J. Cosmet. Sci., 58, 11-17 Oanuary/February 2007)
Structural analysis of the cell membrane complex in the
human hair cuticle using microbeam X-ray diffraction:
Relationship with the effects of hair dyeing
TAKAFUMI INOUE, YOSHIMICHI IWAMOTO,
NOBORU OHTA, KATUAKI INOUE, and NAOTO YAGI,
Basic Research Laboratory, Kanebo Cosmetics, Inc., 5-3-28 Kotobuki-cho,
Odawara, 250-0002, Japan (T.I.), Beauty Care Laboratory, Kanebo
Home Products Ltd., 134 Goudo-cho, Hododaya-ku, Yokohama,
240-0005, Japan (Y.I.), Japan Synchrotron Radiation Research
Institute UASRI/SPring-8)1 Hyogo, 679-51981 Japan (N.0.1 K.I.,
N.Y.)
Accepted for publication September 20, 2006.
Synopsis
This article deals with the structure of the cell membrane complex (CMC) in the human hair cuticle. The
microbeam X-ray provided a pattern of small-angle scattering from the CMC in the cuticle with no sample
preparations, including slicing and pre-staining of hair. The thickness of the 13- and 8-layers, substructure
in CMC, was estimated by analysis of the scattering pattern. We used hair samples extracted with several
solvents, and found that solvent extraction changed the thickness of the 13- and 8-layers in a manner
dependent on the type of solvent. Extraction of hair with solvent was also shown to have effects on the extent
of dyeing. There was a high correlation between the extent of dyeing and the thickness of the 8-layer, i.e.,
a thin layer tended to show a high amount of dyeing, whereas there was no significant correlation between
the thickness of the 13-layer and the extent of dyeing.
INTRODUCTION
The cuticle is the outermost layer of a hair fiber and is made up of a stack of approxi-
mately ten sheet-like cells that is 0.5 µm thick and roughly 60 µm square. The surface
of each cell is covered by a thin layer of lipids called the 13-layer (2.5 to 4.0 mm thick),
and these lipids are separated between adjacent cells by the 8-layer (15 to 18 nm thick),
which acts as an intercellular cement (1). The exact composition of the 8-layer is still
unknown however, it has been proposed to contain non-keratinous proteins (2). This
lipid-protein cement-lipid structure is called the cell membrane complex (CMC) and is
the only substructure that continuously fills the intercellular spaces of hair fibers.
Address all correspondence to Takafumi Inoue.
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12 JOURNAL OF COSMETIC SCIENCE
Therefore, it is considered to be an important pathway for the penetration of substances
into the inner parts of the hair (3 ).Observations of cuticles by transmission electron
microscopy have shown evidence of a penetration pathway through the CMC (4,5)
however, that observation method requires the use of heavy metals and thin-sliced hair
samples. We considered that a less invasive technique is needed to understand the
penetration properties of the cuticle.
In the present study, we utilized microbeam X-ray diffraction for structural analysis of
the CMC in the hair cuticle. With this method, we were able to elucidate the structure
of the CMC in whole hair samples under ambient conditions without pretreatment, such
as staining with heavy metals. The pioneering experiment on the structure of the cuticle
using a microbeam X-ray technique was performed by Kreplak et al. (6), in which they
observed small-angle X-ray scattering (SAXS) patterns of cuticles, and determined the
average thickness values of the r,3- and 8-layers in the CMC. Thereafter, a mathematical
model for precise estimation of the thickness of those layers was proposed by Ohta et al.
(7). Using that model, we determined the thickness of the r,3- and o-layers in hair fibers
treated with various solvents. Our results showed a relationship between the CMC
structure and the extent of hair dyeing as a possible indicator of penetration.
MATERIALS AND METHODS
HAIR SAMPLES
For SAXS-pattern experiments, hair strands were obtained from Japanese women who
had not used any chemical treatments and were then washed with a 0.2% sodium laureth
sulfate solution. After being rinsed with deionized water and drying on a paper towel,
the strands were subjected to extraction with four different solvents, methanol, acetone,
hexane, and a chloroform/methanol mixture (2: 1 v/v) at 3 7°C for six hours. After cutting
to a length of 8 cm, each hair fiber was fixed on a hair holder and subjected to SAXS
analysis.
DYEING EXPERIMENTS
For the dyeing experiments, strands of gray hair (1 gram, 10 cm BM-W, Beaulax,
Tokyo, Japan) were used and subjected to extraction with the four solvents, using the
same method noted above. The hair tresses were then soaked in a solution containing 3.0
g of acid orange 7, 9.0 g of citric acid, and 1.0 g of sodium citrate per liter (pH 2.7) at
35°C for five minutes. After rinsing the tresses with deionized water and drying, the
color index [light (1), red (a), and yellow-blue (b)} of each hair strand was determined
using a chromometer (CR-400, Konica Minolta Sensing, Osaka, Japan). The extent of
dyeing was then determined by calculating the differences in the indexes between
untreated and dyed hair samples (i.e., Lil, Lia, db) using the following formula: dyeing
extent (dE) =SQRT{(d1)2 +(Lia)2 +(Lib)2 }.
SAXS EXPERIMENT
SAXS experiments were performed with a 5-µm high-flux beam (BL40XU High Flux
Beamline) at the SPring-8 facility (Hyogo, Japan) using human hair fibers under an