component (58% of total fatty acids). This fatty acid was subsequently reported to be liberated from wool fabrics

when they were treated with sulphite in aqueous/organic solvents (Erra et al., 1986). Hairs from most mammalian species were subsequently analysed by Wertz and Downing (1988, 1989) and Peet et al. (1992), with the resulting conclusion that this unusual branched chain fatty acid was indeed diverse in nature. The presence of a hydrophobic surface on wool fibres, that was rapidly removed with alkali, was demonstrated using wettability tests, and the presence of a bound fatty layer, termed the F-layer, was postulated by Leeder and Rippon (1985); Leeder et al. (1985); Leeder (1986). It is interesting to note that a shrinkproofing process for wool which was developed by Freney and Lipson (1940) relied on treating the wool with a solution of sodium hydroxide in alcohol. These authors provided no evidence of how their process worked, but it almost certainly removed the bound lipid layer from the cuticle surface. Further investigation confirmed the presence of a class of lipids in wool fibres that were resistant to extraction with lipid solvents, but were liberated by mild alcoholic alkali treatments. Total alkali digests of wool, pretreated to remove all extractable lipids, liberated 0.8-1.3 mg of lipid per gram of fibre, composed predominantly of fatty acids (Rivett et al., 1987; Logan et al., 1989; Kalkbrenner et al., 1990; Negri et al., 1991). The composition of the fatty acids is given in Table 1, with a 21-carbon branched chain fatty acid comprising about 50% by weight of the total bound fatty acids. In analyses using mass spectrometry and nuclear magnetic resonance spectroscopy the fatty acid was conclusively identified as 18-methyleicosanoic acid (Wertz and Downing, 1988, 1989; Negri et al., 1991). Convincing evidence that the ester-linked fatty acids were predominantly and maybe exclusively confined to the cuticle was provided by studies of covalently bound fatty acids from isolated cuticle cells of fibres (Kalkbrenner et al., 1990; Peet, 1994). Wertz and Downing's estimation of the amount of bound lipid in wool fibres was significantly higher than any other recorded values (3.6 mg per gram of wool), with cholesterol sulphate, cholesterol and ceramides also being significant components (Wertz and Downing, 1989). The absolute amount and composition of the bound fatty acids were tbund to be dependent on the type of fibre and the fibre diameter, with an increase in fibre diameter leading to a decrease in total bound fatty

18-Methyleicosanoic Acid in Hair

471

(a) 18 - methyleicosanoic acid

19 17 20~~VVVVV 21

0.8

0.6

/0 V ~OH 2.39nm

,•

(b) stearic acid

"~ 0.4 (0 "(3 ¢-

2.14nm

(c) palmitic acid

0.2

?

0

OH 14

18

22

26

30

Fibre d i a m e t e r

34

38

42

(FLm)

Fig. I. Amount of MEA/g of wool compared with the diameter of the fibre. The curve indicates the theoretical amount of bound MEA required to cover the surface of the cuticle cells.

acids (Negri et al., 1993a) (Fig. 1), highly suggestive of a surface phenomenon. An interesting observation was that with fine fibres, more MEA is covalently bound than that theoretically required to form a monolayer on the surface. In fine (16#m) Australian Merino wool fibres, the relative proportion of MEA is approximately 70% by weight of the bound fatty acids. Negri (1993) also reported that as the fibre diameter increases (for a single-cuticle fibre) the amount of bound MEA as a proportion of total bound fatty acids decreases, approximately in a linear relationship, to 55% by weight for a 35/~m fibre. Branched-chain fatty acids are common in biological systems, although they are usually present as minor components in a mixture of straight-chain saturated and unsaturated fatty acids (Abrahamsson et al.~ 1963; Ahern and Downing, 1973; Dasgupta et al., 1984; Garton. 1985; Valero-Guillen et al., 1987). The main exception is sebaceous lipids which typically contain predominently branched chain fatty acids, however the exceptionally high proportion of a single branched-chain fatty acid, as found in the surface membrane, is unusual. The function of the MEA is uncertain, but considering its unusual occurrence together with the additional energy required by the cell to produce such a unique branched-chain rather than an abundant straight-chain fatty acid (e.g. palmitic acid), a specific role for this fatty acid is expected. In Fig. 2 the structures of MEA, stearic and palmitic acids are compared.

C. M o d e o / a t t a c h m e n t o f the bound f a t t y acids'

The resistance to extraction by lipid solvents and the rapid liberation by alkali of the bound fatty acids strongly suggests that they are covalently bound to some macromolecular structure in the fibre. Fatty acids and other lipids are commonly bound to protein and other

H

1.89nm

~•

Fig. 2. The structure and approximate length of 18methyleicosanoic acid (a) compared with that of the more usual membrane fatty acids, stearic acid (b) and palrnitic acid (c). Calculations of molecular lengths are based on the atomic spacings as quoted by Wong (1991). Only the carbon/carbon spacings are taken into account. The carbon/stdphur bond length is not included.

macromolecules in living and keratinized cells by oxygen ester, thioester, thioether or amide bonds, including palmitoylation (Schultz et al., 1988: Grand, 1989), myristoylation (Schultz et al., 1988: Grand, 1989), prenylation (Giannakouros and Magee, 1993), glypiation (Low, 1989) and glycolipid or other ceramide linkages (Downing, 1992). On the basis of the rapid liberation by anhydrous alkali, it has been postul'ated that the fatty acids in wool fibres are linked directly to protein via an oxygen ester or thioester bond, analogous to palmitoylation (Evans et al., 1985: Wertz and Downing, 1988; Logan et al., 1990; Negri et al., 1991!, 1992). Palmitoylation is the covalent attachment of fatty acids, predominantly palmitate (with 10w levels of stearate and oleate also common) directly to proteins (Schultz et al., 1988; Towler et al., 11988; Grand, 1989: Schmidt, 1989; McIlhinney, 1990i Schlesinger et al., 1993). The fatty acids are linked through a thioester bond to cysteine residues, such as i the cysteinebound palmitate on the insulin receptor (Omary and Towbridge, 1981), or less commonly through an oxygen ester bond to serine or threonine residues such as the serine-bound palmitate in the gp37 protein of Rous sarcoma virus (Gebhardt et al.. 1984). Aithough both thioester and oxygen ester linkages are labile to mild alkali, in contrast to myristoylation (wl~ere myristic acid is linked through an amide bond directly to amino terminal glycine residues), they a~e commonly distinguished by the greater lability of the thioester bonds to hydroxylamine at neutral pH and to reducing agents. Palmitoylation of proteins is relatiyely common in eukaryotic biological systems, from fungi and yeast through to mammals (Schultz et al., 1988: Towler et al., 1988; Grand, 1989: Schmidt, 1989; McIlhinney, 1990: Schlesinger et al., 1993). Most palmit0ylated proteins are intimately associated with cellular or viral membranes (McIlhinney el al.. 1987: Schl~singer et al.,

472

L.N. Jones and D. E. Rivett

1993), and contain less than three molecules of fatty acid, although some contain at least six (Hoeg et al., 1986; Huang, 1989). Palmitoylated proteins have widely diverse biological functions, and can also be myristoylated, glycosylated and prenylated, making them extremely hydrophobic. They are also difficult to purify due to their hydrophobic nature, making direct structural analysis of the acyl linkage extremely difficult. Palmitoylation is a post-translational event occurring between the endoplasmic reticulum and the Golgi, although it has also been reported to occur at other sites in the cell, including the plasma membrane (Schlesinger et al., 1993). There are thought to be a number of palmitoyl transferases responsible for the palmitoylation of proteins, with a broad specificity for acyl chains of 12-18 carbon atoms long (but with a preference for palmitate, and to a lesser extent stearate and oleate). It is also believed that the palmitoylation of some proteins may be autocatalytic (Bizzozero et al., 1987; O'Brien et al., 1987; Pangburn, 1992). The rates of fatty acid turnover vary greatly with different proteins, but are generally greater than the turnover rate of the protein (Schlesinger et al. 1993). In the majority of proteins studied, palmitoylation is believed to be crucial for the structure and function of the protein with respect to membrane association, aiding the anchoring of the protein to the membrane (Schultz et al., 1988; Grand, 1989), disrupting the immediate lipid bilayer (Schmidt, 1989; Schlesinger et al., 1993), or even interacting with the hydrophobic portions of other protein (Bourguignon et al., 1991). Bound fatty acids from wool fibres, in particular MEA, have also been reported to be present in the polar lipid extract of enzymatically prepared cell membrane complex (K6rner et al., 1992). This suggests that the bound fatty acids may be attached to the fibre through hydroxyceramides that are in turn linked directly to protein, as occurs on the surface of stratum corneum cells. The rapid liberation of the bound fatty acids in wool and hair with mild alkali is not consistent with an amide bond to the fibre, as for myristoylation, and the absence of significant levels of phospholipids in mature fibres discounts the possibility of a phospholipid or glycosylphosphatidylinositol linkage. There does exist the possibility of a relatively unusual attachment, such as the thioether-diacylglycerol linkage found in bacterial lipoproteins (Hantke and Braun, 1973; Braun, 1975; Sankaran and Wu, 1993). Another possibility is the non-covalent entrapment of the fatty acids in a milieu of macromolecular matrix that is resistant to treatment with lipid solvents, but is sufficiently disrupted by mild alkali to allow the release of the fatty acids. Investigations into the location of the fatty acids in the fibre by treatment with anhydrous t-butoxide in t-butanol rapidly liberated approximately 33% by weight of the bound fatty acids, and then slowly liberated about another 33% (Negri, 1993; Negri et al., 1993a, 1993b). Under the non-swelling conditions used, the t-butoxide would be confined to the surface of the

fibre. The bulk of the fatty acid liberated is thus likely to be situated on the fibre surface. This conclusion was supported by earlier studies that demonstrated the large increase in the wettability of wool fibres after similar surface treatments with alkali under non-swelling conditions, (Leeder and Rippon, 1985; Leeder et al., 1985). Kalkbrenner et al., (1990) also showed that the fatty acids from isolated cuticle cells contained a higher proportion of MEA than those from the cortex of wool fibres, although the cuticle and cortical preparations used were not pure and the absolute amount of bound fatty acids in each was not determined. Methanolic KOH removes 50% of the bound fatty acids within 15 min, with the remainder only fully removed after 2 h. This suggests that this portion of the bound fatty acid is easily accessible on the surface of the fibre, with the remainder being significantly more inaccessible, or attached by a less labile bond to the fibre. It thus appears that the bound fatty acids play a crucial role in maintaining the hydrophobic surface of wool fibres in the absence of the wool grease and other extractable lipids. Bound lipids from the hair fibres of other mammals, including pig, dog, cow and human hair, have been analysed and compared with those from Merino wool fibres (Wertz and Downing, 1989). All of these bound lipids were found to be composed predominantly of fatty acids, with cholesterol sulphate, ceramides and cholesterol present as minor components. MEA accounted for approximately 40% of the total fatty acids in all samples, whereas C16:0, C18:0 and C18:1 were the other major components. There were two additional anteiso branched-chain fatty acids present in relatively low amounts, 14-methylhexadecanoic and 16methyloctadecanoic acid. The authors also proposed that the bound lipids may be covalently bound through ester or thioester linkages directly to the fibre protein. This research was extended (Peet et al., 1992) to the investigation of the bound lipids in the hair or fur of a broader range of animals. They reported that MEA was a major component in all mammalian hair and fur samples investigated, with the exception of monotreme fur, in which 26 carbon fatty acids predominated and MEA was either absent or present in only trace amounts. Recently, Peet (1994) has reported two other mammals which do not contain MEA in their bound lipids, the elephant and the mammoth. The high degree of evolutionary conservatism in the distribution of bound fatty acids suggests that they play an important role in mammalian hair fibres. Peet et al. (1992) calculated that the results obtained from wool fibres indicated that the surface membrane of the fibre cuticle consisted of 31% fatty acid by weight, which would require one in 10 of the amino acid residues in the fibre cuticle membrane to be acylated. If the mode of attachment is as a thioester, it would require almost all of the cystine in the cuticle membrane to be involved in fatty acid acylation. Peet (1994) also confirmed that the majority of the bound fatty acids were situated on the fibre surface, and

18-MethyleicosanoicAcid in Hair

473

after analysis of serial sections obtained from wool and With structures similar to those of normal fatty acids the rat vibrissae follicles demonstrated that the bound MEA inclusion of a branch between the carbon chains causes originated in the developing fibre cells near the base of a severe reduction in the density of packing. Even the the follicle, well below the level of the sebaceous gland. smallest branch (the methyl group) requires so much The liberation of bound fatty acids by reducing agents, space as to make normal fatty acid-like structures hydroxylamine at neutral pH, chlorine and alkali all impossible (Abrahamsson et al., 1963). supported the presence of a thioester linkage of the fatty Some branched-chain fatty acids display strong acid directly to protein. The absence of hydroxyacids bacteriostatic effects, such as 14-methyl eieosanoic acid and ceramides in extracts following extensive acid or (Roze et al., 1990), but there is no evidence for such alkali treatments of the fibres did not support the effects with MEA. The anti-bacterial activity of presence of a ceramide linkage to the fibres, as found in branched fatty acids have been studied fc~r some time. the stratum corneum. Liberation of the bound fatty Weitzel (1951) investigated the inhibiting effect of acids in isolated cuticle cells by hydroxylamine, com- several series of monomethyl-substituted acids on the bined with alkylation using iodoacetic acid, resulted in growth of different strains of tubercle bacilli in vitro, and the formation of one mole of S-carboxymethylcysteine found that the inhibitory effect depended o n the position for every mole of fatty acid liberated, thus provid- of the methyl group; acids with the methyl group at ing strong evidence for a thioester linkage through odd-numbered carbon atoms had the stronger effect. cysteine residues for at least 90% of the bound fatty Negri (1993) examined the effect of soil and Trichoacid. Radiolabelled iodoacetic acid in the presence of phyton mentagrophytes on wool and hair that had hydroxylamine was used to confirm that the formation been treated to remove bound fatty acidsi He showed of free cysteines and subsequent alkylation was hydroxy- that fungal growth was more prolific on ireated fibres lamine dependent. Hydroxylamine was also used in a compared with untreated fibres, but proposed that this similar study by Naito et al. (1996), where these findings was likely to be as a result of the hydropholbic nature of were largely confirmed. Hydroxylamine had been the untreated fibres rather than intrinsic antifungal used previously by Negri et al. (1993b) to remove the properties of MEA. covalently bound fatty acid in studies of the modification of the surface diffusion barrier of wool. Fatty acid E. Physical measurements oJ bound lipids on hydroxamates (RCOHNOH) were identified as the major product from the reaction of wool with hydroxykeratin fibres lamine (Evans and Lanczki, 1997). Alk~lamines have After the discovery of bound lipids on keratin fibres also been shown to cleave the bound lipids from the surface of hard keratins, the major product being (Evans et al., 1985), several groups of researchers attempted to use a variety of physical techniques to N-alkyl 18 methyleicosanamide. confirm the observation. Carr et al. (1986i used X-ray photoelecton spectroscopy (XPS) to measure ratios of carbon to nitrogen on the fibre surface arid confirmed D. Biosynthesis o[anteiso f a t t y acids that wool fibres did have a hydrocarbon surface. K6rner Some species of bacteria synthesize anteiso methyl- et al. (1990) confirmed this observation, and went on to branched fatty acids, including 18-methyl-eicosanoic show that when the bound fatty acid was lremoved by acid, by utilizing the amino acid isoleucine which, fol- alcoholic alkali treatments, the proportion of carbon lowing deamination and decarboxylation, give rise to atoms on the surface was reduced. Secondary ion mass 2-methylbutyric acid. This can act in the form of its spectrometry (SIMS) and photoelectron ~pectroscopy Coenzyme A (CoA) ester as a primer unit for chain indicated that the surface of wool fibres is almost elongation by the fatty acid synthetase enzyme system, exclusively hydrocarbon in nature (Ward et al., 1993), by the successive addition of methylene groups derived thus confirming the results of Carr et al. (1986) and from malonate, to give anteiso long-chain fatty acids K6rner et al. (1990) and the calculations of Negri et al. (1993a). Ward et al. (1993) reported three c~istinct peaks (Kaneda and Smith, 1980). Bacteria which produce such acids are present in the from a negative ion static secondary ion m~ss spectrum, forestomachs of ruminant animals and act as the source using a time of flight mass spectrometer, at 311,325 and of most of the small amounts (2 3%) of iso and anteiso 341 a.m.u., which they assigned as Cli9H39C00-, acids found in their tissue and milk lipids. The same C20H41COO and C20H40OHCOO . Thelpeak at 325 biosynthetic pathway as that in bacteria has been shown was assumed to originate from oxygen;ester-bound to account for the presence of iso and anteiso acids in MEA whereas the peak at 341 was reported as originatthe sebum of animals (Garton, 1985). The specific ing from the hydroxy fatty acid indicated. These function of a methyl branch is unclear, the major conclusions were disputed by Peet et al. i(1994), who difference to a corresponding straight-chain fatty acid is postulated that the most likely identity of Ithe peak at the reduction in melting point while remaining stable to 341 was that of the thiolate ion, C20H41CON- originatoxidation. The molecular arrangement of branched ing from thioester-bound MEA, because n]o covalently chain fatty acids is complicated by the space require- bound hydroxy acids have been found by chemical ments of a branch projecting out from the carbon chain. analysis. i

474

L.N. Jones and D. E. Rivett

The interpretation of Peet et al. (1994) was supported by the static secondary-ion mass spectrum results of Naito et al. (1996), who found that the major peak was 341 a.m.u, and also assigned the peak as the thiolate ion C2oH41COS-. These authors reported that the peak found by Ward et al. (1993) at 325 a.m.u, was barely detectable. It would appear that there may be variable amounts of serine-ester-bound MEA between fibre sources, or that the removal of unbound fatty acids during sample preparation is sometimes not complete.

II. I N V O L V E M E N T IN FINE S T R U C T U R E A. Structure o f the mammalian hair fibre

The physical characteristics of mammalian fibres (diameter, length, transverse shape, surface characteristics and contours) vary widely between species and are generally influenced by the nutritional and metabolic state of the animal. Mammalian hairs are produced during the so-called anagen or active phase of hair follicle growth, and fibre length can differ markedly between species depending on the duration of anagen. Chemical differences in fibres such as pigmentation, protein and lipid compositions also show wide variation. All these differences can be manifested within a single fibre. Mammalian fibres are composed of cells in which an outer layer of overlapping flattened cells (cuticle) surrounds an inner core of cortical and medullary cell types (Fig. 6). The cuticle cells perform a protective function for the spindle-shaped cortical cells. The cortical cells in turn surround a central stream of vacuolated medullary cells. Cells occupying the medullary region are unusually insoluble and are often present in coarser fibres (Fraser et al., 1972; Montagna and Parakkal, 1974; Rogers and Harding, 1976; Lindley 1977; Zahn, 1977; Orwin, 1979a, 1979b; Fraser et al., 1980; Marshall et al., 1991; Jones et al., 1997).

B. Fibre cuticle

The number and thickness of cellular layers comprising the hair fibre cuticle also vary widely between species. For example, in fine wools, the cuticle is usually one layer thick. In contrast, cuticle thickness in human hair and pig bristle may be up to 10 or 35 layers, respectively. When stained sections of cuticle cells are viewed in transmission electron microscopy (TEM) studies a series of internal laminations are observed. These laminations consist of outer sulphur-rich bands known as the exocuticle and inner regions of lower sulphur content called the endocuticle (Fig. 3). Cuticle cells are responsible for the surface properties of fibres that are important in a variety of applications, such as textile processing or protection of the fibre components from environmental damage (Rivett, 1991).

Fig. 3. Transmissionelectronmicrograph of fibre cuticle(FCU) cells in a section of human hair. The various regions of the FCU include the exocuticle (exo) which contains an outer densely stained band or a-layer (a), the endocuticle (endo), cytoplasmic remnants (Cr) and the cell membrane complex (CMC). The darkly stained bands of the exocuticle form the protective proteinaceous layers which are rich in disulphide bonds. The specimen was reduced and fixed in osmium tetroxide. Sections were post-stained with uranyl acetate and lead citrate. Bar equals 0.1/tm. Hence the surface components of cuticle cells form a barrier to penetration of chemical and biological agents (Speakman, 1984; Mansour and Jones, 1988). However, the cuticle also performs other important functions such as the regulation of ingress and egress of water, which helps to maintain numerous physical properties of fibres. The ultrastructural mechanisms of water transport in the hair and wool fibre are still not entirely clear (Feughelman, 1959; Spei and Zahn, 1979; Fraser and MacRae, 1980) but the surface and intercellular material obviously perform key functions in this regard.

C Resistant membranes

On the exposed fibre cuticle surface a thin membranelike non-staining band is sometimes observed in TEM studies. This band, known as the fibre cuticle surface membrane (FCUSM), is unique in nature and is composed of protein and lipid components. It functions as a highly resistant, hydrophobic, surface-protective barrier to water, and to attack from other chemical and biological agents (Fig. 4). The protein component is apparently stabilized by iso-peptide bonds such as e(7-glutamyl) lysine crosslinks. In many respects this protein layer corresponds to the so-called epicuticle (Zahn, 1951; Negri et al., 1993a). The term epicuticle is sometimes used to include the entire FCUSM. However, it must be remembered that the epicuticle was originally defined as the membrane raised from the fibre surface by chlorine treatment and should strictly be considered as a "membranous residue". In being highly resistant due to stabilization by isopeptide bonds, the epicuticle is raised as bubbles or sacs from the underlying material after treatment with powerful oxidizing agents such as

18-MethyleicosanoicAcid in Hair

475

CH3 CH3

CH~-- CH Isoleucine

CO0 CH NH~ +

O

CH~ Fig. 4. The fibre cuticle (FCU) surface membrane (FCUSM) at high magnification (TEM) in transverse section after specimens were reduced and fixed in osmium tetroxide (section poststained with potassium phosphotungstic acid). The FCUSM contains lipids (fatty acids, predominantly MEA) and proteins and is approximately 6 nm in thickness. The FCUSM is directly apposed to the a-layer (a) of the exocuticle (exo). Bar equals O. 1/lm.

chlorine/water mixtures (yon Allwarden, 1916). Most studies agree that the residual epicuticle obtained after these treatments is predominantly proteinaceous (Zahn, 1951), although some authors have found lipids associated with their epicuticle preparations (King and Bradbury, 1968). In order to explain the hydrophobic nature of the FCUSM and the role of lipids in this "membrane", it has been suggested that these lipid moieties are covalently bound to an underlying protein matrix (Elliot and Manogue, 1952: Lindberg, 1953). In a later study K6pke and Nilssen (1960) postulated that the surface of the fibre cuticle was studded with long-chain fatty acids linked to underlying protein by ester linkages. These authors reasoned that the existence of ester bonds would account for the effect observed after treatment of fibres in alkaline solutions. A postulate confirmed by the recent analytical studies discussed earlier in this review (Figs 5 and 6). On the basis of the analyses mentioned earlier, a model for the FCUSM was proposed, which consisted of fatty acids linked to the underlying layer of heavily crosslinked protein, forming a lipid monolayer surrounding each cuticle cell (Negri et al., 1993a) (see Fig. 7). This model assumes a thickness of the surface layer (FCUSM) of approximately 6nm. Despite the wide range of estimates, it is generally agreed that the thickness of this surface layer is between 2 and 7 nm (Lindberg et al., 1948; Swift and Holmes, 1965; King and Bradbury, 1968: Jones et al., 1997). A number of studies using static secondary-ion mass spectrometry (SSIMS) and XPS have been undertaken to determine the physical and chemical nature of the cuticle/air interface (Carr et al., 1986; K6rner et al., 1990; Ward et al., 1993). Ward et al. (1993) used atomic ratios fi'om XPS to estimate the thickness of the surface lipid layer as 0.9 nm, which is less than half of that tkmnd by Negri et aL (1993a) from the length of a

CH~-,--CH

C SCoA

2-Methylbutyryl CoA

CH3

CH3-- CH~--CH

-(CH~)o--COOH

Anteiso acids Fig. 5. Synthesis of the anteiso methyl-branched fany acids (including MEA) is achieved by utilizing isoleucine as a substrate. After deamination and decarboxylation,2-methylbutyric acid is formed which can act in the form of its koenzyme A (CoA) ester as a primer unit for chain elongation b~' the normal fatty acid synthetase enzyme system. This is achleved by the successive addition of methylene groups d~rived from malonate, to give the anteiso long-chain fatty acids.

Fig. 6. Human hair fibre in transverse section show~ a relatively

thick fibre cuticle (FCU) surrounding a fibre @rtex (FCO) which contains darkly stained melanin granuk{s. MEA is located in the surface membranes of FCU cells. The medulla (M) occupies the central region. Light microgrNgh × 400.

20-carbon chain. An explanation for this difference was given by Zahn et al. (1994). They suggested that MEA may be folded back in the direction of the surface. However, Peet et al. (1994) subsequently indicated that the apparent contradiction in the thicknes i of the lipid layer may be an artefact of the anhydrous, high-vacuum conditions used in the XPS.

476

L.N. Jones and D. E. Rivett

D. Proteinaceous resistant barriers

FIBRE SURFACE

III0 iliIIi !.:..i.j...-....,

t

Fatty acid monolayer (approx 3nm]

I]

...*.'.*. ,-, g O ~ ,"

PROTEIN

I

i

I

:

I I I

Proteolipid membrane (approx 3nm)

:

O0 e O

" 0%"

"0",

•

a - layer [cuticle cell]

, •

• " ~0

O

LIPID

Fig. 7. Proposed model showing arrangement of the protein and lipid moieties in the fibre cuticle surface membrane. The exterior surface of the fibre cuticle consists of a monolayer comprising C2h0a branched chain fatty acids (MEA) linked via thioester bonds to the proteins comprising the Allw6rden reaction-induced resistant membrane (epicuticle). The epicuticle consists of an inert protein matrix containing isopeptide crosslinks. The epicuticle is in turn attached to the exocuticular a-layer by an unknown mechanism.

This suggestion that the hair fibre surface could be different in vacuum conditions compared with other environments (aqueous or solvent) needs to be carefully considered. Horr (1996) studied contact angles and surface energy values previously determined for wool (Bateup et al., 1976; Brooks and Raman, 1986) as they related to the surface components existing on the wool surface (methyl-, methylene-, keratin and absorbed vapours). Horr's findings (Horr, 1996) indicated that the outermost region of the fibre surface cannot be exclusively composed of methyl groups as had been indicated in the model of Negri et al. (1993a). Of particular note is that the method of measurement of contact angle and surface energy of mammalian hair fibres necessarily includes the region between cuticle ceils (scale edges) in addition to the cuticle surface itself. Horr (1996) further suggested that vapour absorption due to capillary condensation may occur at the fibre cuticle scale edges, and that the phenomenon may contribute to the above interpretation that the fibre surface is not entirely methyl (hydrocarbon). He also found that the possible composition of the fibre surface may even vary depending on the liquid with which it is in contact (e.g. water, methylene iodide). It must be emphasized that in spite of the FCUSM being a relatively minor component of mammalian fibres, its function in protection, wettability, friction and surface tension are crucial to the successful performance of fibres in the natural environment. Furthermore, this membrane is of increasing importance in industrial aspects of wool processing and fibre-damage considerations of human hair in the cosmetic industry. In the cosmetic industry, fibre damage in human hair is often associated with this membrane. Consequently there is a need to increase our understanding of the structure and composition of the FCUSM in mammalian fibres.

In positively stained sections of hair fibres examined by TEM, well stained laminated regions are observed in association with the FCUSM. In particular, the exocuticular a-layer nearer the surface is more intensely stained than the lower exocuticular band. These bands together form the proteinaceous resistant barriers of cuticle cells (Fig. 3 and Fig. 4). The remainder of the cell contents form an inner lining known as the endocuticle. This region has low stain uptake and contains remnants of cell organelles (Rogers, 1959; Jones et al., 1997). No evidence exists for the presence of lipids, particularly MEA, being associated with these bands. However, a brief description of the structure/function of cuticle ultrastructural components seems warranted due to their association with the fibre surface and role in the fibre cuticle. On the basis of their relative affinity for heavy metal stains, the exocuticular and a-layer bands are usually considered to be richer in cysteine than the remaining exocuticular region (Fraser et al., 1972). Prior reduction of fibres apparently enhances the uptake of heavy metals by the proteins in the a-layer, suggesting that cleavage of disulphide disulphide bonds enhances reaction with osmium (Rogers, 1959; Kassenbeck, 1961). This interpretation is complicated by the assumption that chemical specificity is uncertain and other factors, such as increased accessibility of the regions, must be considered (Swift, 1977). The development of microanalytical techniques have provided direct evidence about the location and distribution of protein-bound sulphur in the fibre cuticle (Fig. 8). Recent studies have demonstrated that the a-layer is relatively richer in sulphur (cystine, cysteine) than the remaining exocuticle and endocuticle (Jones et al., 1990, 1993; Sideris et al. 1992; Hallegot and Corcuff, 1993). In addition to disulphide bonds in the exocuticular layer, chemical analyses show that this region has relatively high contents of lysine and glutamic acid, suggesting the presence of isopeptide crosslinks (e-[~-glutamyl]lysine) (Schwan and Zahn, 1980). These types of crosslinks exist in various epithelial cells such as the hair medulla, inner root sheath and envelope proteins of the stratum corneum (Banks-Schlegel and Green, 1981; Rogers, 1983: Zettergren et al., 1984).

E. Fibre cortex

Cortical cells are highly elongated (spindle-shaped) cells with a polyhedral cross-section and are aligned in the same direction as the fibre axis. These cells form the main bulk of the fibre and play a major role in determining its mechanical properties and intrinsic strength. The ultrastructural texture of cortical cells appears in the TEM as one of unstained fine filaments contained within a densely stained matrix (Birbeck and Mercer, 1957a; Rogers, 1959). The filaments were originally called microfibrils, but in modern terminology are

18-Methyleicosanoic Acid in Hair

477

Fig. 9. High magnification micrograph (TEM) of~t human hair cortical cell in transverse section. Hard keratiniintermediate filaments (IFs) show an annular substructure co~sisting of an unstained outer ring and core. The lFs are emt)edded in an interfilamentous dark stained sulphur-rich matriX. The diameter of IFs are approximately 7.5 nm. Fatty acids ~uch as MEA or other lipids have so far not been detected in th~ hair cortex. Specimens were reduced and fixed in osmium t{troxide. Sections were post-stained with uranyl acetate and lea/dcitrate. Bar equals 10 nm.

BF STEM

Fig. 8. Sulphur X-ray maps (A) showing the relative distribution of this element in the proteinaceous bands of fibre cuticle cells (FCU). The outermost a-layer (a) is richer in sulphur than the remaining exocuticle (exo). The endocuticle (endo) appears to have a low sulphur content. The bright field (STEM BF) in B is included for comparison. Bar equals 0.1/~m.

widely distributed in eukaryotic cells and iplay a major role in regulating mechanistic behaviour of the cytoskeletal structure in these cells (Lazaridei, 1980). The hard keratin IFs are similar in structural and chemical properties to the other types of 1Fs such as desmin, neurofilaments and vimentin. Together with the wide range of epidermal IFs, the hard keratin iFs appear to be more complex with respect to polypepiide composition and assembly mechanisms (Jones andl Pope, 1985). In transverse sections of hard keratir~s, the interfilamentous matrix appears to be relatively amorphous. M a n y of the sulphur-rich proteins of m a m m a l i a n fibres are thought to be located in the matrix, iand it seems likely that these proteins generally contain intra-chain disulphide bonds (Parry et al., 1979). Such ~rrangements are the most likely explanations of matrilx swelling of fibres (Fraser and MacRae, 1980). It is generally believed that the cortex is mostly proteinaceous in nature and lipids, part!cularly fatty acids, are not associated with the IF/matriN components. However, dedicated studies in this respect have not been performed because of the difficulties iin assigning particular extracted components to ultrastructural or cellular regions of the fibre.

F. Intercellular regions (cell membrane complex) known as hard keratin intermediate filaments (IFs). The generally agreed diameter of these IFs from published results is 7 7.5 nm, and this diameter is considered constant across m a m m a l i a n species (Fig. 9) (Fraser et al., 1972: Jones, 1975, 1976). The characteristic a-type, X-ray diffraction patterns of hard keratinous materials, such as hair, arises from the IF moieties (Fraser et al., 1965). These patterns are similar to those obtained from other fibrous protein systems including muscle, elastin and fibrin. IFs are

In transverse sections of m a m m a l i a n fibres examined by T E M (Fig. 10), intercellular conr~ections exist between the main cell types (cuticle/cUticle, cuticle/ cortex, cortex/cortex). The connections areicharacterized by densely stained intercellular bands (6!layers) sandwiched between two non-staining thinner bands (fllayers) (Swift and Holmes, 1965: Leeder, 1986). These three bands normally constitute the ~o-called cell membrane complex (CMC) based on the! definition of

L. N. Jones and D. E. Rivett

478

....... ......... ,

.....

L~ ~

determine the location and distribution of branched chain fatty acids such as MEA in fibre cuticle cells (Jones and Rivett, 1995; Jones et al., 1996).

G. The mammalian hair follicle

Fig. 10. Stained transverse section (TEM) showingthe ultrafine structure of the cell membrane complex (CMC) between apposed fibre cuticle (FCU) cells of human hair. The CMC consists of two unstained modified membranes (fl-layers)and an intercellularc~-layerof higher staining intensity• Note that the surface fl-layerappears to be wider than the fl-layeron the cuticle cell underside. The g-layer in turn contains internal laminae of thickness approximately 5 nm. An intracellular membrane-associated layer (i) forms a narrow band on the underside of a fibre cuticlecell. The dark-stained band (a) is the surface a-layer of exocuticle(exo) in an underlyingcuticle cell. The specimen was reduced and fixed with osmium tetroxide after which the section was post-stained with uranyl acetate and lead citrate• Bar equals 100 nm. Leeder (1986). The intercellular 6-layer is thought to be composed mostly of proteinaceous material with low crosslink density. In the //-layers, bilayers of lipids coupled to inert proteinaceous resistant membranes are normally assumed. However, the lack of phospholipids found in fibre extracts (Orwin, 1979a, 1979b; Schwan and Zahn, 1980; Rivett, 1991) indicates that the conventional plasma membrane lipid bilayer structure must be markedly modified as hair-forming cells convert from the live state in the follicle to the inert keratinized cells found in fibres. An additional densely stained intracellular band, known as the i-layer, directly apposes the fl-layers and some workers have suggested that this band should be included as part of the CMC (Fraser et al., 1972, 1980). Despite the fact that this intracellular band is an internal component of the cell, it may play a role in the overall stabilization of the CMC. In the context of this review, the fl-layers of the fibre cuticle are particularly relevant with respect to their fatty acid composition, structure and function. The micrograph of human hair shown in Fig. 10 indicates that the non-staining//-layer (arrowed) at the surface of the fibre cuticle cell is clearly different from the//-layer on the underside. An apparent difference (which needs to be verified) is exhibited in the greater thickness of the surface //-layer. These observations have initiated a range of studies in order to understand the structure and composition of surface and underside membranes in fibre cuticle cells. The location of MEA and other fatty acids in these membranes could thus only be explained after high resolution T E M studies were able to demonstrate the mechanism underlying formation of the surface membrane in fibre cuticle cells (Jones et al., 1994). Further studies of mutant hairs in patients with maple syrup urine disease (MSUD) subsequently enabled us to

Hair fibres form from differentiated streams of concentrically arranged germinal epithelial cells originating on the basement membranes of hair follicles (Fraser et al., 1980). These cell streams constitute the various layers of the developing hair shaft, together with those of the so-called inner (IRS) and outer root sheaths (ORS) (Birbeck and Mercer, t957a, 1957b, 1957c: Rogers, 1964). In the follicle, the developing fibre surface of cuticle cells surrounds cortical and medullary (optional) cells, whereas three distinct cell layers comprise the surrounding IRS. The inner cell lining of the IRS apposing the developing fibre cuticle (FCU) is called the IRS cuticle (IRSCU). These FCU and IRSCU cells are surrounded by the so-called Huxley (HU) and Henle (HE) cell layers. In the ORS two cell types (ORS and companion cells) have been identified (Orwin, 1971 ).

H. Formation of fibre-cuticle surface membranes The early TEM studies of developing hair follicles demonstrated that presumptive fibre-cuticle cells flatten longitudinally and circumferentially to form interlocking arrangements with IRSCU cells. These interlocking arrangements provide the basis of the so-called scale edges characteristic of the mammalian fibre surface (Woods and Orwin, 1982). Subsequent changes in the presumptive fibre cuticle are the appearance of densely stained globular particles in intracellular regions. These particles migrate to the outer regions of the cells where they aggregate to form a multiplate-like or lamellated network (Fig. 11) (Orwin, 1979b). The exocuticular bands observed in mature fibre cuticle cells (Fig. 2) arise from this densely stained network. In association with the formation of exocuticular material changes also occur in the cellular plasmamembranes and intercellular regions of developing fibre and inner root sheath cells (Orwin and Thompson, 1973). Other modifications in the region result from intercellular acid phosphatase activity between apposed presumptive fibre cuticle and fibre cortex cells. Changes have also been observed in the membrane-associated polysaccharides presenting at the surfaces of keratinizing cells (Orwin, 1970). These earlier studies were not able to determine the mechanisms of fibre-surface membrane development, partly due to the limited resolution of conventional T E M imaging. In a more recent study by Jones et al., (1994) high resolution T E M incorporating energyfiltered imaging enabled observation of the ultra-fine structural changes taking place in the surface and intercellular regions. This investigation followed the developing fibre cuticle surface through its formation until it entered the pilary canal.

18-Methyleicnsanoic Acid in Hair

Fig. 1l. Transverse section through a wool follicle shows formation of the fibre cuticle. The micrograph (TEM) shows developing fibre cuticle (FCU) cells where they appose inner root sheath cuticle (IRSCU) cells. The FCU cells show formation of the exocuticle (exo). This exo structure appears to be composed of lamellated components which show dual staining. Above the lamellae, the former plasma membrane is absent and four intercellular laminae occupy the intercellular region between FCU and IRSCU cells. These intercellular laminae presumably contain lipids such as MEA and proteins which form the mature fibre cuticle surface membrane. The cleavage plane where FCU and IRSCU cells are separating is indicated by a narrow light-stained band (arrows). Section was stained with osmium tetroxide, uranyl acetate and lead citrate. Bar equals 0.1/*m.

D u r i n g the f o r m a t i o n o f e x o c u t i c u l a r l a m i n a e in d e v e l o p i n g fibre cuticle cells the p l a s m a - m e m b r a n e on the o u t e r surface a p p e a r s to be d i s r u p t e d . In place o f these m e m b r a n e s p a i r e d l a m i n a e o f p r e s u m e d lipid bilayers are d e p o s i t e d o n the fibre cuticle surface a n d o c c u p y the intercellular regions between j u x t a p o s e d IRS C U a n d fibre cuticle cells (Fig. 11). A f t e r cleavage o f I R S C U a n d fibre cuticle cells takes place a l o n g a densely stained n a r r o w b a n d between two p a i r s o f intercellular laminae, one p a i r o f l a m i n a e is d e p o s i t e d on the densely stained exocuticle b a n d o f the fibre cuticle surface. T h e r e m a i n i n g p a i r is s u b s e q u e n t l y l o c a t e d on the I R S C U surface (Fig. 12). In a d d i t i o n , cleavage between F C U a n d I R S C U cells leads to f o r m a t i o n o f the o v e r l a p region (scale edge) between a p p o s e d fibre cuticle cells. T h e i n t r a c e l l u l a r exocuticle layers extend a r o u n d the scale edge c o i n c i d i n g with an intercellular g a p o f a p p r o x i m a t e l y 1 2¢tm length between o v e r l a p p i n g fibre cuticle cells. T h e p l a s m a m e m b r a n e s on the u n d e r s i d e o f fibre cuticle cells a p p e a r to have m a i n t a i n e d their original m o r p h o l o g y a n d staining characteristics t h r o u g h o u t the differentiation process. Intercellular m a t e r i a l between a p p o s e d fibre cuticle cells d e m o n strates an u n s t a i n e d central b a n d s a n d w i c h e d between relatively lightly stained single l a m i n a e (d-layer). A m o d e l d i a g r a m o f the e m e r g i n g fibre cuticle cell is shown in Fig. 13. The f o r m a t i o n o f a new fibre cuticle m e m b r a n e o n the o u t e r surface seems r e a s o n a b l e b e c a u s e m a m m a l i a n fibres are a d a p t e d to p e r f o r m a p r o t e c t i v e function in their new e n v i r o n m e n t . T h e r e f o r e the new fibre surface is uniquely h y d r o p h o b i c a n d w a t e r resistant. H o w e v e r , the n o r m a l fibre function o f the internal fibre cortex requires a m e c h a n i s m e n a b l i n g the ingress o f water. T h e

479

Fig. 12. The developing fibre cuticle (FCU) and{urface layers at the region of the FCU scale edge. The cleavage process between FCU and inner root sheath cuticle~RSCU cells (liberation of fibre into pilary canal) is well advaOced (arrowl. Note that the cleavage extends between overlappiOg FCU cells and terminates together with the enveloping exocqticular layers on the underside of FCU cells {arrow). Cleavag~ takes place along the central dark stained band between t~e four intercellular laminae between apposed FCU and I]RSCU cells. Section stained with osmium tetroxide uranyl ace~tateand lead citrate. Bar equals 0.1um. IRSCU

W

Fig. 13. Diagram showing the formation of thei fibre cuticle surface membrane (FCUSM), the cell membrane complex (CMC) between apposed fibre cuticle cell (FQU) and the exocuticle (exo). The FCU has separated from toe inner root sheath cuticle (IRSCU) to enter the pilary canal (PC). The plasma membrane (PM) of the IRSCU is coated ,,yith a pair of intercellular laminae (PIL). The plasma membrane of FCU cells has been replaced by a pair of intercellular l~iminae (PIL) and these are in contact with the a-layer (a) ~f the FCU exocuticle (exo). The endocuticle is denoted endo ~nd the CMC comprises two/,~-layers ([]) and an intercellular d#lta layer (,5). MEA is a constituent of the paired intercellular l~rninae of the FCUSM but its precise distribution is unknown al~this stage of development. m e m b r a n e s a n d intercellular m a t e r i a l between a p p o s e d fibre cuticle cells are p r e s u m a b l y a d a p t e d f6r p e r f o r m i n g this function. O u r m a i n interest in this regiew c o n c e r n s the o u t e r fibre surface m e m b r a n e s o f fibr~ cuticle cells a n d the role o f M E A in the v a r i o u s functions p e r f o r m e d by these m e m b r a n e s .

I. Mutation involving M E A T o d a t e one inherited defect involving the b r a n c h e d chain fatty acid M E A has been described in two

480

L.N. Jones and D. E. Rivett

mammalian species. M S U D or branched chain ketoaciduria has long been known to affect human populations, but other studies have shown that a related mutation occurs in Poll Hereford cattle (Harper et al., 1989). M S U D is caused by an inherited metabolic deficiency in the enzyme, branched chain 2-oxo acid dehydrogenase, which leads to accumulation of the branched chain amino acids, leucine, isoleucine and valine, together with their respectively derived ~-keto acids ( Z h a n g et al., 1990) in the blood, urine and cerebrospinal fluid (Gennaro et al., 1979; Tanaka and Rosenberg, 1983). The synthesis of the anteiso methyl branched chain fatty acids (Fig. 4) including MEA are believed to be derived from 2-methylbutyric acid following oxidative decarboxylation of isoleucine by branched chain 2-oxo acid dehydrogenase (Garton, 1985). A deficiency of branched chain 2-oxo acid dehydrogenase should lead to the elimination of branched chain fatty acids derived from the branched chain amino acids. Yorimoto and Naito (1994) and Naito et al. (1996) found that M S U D patients' hair lacked the MEA but contained approximately the same amounts of the other major fatty acids (palmitic, oleic and stearic acids) as normal hair. As a result of this deficiency, hairs from patients with M S U D were found to provide valuable information regarding the location and distribution of MEA, together with its role in the function of the fibre cuticle surface membrane (Jones, 1994; Jones and Rivett, 1995; Jones et al., 1996). In their detailed observations using TEM, Jones et al. (1996) observed that the structural defect in M S U D was mostly confined to the surface r-layers of F C U cells. Figure 6 shows the arrangement of proteins and lipids in the model proposed for the fibre cuticle surface membrane (Negri et al., 1993a). The hydrophobic surface results from a monolayer of fatty acids linked to cysteine residues of an underlying proteinaceous band (epicuticle) (Kalkbrenner et al., 1990) via thioester bonds. As indicated previously, recent research has shown that M E A is the major fatty acid component of the surface membrane. MEA is apparently synthesized in the developing follicle and is not derived from sebum (Peer, t994). Downing and Lindholm (1982) also found in an analysis of cow sebum that no branched chain fatty acids were present, although they were present in the hair. Because M E A is present in the anteiso form, the most probable metabolic precursor in animal cells is the amino acid isoleucine (Wheatley et al., 1967; Downing, 1976; Garton, 1985). The other branched chain amino acids such as leucine and valine would not be substrates in the synthesis of MEA. It was concluded from the T E M observations (Jones and Rivett, 1995; Jones et al., 1996) that the structural defect associated with the r-layers of fibre cuticle cells in hair correlates with disruption in the synthesis of MEA, and that in normal hair MEA is present in the r-layers of fibre cuticle cells. Furthermore, it would also seem that M E A is confined to the upper surfaces of fibre cuticle r-layers because the r-layers on the fibre cuticle

Fig. 14. (A) Transmission electron micrograph (TEM) of the fibre cuticle (FCU) and fibre cortex (FCO) in a hair from a patient with maple syrup urine disease (MSUD). A structural defect (arrow) in MSUD appears to be located in the outer surface of fibre cuticle (FCU) cells when they directly appose the cell membrane complex (CMC). The FCU comprises the exocuticle (exo), a-layer (a) and endocuticle(endo). Specimens were reduced and heated with osmium tetroxide. Sectionswere post-stained with uranyl acetate and lead citrate. Bar equals 0.1/~m. (B) High magnificationTEM of the defectiveregion in the FCU found in MSUD hairs. The transverse section shows that the structural defect is confined to the surface r-layer between the a-layer (a) and the intercellular delta layer (3). Adhesion betweenthese layersis disrupted (arrow) where MEA deficiency apparently exists in MSUD. Specimens treated as above. Bar equals 10 nm. underside of M S U D hairs are not affected. Therefore the upper and lower surfaces of fibre cuticle cells are different with regard to structure and composition. Earlier studies by Jones et al. (1994) had indeed shown that a newly derived membrane on the upper surface of fibre cuticle cells had replaced the original plasma membrane (Figs 11, 13 and 14).

18-Methyleicosanoic Acid in Hair

Fig. 15. Scanning electron micrographs of a normal control h u m a n hair fibre cuticle surface (A) compared with a preparation of hair from a patient with maple syrup urine disease (MSUD), (B). M E A synthesis is aberrant in M S U D patients and this deficiency is manifested in the fibre cuticle surface membrane ( F C U S M ) of the hair fibre cuticle. The normally smooth surface appearance observed in control hairs is disrupted in the M S U D hair cuticle surface. Bars equal 1 l~m.

During development of fibre cuticle cells, the plasma membrane on the outer-surface is disrupted during the formation of the exocuticular layers. A pair of inter-

481

cellular laminae deposit on the fibre cuticle surface after cleavage takes place between the fibre cuticle and inner root sheath cuticle cells. These surface laminae are presumably composed of the lipids and proteins that in turn form the mature fibre cuticle surface membrane. It seems likely that MEA is a component of the lipid moieties as the fibre is liberated into the pi!ary canal. In support of these observations Peet (1994) i has demonstrated that the bound MEA originates from the developing fibre cells at the base of the fbllicle, quite independent of sebaceous gland secretions.i The studies of Negri et al. (1993a) had led to the conclusion that MEA extended around th~ entire fibre cuticle boundary, which was in contrast [to the more recent observations by Jones et al. (1996) c~ncerning the developing fibre cuticle surface membraoes and the nature of the structural defect in MSUD. This discrepancy could be explained if approximatelN 30% of the covalently bound lipid were trapped within the protein layer (see Fig. 7). Surface studies of MEA-deficient hair such as in MSUD have so far not been undertakel~, and future research using atomic force microscopy an d XPS should provide valuable surface molecular data. The scanning electron micrographs (D. Watson, unpublished communication) clearly show differences i n the surface structure of MEA-deficient hair and norma ! human hair (Fig. 15). In fatty acid analyses of MSUD and qormal hairs, differences were found in the ratio of covalently bound fatty acids. Comparisons between MSUD] and normal hairs (see Table 2) showed that the most o~vious difference was the 90'70 reduction in MEA and the concomitant three-fold increase in the C20:0 (eicosanoic acid) of MSUD hair (Jones et al., 1996): [other minor branched chain fatty acids (C17:0 and C19i:0) were also significantly reduced. Eicosanoic acid is a relatively minor constituent of the bound fatty acids in normal

Table 2. Comparison of published analyses of the fatty acid composition of hair from M S U D patients i

Fatty acid as percentage of total bound fatty acids Jones et al. (1996) Naito el al. (1996)*

Fatt 5 acid 1

Cl4:0 C15:11 Cl6:l C16:0 Cl7:Obr CI7:(t C18:1 C18:0 C 19:Obr C20:0 C21:0a C2 I:0 C22:0 C24:0

3.6 1.5 1.6 38.0 0.6 3.0 2.7 20.8 0.1 18.8 1.8 1.1 3.3 3.2

Normal hair Jones et a/. (I 9961

2

5.7 2.3 2.4 38.5 0.6 3.0 7.0 17.3 0.0 15.6 1.6 1.3 2.6 1.9

Trace ND Trace 43 ND ND 7 32 ND Present** Trace ND ND ND

3.2 2.4 1.3 27.8 4.5 3.6 2.6 10.4 2.(1 5.2 31.7 1.3 I.I 1.0

br, a branched chain fatty acid with the location of the branch uncertain: a, an anteiso-branched fatty acid. ND, not detected. *The percentages included in this table have been estimated from the text of the paper as the actual values were not reported. **(3as chromatographic trace showed a significant a m o u n t but the quantity was not estimated by the authors.

482

L.N. Jones and D. E. Rivett

hair fibres, usually around 5%, but in MSUD hair the C20:0 was found to be one of the major fatty acids (15-19% of the total) and was suggested to replace MEA to some extent (Jones et al., 1996). These authors also noted that in MSUD hair there appeared to be a general increase in all the linear saturated fatty acids, particularly C16:0, 18:0 and 20:0 (Table 2); the increase in C20:0 being the most pronounced. In contrast, Naito et al. 1996, commented that the deficiency in MEA was not compensated by other fatty acids. However, although they did not give analytical percentages, their published gas chromatographic traces showed a significant increase in C20 but not to the extent reported by Jones et al. (1996). In addition, Naito et al. (1996) did not give analytical values for the minor fatty acids but they did indicate that small amounts of 14methylhexadecanoic acid were present in normal hair but absent in hair from patients with MSUD. The functions of MEA in mammalian fibres are still unclear but the findings from studies of MSUD hair suggest that it plays an important role in cell adhesion. Jones et al. (1996) reasoned that the major physical differences between eicosanoic acid and MEA is that the introduction of a methyl group lowers the melting point from 77°C (eicosanoic acid) to 56°C (MEA). Therefore the loss of MEA in the cuticle membrane and its replacement with eicosanoic acid could possibly affect the fluidity of the membrane, but the significance of these temperatures, considering that the physiological temperature is 37°C, needs to be considered. It was further suggested by these authors that this substitution would lead to greater stiffness or rigidity, leading to a possible loss of adhesion between fibre cuticle cells. The conclusion reached by Jones et al. 1996 in their studies of MSUD hairs was that MEA plays a specific structural or biological role in the function of fibre cuticle surface membranes. This view was reinforced by other findings which have shown that significant amounts of this uncommon fatty acid (MEA) are covalently linked to the fibre cuticle of hair fibres from most mammals (Wertz and Downing, 1988; Peet et al., 1992).

1II. CONCLUSIONS Although much data has been accumulated on the biological distribution of 18-methyleicosanoic acid and its specific localization in the hydrophobic barrier layers of mammalian hair fibre cuticle cells, more research is needed on the details of the distribution within the boundary layers of the cuticle cells. The raison d'6tre of this uncommon fatty acid has also yet to be established. Three possibilities have been considered: (1) Inhibition of bilayer (micelle) formation due to the presence of the branched chain; (2) The methyl branch near the terminal (i.e. at the hydrophobic end) of the fatty acid could aid in improving the hydrophobic "umbrella" at the

surface, especially given that a limited number of cysteine residues are available for covalent bond (thioester) formation in the underlying protein layer. (3) The existence of the branch may render the hydrophobic layer more resistant to biological degradation. Acknowledgements--The authors are grateful to Ms Melinda Cairns for excellent assistance in preparing the manuscript. Much of the work concerning MEA reviewed here has been supported by the International Wool Secretariat and the Australian Government through the International Wool Secretariat and the Cooperative Research Centre for Premium Quality Wool.

REFERENCES Bateup, B. O., Cook, J. R., Feldtman, H. D. and Fleichfresser, B. E., 1976. Wettability of wool fibres. Textile Research Journal, 46, 720-723. Brooks, J. H. and Raman, M. S., 1986. Surface energy of wool. Textile Research Journal, 56, 164-171. Abrahamsson, S., Stallberg-Stenhagen, S. and Stenhagen, E., 1963. The higher saturated branched chain fatty acids. In Progress in the Chemistry of Fats and Other Lipids, eds R. T. Holman and T. Malkin, Vol. 7, pp. 1-164. Pergamon Press, New York. Ahern, D. G. and Downing, D. T., 1973. Skin lipids of the Florida indigo snake. Lipids, 9, 8 14. Banks-Schlegel, S. and Green, H., 1981. Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured epithelia. Journal of Cell Biology, 90, 732-737. Birbeck, M. S. C. and Mercer, E. H., 1957. The electron microscopy of the human hair follicle. 1. Introduction and the hair cortex. Journal of Biophysics, Biochemistry and Cytology, 3, 203-214. Birbeck, M. S. C. and Mercer, E. H., 1957. The electron microscopy of the human hair follicle. II. The hair cuticle. Journal of Biophysics, Biochemistry and Cytology, 3, 215 221. Birbeck, M. S. C. and Mercer, E. H., 1957. The electron microscopy of the human hair follicle. IlL The inner root sheath and trichohyaline. Journal qf Biophysies. Biochemistry and Cytology, 3, 223 229. Bizzozero, O. A., McGarry, J. F. and Lees, M. B., 1987. Autoacylation of myelin proteolipid protein with coenzyme A. Journal o[' Biological Chemistry, 262, 13550-13557. Bourguignon, L. Y. W., Kalomiris, E. L. and Lokeshwar, V. B., 1991. Acylation of the lymphoma transmembrane protein, gp85, may be required for gp85-ankyrin interaction. Journal o[' Biological Chem&try, 266, 11761 11766. Braun, V., 1975. Covalent lipoprotein from the outer membrane of Escheriehia coli. Biochimica Biophysica Aeta. 415, 335 377. Carr, C. M., Leaver, 1. H. and Hughes, A. E., 1986. X-ray photoelectron spectroscopic study of the wool fiber surface. Textile Research Journal, 56, 457~161. Crabtree, H. E. and Truter, E. V., 1974. Constitution of some steroid esters of wool wax. Journal of the Science of Food and Agriculture, 25, 1441 1449. Currey, K. V. and Golding, S., 1971. Hair lipids. 1. The extraction of fatty materials from hair clippings. Journal of the Society of Cosmetic Chemists, 22, 681 689. Dasgupta, A., Ayanoglu, E. and Djerassi, C., 1984. Phospholipid studies of marine organisms: new branched fatty acids from Strongylophora durissima. Lipids, 19, 768-776. Downing, D. T., 1963. The fatty acid composition of vernix caseosa. Australian Journal of Chemistry, 16, 679-682. Downing, D. T., 1976. Mammalian waxes. In Chemistry and Biochemistry q]" Natural Waxes, ed. P. E. Kolattukudy, pp. 17~8. Elsevier, Amsterdam. Downing, D. T., 1992. Lipid and protein structures in the permeability barrier of mammalian epidermis. Journal of Lipid Research, 33, 301 313. Downing, D. T. and Lindholm, J. S., 1982. The skin surface lipids of the cow. Comparative Biochemist O, and Physiology, 73B, 327 330.

t8-Methyleicosanoic Acid in Hair Downing, D. T. and Stewart, M. E., 1987. Skin surface lipids of the mole Sealopus aquaticus. Comparative Biochemist O' and Physiology, 86B, 667 670. Downing, D. T., Kranz, Z. H. and Murray, K. E., 1960. An investigation of the aliphatic constituents of hydrolysed wool wax by gas chromatography. Australian Journal of Chemistry, 13, 80 94. Elliot, R. L. and Manogue, B., 1952. An electron microscope study of the surface structure of wool. Journal of the Socie b, o!" Dyers and Cohmrists, 68, 12 14. Erra, P., Codercfi, L., Julia, R., Infante, R. and Garcia Dominguez, J.. 1986. Shrinkage modifications of wool fabrics by sulphate treatments in aqueous organic solvent media; effect of the organic solvent on internal lipids. Textile Research Journal, 56, 611 616. Evans, D. J. and LanczkL M., 1997. Cleavage of integral surface lipids of wool by aminolysis. Textile Research Journal, 67, 435M.44. Evans, D. J.. Leeder, J. D., Rippon, J. A. and Rivett, D. E., 1985. Separation and analysis of the surface lipids of the wool fibre.

Proeeeding.~ 7th hTternational Wool Textile Research Con/i'renee. I, 135 142. Feughelman, M., 1959. A two-phase structure for keratin fibres. Te ~tile Research Journal. 29, 223-228. Fraser, R. D. B. and MacRae, T. P., 1980. Molecular structure and mechanical properties of keratins In the mechanical properties of biological materials. In Symposiunl o/the Society.fi)r Exper#nental Bhdogy No. 43, ed. J, F. V. Vincent and J. D. Curry, pp. 211 246. Cambridge University Press. Fraser, R. D. B.. MacRae. T. P. and Miller, A., 1965. X-ray diffraction patterns of ,-fibrous proteins. Journal q/Molecular Biology. 14, 432 442. Fraser, R. D. B., MacRae, T. P. and Rogers, G. E., 1972. Keratins, Their Composition. Structure and Biosynthesis. Charles 121. Thomas, Springfield, Illinois. Fraser, R. D. B., Jones, L. N., MacRae, T. P., Suzuki, E. and Tulloch, P. A., 1980. The fine structure of the wool fibre. Proceedings q/the Sixth hlternatiomd Wool Textile Con]erence (Pretoria), 1, 1 33 Freney, M. R. and Lipson, M., 1940. Effects of concentrated acqueous and certain non-acqueous solutions of alkali upon wool. Nature, 145, 25 26. Garton, G. A., 1985. Aspects of the chemistry and biochemistry of branched-chain fatty acids. Chemistry and lndusto' (London), 9, 295 300. Gebhardt. A., Bosch, J. V., Ziemiecki, M. A. and Friis, R. R., 1984. Rous sarcoma virus p19 and gp35 can be chemically crosslinked to high molecular weight complexes. Journal of Molecular Biolo
483

Jones, L. N., 1994. Surface membranes in developing mammalian hair follicles. Journal of lnvestigative Dermatology, 102, 559. Jones, L. N. and Pope, F. M., 1985. Isolation of intermediate filament assemblies from human hair follicles. Journal q/Cell Bioh;gy. 101, 1569 1577. Jones, L. N. and Rivett, D. E., 1995. Effects of branched chain 2-oxo acid dehydrogenase deficiency on hair in maple syrup urine disease. Journal qf lnvest~ative Dermatology, I0~, 688. Jones, L. N., Cholewa, M., Kaplin, 1. J., Legge, G. J. ~. and Ollerhead, R. W., 1990. Elemental distributions in keratin fibre/follicle sections. Proceedings of the Eighth hTternatiomd 14~ol Te.vtilc Research CanI~,renee. CJwistehureh, 1,246 255. l Jones, L. N., Kaplin. I. J. and Legge, G, J. F.. 19~3. Distributions of protein moieties in a-keratin sections..]ourmd q! Conll~uter Assisted Micro.scopy, 5, 85 89. Jones, L. N., Horr, T. J. and Kaplin, 1. J., 1994. Fornaation of surlace membranes in developing mammalian hair fibres. Micron. 24, 589 595. Jones, I. N., Peet, D. J., Danks, D. M., Negri. A. P. ai]d Rivett, D. E.. 1996. Hairs from patients with maple syrup urin~ disease show a structural defect in the fibre cuticle. Journal !q/ Investigative Dermatoloe.' 106, 461M-64. Jones, k. N., Rivett, D. E., Tucker, D. J., 1997. Wbol and Related Mammalian Fibres. In Handhook q/ Fibre Science and Technology, eds E. M. Pearce and M. Lewin, Marcel Dekker, New York, 355 413. Kalkbrenner, U., K6rner, A.. H6cker. H. and Rivdtt, D. E., 1990. Studies on the composition of the wool cuticle, l~a Proceedings o! 81h International Wool Textile Research Con!~'r~,nee, ed. G. H. Crawshaw, Vol. 1, pp. 398 407. Wool Research !Organisation of New Zealand, Christchurch. Kaneda. T. and Smith, E. J., 1980. Relationship of primer specificity of fatt,,, acid denovo synthetase to latty acid cornposlnon m ten species of bacteria and yeasts. Canadian Journal !?! Micr~)hiolo~l'. 26, 893 898. Karkkainen, J.. Nikkari, T., Ruponen, S. and Haati, iE., 1965. Lipids of vemix caseosa. Journal q/ htvestiL,ative D~,rmatoh~gy, 44, 333 338. Kassenbeck, P., 1961. Applications de I'ultra-microtobmie a l't?tude de la structure des fibres k6ranitiques. In Structm'e d~~]a Laine. p. 50. Institut Textile de France, Paris. King, N. L. R. and Bradbury, J. H., 1968. The chemical composition of wool. V: The epicuticle. 4ustralian ,hmrnM q/ Bioloeieal Sciences. 21,375 384. K6pke, V. and Nilssen, B., 1960. Wool surface properties and their influence on dye uptake--a microscopical study i. Journal q[ the Textile h~stitute, 51, T1398 T1413. K6rner, A , Kalkbrenner, U., Kaufmann. R.. H6cker i H. and Rivett, D. E., 1990. X-ray photo electron spectroscopy tXPS) measuremcnts of the wool fibre surface. In hTternation!d Wool Textile Organi.sathm Technical Committee, Report No. ~, Nice Meeting, pp. 1 5. Korner, A.. Hocker, H. and Rwett, D. E., 1)92. i The ratty acid composition of lipids from the wool cell memlbrane complex. Fresenius Journal o/ Analvtieal (7wntistry, 344, ~'~5pl 509. Lazarides, E., 1980. Intermediate filaments as mechanical integrations of cellular space. Nature, 283, 249 256. Leeder, J. D. and Rippon, J. A., 1985. Changes [induced in the properties of wool by specific epicuticle modific',Jtion. Journal o[ the Society o/Dyers and Colourista. 101, 11 16. Leeder, J. D., Rippon. J. A. and Riven. D. E.. 1985. iModification of the surface properties of wool by treatment with a~ahydrous alkali. In Pro<'eedings o/ the 7th International ~ m l Textile Research Cot!/brenee, ed. M. Sakamoto, Vol. IV. pp. 312 J21. The Society of Fiber Science and Technology (Japan), Tokyo[. Leeder, J. D., 1986. The cell membrane complex abd its influence o n the properties of the wool fibre. 11"ool Scie!tce Review, 63, 3 35. Lindberg, J., 1953. Relationship between various pr6perties of wool fibre, Textile Researeh Journal, 23. 67-76. 225 2~6 and 573 588. Lindberg, J.. Philip, B. and Gralen. N., 1948. OccUrrence of thin membranes in the structure of wool..Vatur¢,. 162!/458~,59. Lindley. H., 1977. The chemical composition and str~ucture of wool. In Chemistry o! Natural Protein Fihrc~', ed. R. S. Asquith. pp. 147 191. Plenum Press, New York. Logan, R. I., Rivett, D. E., Tucker. D. J. and Huds6n, H. F., 1989. Analysis of the intercellular and membrane lipi~ts of wool and other animal fibers, fextih" Re.search .hmrnal. 59,1109 113. .

.

.

.

"

(

i

-

•

484

L.N. Jones and D. E. Rivett

Logan, R. I., Jones, L. N. and Rivett, D. E., 1990. Morphological changes in wool fibres after solvent extraction. In Proceedings of the. 8th International Wool Textile Research Conference, ed. G. H. Crawshaw, Vol. I, pp. 408-418. Wool Research Organisation of New Zealand, Christchurch. Low, M. G., 1989. The glycosyl-phosphotidylinositol anchor of membrane proteins. Biochimica Biophysica Acta, 988, 427454. Mansour, M. P. and Jones, L. N., 1988. Morphological changes in wool after solvent extraction and treatment in hot acqueous solvent. Textile Research Journal 42, 530-535. Marshall, R. C., Orwin, D. F. G. and Gillespie, J. M., 1991. Structure and biochemistry of mammalian hard keratin. Electron Microscopy Review, 4, 47 83. Mcllhinney, R. A. J., 1990. The fats of life: the importance and function of protein acylation. Trends in Biochemical Science. 15, 387-391. Mcllhinney, R. A. J., Chadwick, J. K. and Pelly, S. J., 1987. Studies on the cellular location, physical properties and endogenously attached lipids of acylated proteins in human squamouscarcinoma cell lines. Biochemical Journal 244, 109 115. Montagna, W. and Parakkal, P. F., 1974. The Structure and Function of Skin, 3rd edn. Academic Press, New York. Motiuk, K., 1979. Wool wax acids: a review. Journal of the American Oil Chemists Society, 56, 91-97. Naito, S., Yorimoto, N. and Kuroda, Y., 1996. The structure of bound lipids of human hair fibres and its physical properties. Proceedings of" the 9th International Wool Textile Research Conference, Biella, Italy, 2, 367-374. Negri, A. P., 1993. Covalently bound fatty acids in the cell membrane complex of keratin fibres. PhD Thesis, Royal Melbourne University of Technology, Australia. Negri, A. P., Cornell, H. J. and Rivett, D. E., 1991. The nature of covalently bound fatty acids in wool fibres. Australian Journal of Agricultural Research, 42, 1285 1292. Negri, A. P., Cornell, H. J. and Rivett, D. E., 1992. Effects of processing on the bound and free fatty acid levels in wool. Textile Research Journal 62, 381-387. Negri, A. P., Cornell, H. J. and Rivett, D. E., 1993. A model for the surface of keratin fibres. Textile Research Journal 63, 109 115. Negri, A. P., Cornell, H. J. and Rivett, D. E., 1993. The modification of the surface diffusion barrier of wool. Journal of the Society of Dyers and Colourists, 109, 29~301. Nicolaides, N., Apon, J. M. B. and Wong, D. H., 1976. Further studies of the saturated methyl branched fatty acids of vernix caseosa lipid. Lipids, 2, 781-790. Nunn, J. R., 1951. Wool wax. Part 1, Synthesis of some anteiso-acids. Journal of the Chemical Society, 154, 1740-1744. O'Brien, P. J., St Jules, R. S., Reddy, T. S., Bazan, N. G., Zatz, M., 1987. Acylation of disc membrane rhodopsin may be nonenzymatic. Journal of Biological Chemistry, 262, 5210-5215. O'Donnell, A. G., Nahaie, M. R., Goodfellow, M., Minnikin, D. E. and Hajek, V., 1985. Numerical analysis of fatty acid profiles in the identification of Staphylococci. Journal of General Microbiology, 131, 2023-2033. Omary, M. B. and Towbridge, I. S., 198l. Covalent binding of the transferring receptor in cultured human cells. Journal of Biological Chemistry, 256, 47154718. Orwin, D. F. G., 1970. A polysaccharide containing cell coat on keratinizing cells of the Romney wool follicle. Australian Journal of Biological Sciences, 23, 623-635. Orwin, D. F. G., 1971. Cell differentiation in the lower outer sheath of the Romney wool follicle: a companion cell layer. Australian Journal of Biological Sciences, 24, 989 999. Orwin, D. F. G., 1979a. Cytological studies on keratin fibres. In Fibrous Proteins, Scientific, Industrial and Medical Aspects, eds D. A. D. Parry and L. K. Creamer, Vol. 1, pp. 271-297. Academic Press, New York. Orwin, D. F. G., 1979. The cytology and cytochemistry of the wool follicle. International Review of Cytology, 160, 331-374. Orwin, D. F. G. and Thompson, R. W., 1973. Plasma membrane differentiations of keratinizing cells of the wool follicle. IV. Further membrane differentiations. Journal of Ultrastructure Research, 45, 41-49. Pangburn, M. K., 1992. Spontaneous thioester bond formation in a2-macroglobulin. C3 and C4. FEBS Letters, 308, 280-282. Parry, D. A. D., Fraser, R. D. B. and MacRae, T. P., 1979. Repeating patterns of amino acid residues in the sequences of some high-sulphur proteins from a-keratin. International Journal of Biological Macromolecules, 1, 17 22.

Peet, D. J., Wettenhall, R. E. H., Rivett, D. E. and Allen, A. K., 1992. A comparative study of covalently bound fatty acids in keratinized tissues. Comparative Biochemistry and Physiology, 102B, 363-366. Peet, D. J., 1994. Protein-bound fatty acids in mammalian hair fibres. Ph.D. Dissertation, University of Melbourne. Peet, D. J., Wettenhall, R. E. H. and Rivett, D. E., 1994. The chemistry of the cuticle surface of keratin fibers. Textile Research Journal, 64, 58 59. Rivett, D. E., 1991. Structural lipids of the wool fibre. Wool Science Review. 67, 1 25. Rivett, D. E., Logan, R. 1., Tucker, D. J., and Hudson, A., 1987. The lipid composition of speciality animal fibres. Proceedings of the 1st International Symposium of Specialty Animal Fibres, Deutches Wollforschungsinstitut an der Technischen Hochschule, Aachen, 1, 128 136. Rogers, G. E., 1959. Electron microscope studies of hair and wool. Annals of the New York Academy of Science, 83, 378 399. Rogers, G. E., 1964. Structural and biochemical features of the hair follicle. In The Epidermis, eds W. Montagna and W. C. Lobitz, pp. 179 236. Academic Press, New York. Rogers, G. E., 1983. The occurrence of citrulline in structural proteins of the hair follicle. In Biochemistry and Physiology of the Skin, ed. L. A. Goldsmith, Vol. 1, pp. 511-521. Oxford University Press, New York. Rogers, G. E. and Harding, H. W. J., 1976. Molecular mechanisms in the formation of hair. In Biology and Disease of the Hair, eds K. Toda, Y. Ishibashi and F. Morikawa, pp. 411435. University of Tokyo Press, Tokyo. Roze, U., Locke, D. C. and Vatakis, N., 1990. Antibiotic properties of porcupine quills. Journal of Chemical Ecology, 16, 725 734. Sankaran, K. and Wu, H. C., 1993. Bacterial lipoproteins. In Lipid Modifications of Proteins, ed. M. J. Schlesinger, pp. 163-182. CRC Press, Florida. Schlesinger, M. J., Veit, M. and Schmidt, M. F. G., 1993. Palmitoylation of cellular and viral proteins. In Lipid Modifications of Proteins, ed. M. J. Schlesinger, pp. 1 20. CRC Press, Florida. Schmidt, M. F. G., 1989. Fatty acylation of proteins. Biochimica Biophysica Acta, 988, 411426. Schultz, A. M., Henderson, L. E. and Oroszlan, S., 1988. Fatty acylation of proteins. Annual Review of Cell Biology, 4, 611 647. Schwan, A., Zahn, H., 1980. Investigations of the cell membrane complexes in wool and hair. Proceedings of the Sixth International Wool Textile Research Conference, Pretoria, 2, 2941. Sideris, V., Leaver, I. H., Holt, L. A. and Jones, L. N., 1992. Photomodification of the cell membrane complex of wool--the effect on dyeability. Journal of" the Society of Dyers and Colourists, 108, 436~40. Speakman, P. T., 1984. Wool fibres, In Fibre Chemistry Handbook of Fibre Science and Technology, eds M. Lewin and E. M. Pearce, Vol. IV, pp. 589-646. Marcel Dekker,lnc., New York. Spei, M., Zahn, H., 1979. X-ray small angle examination of swollen fibre keratins. Melliand Textilberichte, 60, 523-525. Swift, J. A., 1977. The histology of keratin fibres. In Chemistry oJ" Natural Protein Fibres, ed. R. S. Asquith, pp. 81 146. Plenum Press, New York. Swift, J. A. and Holmes, A. W., 1965. Degradation of human hair by papain III: Some electron microscope observations. Textile Research Journal 35, 1014-1019. Tanaka, K. and Rosenberg, L. E., 1983. Disorders of branched chain amino acid and organic metabolism. In The Metabolic Basis of Inherited Disease, eds J. B. Stanbury, J. B. Wyngaarden, D. S. Frederickson, J. L. Goldstein and M. K. Brown, 5th edn, pp. 400-473. McGraw-Hill, New York. Towler, D. A., Gordan, J. I., Adams, S. P. and Glaser, L., 1988. The biology and enzymology of eukaryotic protein acylation. Annual Review of Biochemistry, 57, 69-99. Valero-Guillen, P. L., Martin Luengo, F., Larsson, L. and Jimenez, J., 1987. Demonstration of 2-methyl branched-chain fatty acids in some rapidly growing mycobacteria. FEMS Mierobiology Letters, 44, 303 305. von Allw6rden, K., 1916. Die eigenschaften der Schafwolle und eine neue untersuchungs Methode zum nachweis gesch~idigter Wolle auf chemischem Wege. Zeitschrifi fiir Angewandte Chemic, 29, 77 78. Ward, R. J., Willis, H. A., George, G. A., Guise, G. B., Denning, R. J., Evans, D. J. and Short, R. D., 1993. Surface analysis of wool by X-ray photoelectron spectroscopy and static secondary ion mass spectrometry. Textile Research Journal, 63, 362 368.

18-Methyleicosanoic Acid in Hair Weitkamp, 1945. The acidic constituents of degras. A new method of structural elucidation. Journal (?[the American Chemical Society, 67, 447 454. Weitzel, G.. 1951. Antituberkul~ire Wirkung methylverzweigter Fetts/iuren and Alkohole. Zeitschr(ft fiir Physiologische Chemic. 288, 174 188. Wertz, P. W. and Downing, D. T., 1988. Integral lipids of human hair. Lipids, 23, 878 881. Wertz, P. W. and Downing, D. T.. 1989. Integral lipids of mammalian hair. Comparative Biochemistry Physiology, B." Comparative Biochemistry. 92h, 759 761. Wheatley. V., kipkin, G. and Woo, T. H., 1967. Lipogenesis from amino acids in perfused isolated dog skin. Journal o[' Liptd Research. 8, 84 89. Wilkinson, D. I. and Karasek, M. A., 1966. Skin lipids of a normal and a mutant (asebic) mouse strain. Journal c~[' Invest(~ative Dermatology, 47, 449~,55. Wong, S. S., 199t. Chemist o, o['protein conjugation and crosslinking. p. 222. CRC Press Inc., Boca Raton, Florida. Woods, J. L. and Orwin, D. F. G., 1982. The cytology of cuticle scale pattern formation in the wool follicle. Journal o[' l~Ttrastructure Research. 80, 230 242.

485

Yorimoto, N. and Naito, S., 1994. Physical and chemical properties of integral lipids in hair cell membrane complex. Proceedin,es (~/ the International Symposium on Fihre Science and Technolo~,,v, p. 215. Yokohama, Japan. Zahn, H., 1951. Die Epikutikula und die allw6rdensche Reaktion. Melliand Textilberichte. 32, 419 420. Zahn, H., 1977. Fibres in macromolecular chemistry. Lenzmeer Berichte, 42, 19 34. Zahn, H., Messinger, H. and H6cker, H., 1994. Covalently linked fatty acids at the surface of wool: part of the "'cuticle cell envelope". Textile Research Journal, 64~ 554- 555. Zettergren. J. G., Peterson, L. L. and Wuepperi K. D., 1984. Keratolinin the soluble substrate of epidermal iransglutaminase EC-2.3.2.13 from human and bovine tissue. Pt~<)ceedin~,s(?/' the National Academy of Sciences USA, 81~ 238 242. Zhang. B., Healy, P. J., Zhao, Y., Crabb, D. W. and Harris, R. A., 1990. Premature translation termination of the prpe-Elt~subuni! of the branched chain a-ketroacid dehydrogemlsc as a cause of maple syrup urine disease in polled Hereford dalves..Iourna/~?/ Biological Chemi.stry, 265, 2425 2427.