The structure of people’s hair

Fei-Chi Yang, Yuchen Zhang and Maikel C. Rheinstädter

Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, Canada

ABSTRACT
Hair is a filamentous biomaterial consisting mainly of proteins in particular keratin.
The structure of human hair is well known: the medulla is a loosely packed, disor-
dered region near the centre of the hair surrounded by the cortex, which contains
the major part of the fibre mass, mainly consisting of keratin proteins and structural
lipids. The cortex is surrounded by the cuticle, a layer of dead, overlapping cells
forming a protective layer around the hair. The corresponding structures have been
studied extensively using a variety of different techniques, such as light, electron and
atomic force microscopes, and also X-ray diffraction. We were interested in the ques-
tion how much the molecular hair structure differs from person to person, between
male and female hair, hair of different appearances such as colour and waviness. We
included hair from parent and child, identical and fraternal twins in the study to see if
genetically similar hair would show similar structural features.
The molecular structure of the hair samples was studied using high-resolution
X-ray diffraction, which covers length scales from molecules up to the organization
of secondary structures. Signals due to the coiled-coil phase of α-helical keratin
proteins, intermediate keratin filaments in the cortex and from the lipid layers in the
cell membrane complex were observed in the specimen of all individuals, with very
small deviations. Despite the relatively small number of individuals (12) included in
this study, some conclusions can be drawn. While the general features were observed
in all individuals and the corresponding molecular structures were almost identical,
additional signals were observed in some specimen and assigned to different types of
lipids in the cell membrane complex. Genetics seem to play a role in this composition
Submitted 7 August 2014 as identical patterns were observed in hair from father and daughter and identical
Accepted 22 September 2014 twins, however, not for fraternal twins. Identification and characterization of these
Published 14 October 2014 features is an important step towards the detection of abnormalities in the molecular
Corresponding author structure of hair as a potential diagnostic tool for certain diseases.
Maikel C. Rheinstädter,
rheinstadter@mcmaster.ca
Academic editor Subjects Biophysics
Mikko Karttunen Keywords Human hair, Molecular structure, X-ray diffraction, Keratin, Intermediate filament,
Additional Information and Coiled-coil proteins, Alpha helix, Cell membrane complex
Declarations can be found on
page 15
DOI 10.7717/peerj.619 INTRODUCTION
Human scalp hair is a bio-synthesized material that has a complex internal structure.
Copyright
2014 Yang et al. The adult human hair is around 20–180 µm in width, and generally grows to a length of
Distributed under
approximately 90 cm. It consists of many layers including the cuticle, the cortex and the
Creative Commons CC-BY 4.0 medulla. These layers are bound together by the cell membrane complex (Robbins, 2012).

OPEN ACCESS

How to cite this article Yang et al. (2014), The structure of people’s hair. PeerJ 2:e619; DOI 10.7717/peerj.619
 The structure of human hair is well known and in particular X-ray diffraction revealed
details of molecular structure and organization within hair (Fraser et al., 1986; Briki et al.,
2000; Busson, Engstrom & Doucet, 1999; Randebrook, 1964; Fraser, MacRae & Rogers, 1962;
Kreplak et al., 2001b; Wilk, James & Amemiya, 1995; Pauling & Corey, 1951; Ohta et al., 2005;
Astbury & Street, 1932; Astbury & Woods, 1934; Astbury & Sisson, 1935; Franbourg et al.,
2003; Rafik, Doucet & Briki, 2004; James et al., 1999; Veronica & Amemiya, 1998; Briki et
al., 1999; James, 2001). In particular microbeam small angle X-ray scattering techniques
enables the determination of hair structure with a high spatial resolution (Iida & Noma,
1993; Busson, Engstrom & Doucet, 1999; Kreplak et al., 2001b; Ohta et al., 2005; Kajiura et
al., 2006). It is a long-standing question whether changes in the molecular structure of nail
or hair can be related to certain diseases and potentially be used as a diagnostic tool. Such a
technique would in particular be interesting and relevant as simple, non-invasive screening
method for cancer (James et al., 1999; Briki et al., 1999; James, 2001). Abnormal kinky hair
is, for instance, characteristic of giant axonal neuropathy (Berg, Rosenberg & Asbury, 1972).
The purpose of this study is to use X-ray diffraction to analyze the structure of human
scalp hair for individuals with differing characteristics. The 12 individuals in this study
include hair from men and women and hair of different colour and appearance, such as
straight, wavy and curly. In addition to appearance, the study also includes hair from a
father and daughter, a pair of identical and a pair of fraternal twins to include genetic
similarities. All hair was collected from healthy individuals and care was taken that the hair
was not permed or dyed before the experiments.
Signals due to the coiled-coil organization of α-helical keratin proteins and intermediate
filaments in the cortex, and lipids in the cell membrane complex were observed in the hair
of all individuals. While these general features occur independent of gender or appearance
of the hair with a very small standard deviation in the underlying molecular dimensions,
we find significant differences between individuals in the composition of the plasma
membrane in the cell membrane complex. Genetics appear to be the most important
factor that determines membrane composition, as no or little differences were observed in
genetically related hair samples, rather than external factors such as nutrition or hair care
products.

Properties of human hair

The cuticle is the outermost layer formed by flat overlapping cells in a scale-like
formation (Robbins, 2012). These cells are approximately 0.5 µm thick, 45–60 µm long
and found at 6–7 µm intervals (Robbins, 2012). The outermost layer of the cuticle, the
epicuticle, is a lipo-protein membrane that is estimated to be 10–14 nm thick (Swift &
Smith, 2001). Beneath that is the A layer with a high cysteine content and a thickness
of 50–100 nm, the exocuticle with again a high cysteine content and a highly variable
thickness ranging from 50 to 300 nm, and the endocuticle with a low cysteine content and a
thickness also ranging from 50 to 300 nm.
The majority of hair fibre is the cortex which contains spindle shaped cells that lie
parallel along the fibre axis. These cortical cells were found to be approximately 1–6 µm

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 in diameter and 50–100 µm in length (Randebrook, 1964). In wool fibres as well as human
hair, the cortical cells were observed to be divided into different regions termed ortho-
cortex, paracortex and mesocortex (Mercer, 1953). The difference in distribution of these
cell types is an important factor for determining the curvature of the hair fibre (Kajiura et
al., 2006). In particular, straight hair tends to have symmetrical distribution of the ortho-
and paracortices whereas curly hair tends to have a non-symmetrical distribution of these
cortical cells (Kajiura et al., 2006). Most of the cortical cells are composed of a protein
known as keratin (Robbins, 2012).
At the molecular level, keratin is a helical protein (Pauling & Corey, 1950). There are two
types of keratin fibres that exist in hair: type I with acidic amino acid residues and type II
with basic amino residues. One strand of type I fibre and one strand of type II fibre spiral
together to form coiled-coil dimers. In turn, these dimers coil together in an antiparallel
manner to form tetramers (Crewther et al., 1983; Fraser et al., 1988).
When tetramers are connected from head to tail, they are known as protofila-
ments (Robbins, 2012). These tetramers or protofilaments are believed to interact together
to form a single intermediate filament which is approximately 75–90 Å in diameter. The
current model of an intermediate filament was proposed in the 1980’s and it involves
7 protofilaments surrounding a single core protofilament (Robbins, 2012; Fraser et al.,
1988). The intermediate filaments then aggregate together to form macro-filaments with a
diameter of 1000 to 4000 Å (Robbins, 2012; Randebrook, 1964). Between the intermediate
filaments is a matrix consisting of keratin associated proteins, which are irregular in
structure. The macro-fibrils consisting of intermediate filaments and the surrounding
matrix are the basic units of the cortical cell.
The cell membrane complex is the material that glues hair cells together. There exist
various types of cell membrane complexes: cuticle–cuticle, cuticle–cortex and cortex–cotex
depending on the location (Robbins, 2012). The general membrane structure is one
15 nm proteinous delta layer sandwiched by two 5 nm lipid beta layers (Rogers, 1959).
Much speculation still exist regarding the precise structure of the beta and delta layers.
However, it has been determined that 18-methyl eicosanoic acid, a covalently bound
fatty acid, exists in the upper beta layer in the cuticle–cuticle but not in cortex–cortex
membranes (Ward & Lundgren, 1954). In fact, most of the fatty acids in beta layers of
membranes in the cuticle–cuticle are covalently bound and most of the fatty acids in the
beta layers of cortex–cortex are non-covalently bound (Robbins, 2012). Further evidence
suggests that the fatty acids in cuticle–cuticle membranes are organized in a monolayer
whereas the fatty acids in cortex–cortex cell membranes are bilayers (Robbins, 2012). The
cuticle–cortex cell membrane complex is then a mixture of the two, with the side facing
the cuticle similar to cuticle–cuticle membranes and the side facing the cortex similar to
cortex–cortex membranes (Robbins, 2012).

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 Table 1 List of all hair samples in this study. The individuals include men and women and hair of
different appearance, such as thickness, colour and waviness, and also genetically related hair samples
from a father and daughter, a pair of identical and a pair of fraternal twins. Labeling agrees with the data
shown in Fig. 1.

Subject Gender Diameter(µm) ± SD Colour Appearance Special comment

1 F 30 ± 3 light blonde straight daughter
2 M 49 ± 5 brown/grey curly father
3 F 74 ± 7 black wavy –
4 M 50 ± 5 light brown curly –
5 F 49 ± 5 blonde curly –
6 F 43 ± 4 light brown straight –
7 F 61 ± 6 light brown wavy –
8 F 49 ± 5 black wavy –
9 F 31 ± 3 blonde wavy identical twin
10 F 66 ± 7 black straight fraternal twin
11 F 69 ± 7 black straight fraternal twin
12 F 48 ± 5 blonde curled identical twin

MATERIALS AND METHODS

Preparation of hair samples
This research was approved by the Hamilton Integrated Research Ethics Board (HIREB)
under approval number 14-474-T. Written consent was obtained from all participating
individuals. Scalp hair samples were gathered from 12 adults of various age, gender,
ethnicities, hair colour and hair curvature. It is of interest to note that there are 3 pairs
of study participants with genetic relations including a father and daughter, fraternal twins
and identical twins. Characteristics of the samples are listed in Table 1.
The hair samples gathered were cut into strands around 3 cm long. Care was taken
to not stretch or deform the hair strands during this process. For each subject, around
10 strands were taped onto a flexible cardboard apparatus as shown in Fig. 2. The cut-out
at the middle of the apparatus is where scattering occurs on the hair sample. The cardboard
apparatus is then mounted vertically onto the loading plate of the Biological Large Angle
Diffraction Experiment (BLADE) using sticky putty as shown in Fig. 2. All hair samples
were measured at room temperature and humidity of 22 ◦ C and 50% RH.

X-ray diffraction experiment

X-ray diffraction data was obtained using the Biological Large Angle Diffraction Exper-
iment (BLADE) in the Laboratory for Membrane and Protein Dynamics at McMaster
University. BLADE uses a 9 kW (45 kV, 200 mA) CuKα Rigaku Smartlab rotating anode
at a wavelength of 1.5418 Å. Focusing multi-layer optics provided a high intensity parallel
beam with monochromatic X-ray intensities up to 1010 counts/(s × mm2 ) at the sample
position. In order to maximize the scattered intensity, the hair strands were aligned parallel
to the parallel beam for maximum illumination. The slits were set such that about 15 mm
of the hair strands were illuminated with a width of about 100 µm. The effect of this

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 particular beam geometry is seen in the 2-dimensional data in Fig. 1: while it produces
a high resolution along the equator, the main beam is significantly smeared out in the
qz -direction up to qz -values of about 0.5 Å−1 , limiting the maximum observable length
scale to about 13 Å.
The diffracted intensity was collected using a point detector. Slits and collimators were
installed between X-ray optics and sample, and between sample and detector, respectively.
By aligning the hair strands in the X-ray diffractometer, the molecular structure along
the fibre direction and perpendicular to the fibres could be determined. We refer to
these components of the total scattering vector, Q, ⃗ as qz and q∥ , respectively, in the
following. An illustration of qz and q∥ orientations is shown in Fig. 3. The result of an
X-ray experiment is a 2-dimensional intensity map of a large area of the reciprocal space
−1 −1 −1 −1
of −2.5 Å < qz < 2.5 Å and −2.5 Å < q∥ < 2.5 Å . The corresponding real-space
length scales are determined by d = 2π/|Q| and cover length scales from about 3 to 90 Å,
incorporating typical molecular dimensions and distances for secondary protein and lipid
structures.
Integration of the 2-dimensional data was performed using Matlab, MathWorks. By
adding up the peak intensities along the qz and the q∥ directions, 1-dimensional data along
each of the two directions were produced. The qz intensity was integrated azimuthally for
an angle of 25 degrees over the meridian. The q∥ intensity was integrated azimuthally for an
angle of 25 degrees over the equator, as depicted in Fig. 3.
The fitting process is performed on both the 1-dimensional qz and the q∥ data produced
from integration. Distinguishable peaks were observed and fitted with the least numbers of
Lorentzian peak functions with an exponential decay background of the form (a · qb + c)
in the first run. Initial Parameters were chosen based on the observed positions, widths and
heights of the peaks and free to move through the entire q-range. The criterion for the final
parameters was to minimize the mean square of the difference between data intensity and
the fitted intensity. If the fitted intensity cannot conform to the shape of the data intensity,
more peaks will be added in the following runs until a good fit is acquired. This process was
repeated for all 12 subjects and performed with little or no consultation of previous fittings
to minimize bias.
As for the SAXS data, Gaussian functions are used instead. We note that the use of
optical components in the beam path has an impact on the shape of the observed Bragg
peaks: instead of Lorentzian or Bessel peak functions, Gaussian peak profiles were found to
best describe the SAXS peaks. The fitting process was the same as mentioned before: three
Gaussians were fitted to the SAXS data using free-to-move parameters and an exponential
decay background. However, for some subjects, the third peak was noisy and the least
mean square logarithm could not reach a good fit and hence the data was fitted with two
Gaussians, only.

RESULTS
A total of 12 adult subjects participated in this study. Details of gender and appearance of
the hair strands are listed in Table 1. About 10 strands were cut from the scalp, glued onto a

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 Figure 1 Two-dimensional X-ray data of all 12 subjects. The hair strands were oriented with the long
axis of the hair parallel to the vertical z-axis. The (q∥ ,qz )-range shown was determined in preliminary
experiments to cover the features observable by X-ray diffraction. The measurements cover length scales
from about 3–90 Å to study features from the coiled-coil α-keratin phase, keratin intermediate filaments
in the cortex, and the membrane layer in the membrane complex. While common features can easily be
identified in the 2D plots, subtle differences are visible, which are discussed in detail in the text.

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 Figure 2 The apparatus used to mount the hair strands in the experiment. The cardboard apparatus is
mounted vertically onto the loading plate of the Biological Large Angle Diffraction Experiment (BLADE)
using sticky putty.

Figure 3 Schematics of the X-ray setup and example X-ray data. The hair strands were oriented in the X-ray diffractometer with their long axis
along qz . Two-dimensional X-ray data were measured for each specimen covering distances from about 3–90 Å including signals from the coiled-coil
α-keratin phase, the intermediate fibrils in the cortex and from the cell membrane complex. The 2-dimensional data were integrated and converted
into line scans and fit for a quantitative analysis.

sample holder and aligned in the X-ray diffractometer. The resulting 2-dimensional X-ray
intensity maps of the reciprocal space reveal exquisite details of the molecular structure of
human scalp hair, as presented in Fig. 1. The hair strands were oriented with the long axis
of the hair parallel to the vertical z-axis. The displayed (qz ,q∥ )-range was determined to
cover the length scales of the features of interest in preliminary experiments.
The data in Fig. 1 show a distinct non-isotropic distribution of the diffracted intensity
with pronounced and well defined intensities along the long axis of the hair and in the
equatorial plane (the qz and q∥ -axes, respectively), indicative of a high degree of molecular
order in the hair strands. Some features were common in all specimens and assigned to
certain molecular components, as explained in the next section.

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 Figure 4 The hierarchical structure of hair in the cortex and cuticle. The main component of the cortex is a keratin coiled-coil protein phase. The
proteins form intermediate filaments, which then organize into larger and larger fibres. The hair is surrounded by the cuticle, a dead cell layer. The
common features observed in the X-ray data of all specimens are signals related to the coiled-coil keratin phase and the formation of intermediate
filaments in the cortex, and the cell membrane complex. Signal assignment and corresponding length scales are shown in the figure.

Assignment of common scattering signals

Coiled-coil protein phase in the cortex
The keratin proteins in the cortex are known to organize in bundles whose structures are
dominated by α-helical coiled-coils (Pauling & Corey, 1950; Pinto et al., 2014; Yang et al.,
−1
2014). The main features of this pattern are a ∼9.5 Å (corresponding to q∥ ∼ 0.6 Å )
equatorial reflection corresponding to the spacing between adjacent coiled-coils and a
−1
∼5.0 Å meridional reflection (corresponding to qz ∼ 1.25 Å ) corresponding to the
superhelical structure of α-helices twisting around each other within coiled-coils (Crick,
1952; Cohen & Parry, 1994; Lupas & Gruber, 2005). As displayed in Fig. 4, these signals were
observed in the X-ray data in all specimen and assigned to the coiled-coil protein phase. We
note that these peaks are related to generic α-helical coil structures of monomeric proteins,
and not specific to a certain type of protein.
Lipids in the cell membrane complex
The cell membrane complex mainly consists of lipid mono- and bilayers. The correspond-
ing scattering features correspond to a lamellar periodicity of about 45 Å, and rings at
spacings of about 4.3 Å, characteristic of the order within the layers (Busson, Engstrom
& Doucet, 1999). Both these features are observed in the 2-dimensional X-ray data of
all individuals in Fig. 1, as a ring-like scattering intensity at q-values of ∼0.1 Å−1 and a
broad, ring-like scattering at ∼1.5 Å−1 as a result of the lipid order within the membrane
layers. The corresponding diffraction signal has a maximum on the qz -axis, indicating a
preferential orientation of the membrane plane parallel to the surface of the hair.

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 Intermediate filaments in the cortex
The keratin coils organize into intermediate filaments whose structure and packing in
the plane of the hair result in additional scattering signals. The packing of these fibrils
by bundling into macro-fibrils is characterized by X-ray diffraction pattern by three
equatorial spots located at about 90, 45 and 27 Å (Busson, Engstrom & Doucet, 1999). The
corresponding signals are observed in the 2-dimensional data in Fig. 1. The exact position
of the features is, however, best determined in small angle diffraction experiments (SAXS),
which offer a drastically improved resolution, and will be shown below. We note that the
axial packing of coiled-coils within keratin filaments in hair gives rise to a number of fine
arcs along the meridian (z). The typically observed signal on the meridian at 67 Å, which
arises from the axial stagger between molecules along the microfibril (Briki et al., 2000;
Rafik, Doucet & Briki, 2004), could not be observed in our experiments due to the relaxed
resolution of the parallel beam in this direction. While the features observed in scattering
experiments are well known, the molecular architecture of the intermediate filaments is
still under discussion (Rafik, Doucet & Briki, 2004). Supercoiled coiled-coils or models that
involve straight dimers with different numbers of coils are being discussed.
The three features above were observed in all individuals in Fig. 1. The underlying
molecular structures will be quantitatively analyzed in the next section (Quantitative
analysis of scattering results). We note that additional features are seen in some of the
measurements in Fig. 1, mainly in the broad membrane ring at around 1.5 Å−1 which
indicates a difference in molecular composition of the cell membrane complex between
individuals. We will come back to these differences in the Discussion.

Quantitative analysis of scattering results

In order to quantitatively determine the position of the corresponding scattering features,
the 2-dimensional data for all 12 individuals were integrated in the equatorial plane
(q∥ -axis) of the hair fibres, and along the hair fibres (qz -axis). The resulting plots are
shown in Fig. 5. In the direction along the hair fibre axis (qz ), there are two major peaks
that were consistent among all subjects, one narrow peak around 5.0 Å and one broader
peak around 4.3 Å.
In the direction perpendicular to the hair fibre axis (q∥ ), there are also two major peaks
consistent among all subjects, one narrow peak around 9.5 Å and one broad peak around
4.3 Å. The total scattering profile was well fit by two Lorentzian peak profiles (and a back-
ground), whose positions is plotted in Fig. 5. The signals at 5.0 Å and 9.5 Å are in excellent
agreement with signals reported from coiled-coil keratin proteins (Pauling & Corey, 1950),
as depicted in the Figure. The broad signal at about 4.3 Å present in both directions is due
to the ring-like scattering from the lipids in the membrane component. As plotted in Fig. 5,
there is a narrow distribution of the corresponding length scales with standard deviations
of 9.51 ± 0.07 Å and 5.00 ± 0.02 Å for the keratin coiled-coils and 4.28 ± 0.08 Å for the
membrane signal, indicating that the common features observed in all individuals are well
defined with little spread in the corresponding molecular dimensions.
Due to the large length scales involved, the signals from intermediate filaments occur
at small scattering vectors, shown in Fig. 6. The Small Angle X-ray Scattering (SAXS)

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 Figure 5 Integration of the 2-dimensional scattering data in Fig. 1 in the equatorial plane (q∥ ) (A), and along the axis of the hairs (qz ) (C),
respectively, for all subjects. The two signals present in all individuals in the equatorial plane (q∥ ) correspond to the distance between two coiled
coils of 9.5 Å and between two lipid tails in the cell membrane cortex of 4.3 Å. The common meridional signal along the long axis of the hair (qz ) at
5 Å corresponds corresponds to the α-helices twisting around each other within coiled-coils. Average values and standard deviations are in (B).

profile was well fit with three Gaussian peaks at 90 Å, 45 Å, and 27 Å. We note that
the third peak was not observed in all hair samples. The corresponding peak positions
and distributions are shown in the figure. The 90 Å peak has been reported early in
the literature as the distance between intermediate filaments in human hair. As further
elaborated by Rafik, Doucet & Briki (2004), these peaks correspond to the radial structures
of the intermediate filaments and can be well-simulated by assuming parallel tetramers
formed by 2 coiled-coils with a slight disorder in positions and orientations, as depicted in
the figure. Also here, the standard deviations of 90 ± 2 Å, 47 ± 2 Å, 27 ± 1 Å, as shown in
the figure, are small, indicating that the organization of the intermediate filaments on the
nanoscale varies very little between different individuals.

DISCUSSION
All hair used in this study was in its native state, collected from healthy individuals and
not chemically treated prior to the experiments. However, all individuals regularly used
shampoos for cleaning and additional products such as conditioners, wax and gel. These
products function primarily at or near the fiber surface to remove dirt from the hair
surface, for instance, and do not seem to have an impact on the internal keratin structure,
as will be discussed below.
An abnormal signal was previously reported by James et al. (1999) in hair samples
of patients with breast cancer. Such an approach is quite intriguing, as scanning of hair
samples could be used as easy, inexpensive and non-invasive screening techniques in the
diagnosis of cancer. James et al. (1999) observed a ring-like signal at 44.4 Å, at the position

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 Figure 6 Diffraction features at small scattering angles. The small q∥ -range is shown in magnification
in (A). The specimen of most individuals showed 3 distinct reflections at ∼90 Å, 46.5 Å and 27 Å, related
to the properties of intermediate keratin filaments (B).

of the lamellar plasma membrane signal, and assigned this signal to the presence of breast
cancer. The analysis and assignment was questioned later on by Briki et al. (1999) and
Howell et al. (2000), who observed this feature in healthy and cancer patients in equal
measure. The ring-like 45 Å signal is also present in the data for all individuals included in
our study, such that a relation to breast cancer can most likely be excluded.

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 General structural features from the X-ray experiments
From the 2-dimensional X-ray data in Figs. 1 and 4, and the analysis in Figs. 5 and 6, we
identify three features present in all individuals. These signals are related to the coiled-coils
arrangement of the keratin proteins in the cortex, the formation of intermediate filaments
in the cortex, and lipids in the cell membrane complex of the hair. Statistical analysis of
the corresponding molecular dimensions revealed a rather small distribution between
different individuals. These general properties of human hair are observed in all hair
independent of gender, colour or optical appearance of the hair (as listed in Table 1) within
the number of individuals included in this study.
Differences in the X-ray data between individuals were observed in the wide angle
region (WAXS) of the 2-dimensional data in Fig. 1, related to properties of the membrane
component. Figure 7A shows a comparison between individual 3 and 4 to illustrate the
effect. For an easy comparison, the original data were cut in half and recombined, such
that the left half depicts individual 3, and the right half individual 4. While signals from the
coiled-coil protein phase, the diffuse, ring-like intensity from lipids in the cell membrane
complex and the small angle signals due to the formation of intermediate filaments are
observed in both individuals, additional signals occur in Subject 3 around the position
of the membrane-ring. Almost identical patterns are observed in Figs. 7B and 7C, while
differences are seen in Fig. 7D; this will be discussed in detail below.
The additional signals observed between about 1.34 Å−1 and 1.63 Å−1 can be assigned
to fatty acids located within the plasma membrane of the cell membrane complex.
The position of these lipids inside the hair was determined by synchrotron infrared
microspectroscopy (Kreplak et al., 2001a) detecting the corresponding CH2 and CH3
bands. The lipid component of the cell membrane complex consists of three major classes
of lipids: glycerolipids (mainly phospholipids), sterols and sphingolipids (Furt, Simon-Plas
& Mongrand, 2011). The most abundant lipid species are referred to as structural lipids up
to 80% of which are phosphocholine (PC) and phosphoethanolamine (PE) phospholipids.
The position and width of the broad, ring-like intensity observed in all specimens in
Fig. 1 agree well with lipid correlation peaks reported from single and multi-component
phospholipid fluid lipid membranes (Kučerka et al., 2005; Petrache et al., 1998; Kuč,
Tristram-Nagle & Nagle, 2006; Rheinstädter et al., 2004; Rheinstädter, Seydel & Salditt,
2007; Rheinstädter et al., 2008; Pan et al., 2008; Schneggenburger et al., 2011; Harroun et
al., 1999) and diffraction observed in plasma membranes (Welti et al., 1981; Poinapen
et al., 2013). The broad correlation peak is the tell-tale sign of a fluid-like, disordered
membrane structure. It is related to the packing of the lipid tails in the hydrophobic
membrane core, where the lipid acyl chains form a densely packed structure with
hexagonal symmetry (planar group p6) (Armstrong et al., 2013). The distance between
√
two acyl tails is determined to be aT = 4π/( 3qT ) (Mills et al., 2008; Barrett et al.,
2012; Barrett et al., 2013), where qT is the position of the membrane correlation peak.
The average nearest-neighbour distance between two lipid tails is calculated from the peak
position to 4.97 Å. We note that the intensity of the disordered membrane component
is not distributed isotropically on a circle, which would be indicative of a non-oriented,

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 Figure 7 Comparison between hair samples. (A) shows a comparison between individuals 3 and 4.
While the two specimens both show the general features, differences are observed in the region of signal
from the cell membrane complex. (B) Comparison between individuals 1 and 2, father and daughter. The
data in (C) (individuals 9 and 12) are from identical twins. Data in (D) was taken from fraternal twins
(individuals 10 and 11). While different individuals in general show different membrane patterns (A),
features in (B) and (C) perfectly agree. Fraternal twins show slight differences in their pattern in (D).

isotropic membrane phase. The corresponding scattering signal has a maximum along the
qz -axis, indicative that most of the membranes are aligned parallel to the hair surface.
The additional narrow components in Fig. 1 between about 1.34 Å−1 and 1.63 Å−1 ,
which are observed in some hair samples, agree with structural features reported in lipid
membranes of different composition. A correlation peak at ∼1.5 Å−1 was found in the gel
phase of saturated phospholipid membranes, such as DMPC (Dimyristoyl-sn-glycero-3-
phosphocholine) and DPPC (Dipalmitoyl-sn-glycero-3-phosphocholine) (Tristram-Nagle
et al., 2002; Katsaras et al., 1995; Rheinstädter et al., 2004). Unsaturated lipids were
reported to order in a structure with slightly larger nearest neighbour tail distances,

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 leading to an acyl-chain correlation peak at ∼1.3 Å−1 , as reported for DOPC and POPC
(Mills et al., 2009), for instance. Lipids, such as Dimyristoylphosphatidylethanolamine
(DMPE) and the charged DMPS (Dimyristoyl-sn-glycero-3-phosphoserine) with smaller
head groups were reported to order in more densely packed structures (Rappolt & Rapp,
1996). The corresponding acyl chain correlation peaks were observed at Q values of
−1
∼1.65 Å . The observed differences in the X-ray diffraction patterns between different
individuals can, therefore, most likely be assigned to differences in the molecular
composition of the plasma membrane in the cell membrane complex. Genetics plays an
important role in this composition.

Genetic similarity
Some subjects have genetic relations within the subject pool. In particular, Subject 1 and
2 are daughter and father, Subjects 10 and 11 are fraternal twins, and Subjects 9 and 12
are identical twins. The corresponding diffraction data are shown in Figs. 7B, 7C and
7D. While in general, the diffraction patterns in the membrane region were found to be
different (as demonstrated in Fig. 7A), the genetically similar hair of father and daughter
and identical twins show identical patterns within the resolution of our experiment.
It is interesting to note that differences are observed for the fraternal twins in Fig. 7D.
This finding is in agreement with the expectation that individuals with similar genetics
would share similar physical traits such as hair structure. Identical or monozygotic
twins originate from one zygote during embryonic development, and they share 100%
of their genetic material. Fraternal or dizygotic twins develop from the fertilization of two
different eggs and they only share 50% of their DNA on average (Nussbaum et al., 2007).
As expected, the identical twin pair shows almost identical hair structures whereas the
fraternal pair exhibits distinct differences. Offspring receive half of their chromosomes
from each parent, thus the genetic similarity between the parent and child pair is roughly
the same as fraternal twins (Creasy et al., 2013). It is, therefore, surprising that the father
and daughter pair share significantly more similarities than the pair of fraternal twins. This
can be attributed to the fact that the expression of a complex trait such as hair structure
would depend on the inheritance pattern of many phenotype-determining genes, such
as whether they are dominant or recessive traits. Genetic similarity does not guarantee
identical hair structure and similarly, genetic variability does not guarantee differences.
While we can report this finding, the small number of related samples excludes a more
detailed and quantitative analysis of this effect at this time.
The comparison in Fig. 7B between father and daughter also enables the study of the
effect of hair care products, such as shampoo and conditioner on the molecular structure
of hair. While Subject 2 (father) uses soap and shower gel to clean scalp and hair, Subject
1 (daughter) regularly uses shampoo and conditioner. The identical X-ray signals indicate
that these products do not have an effect on the molecular structure of keratin and
membranes deep inside the hair (within the resolution of our experiment).
We note that in order to maximize the scattered signals, the entire hair strand was
illuminated in our experiments using a relatively large X-ray beam. Microbeam X-ray

Yang et al. (2014), PeerJ, DOI 10.7717/peerj.619 14/19

 diffraction on synchrotron sources, which uses small, micrometre sized beams (Iida &
Noma, 1993; Busson, Engstrom & Doucet, 1999; Kreplak et al., 2001b; Ohta et al., 2005;
Kajiura et al., 2006), gives a high spatial resolution. By illuminating selective parts of the
hair, the occurrence of the signals that we observed can be determined as a function of their
location within the hair in future experiments.

CONCLUSIONS
We studied the molecular hair structure of several individuals using X-ray diffraction.
Hair samples were collected from 12 healthy individuals of various characteristics,
such as gender, optical appearance and genetic relation. Signals corresponding to the
coiled-coil phase of the keratin molecules, the formation of intermediate filaments in the
cortex and from the lipid molecules in the cell membrane complex were observed in the
experiment. The corresponding signals were observed in all individuals, independent of
gender or appearance of the hair, such as colour or waviness, within the resolution of
this experiment. Given the small standard deviation of the molecular dimensions of these
general features, anomalies possibly related to certain diseases should be easy to detect.
While all hair samples showed these general features, differences between individuals
were observed in the composition of the plasma membrane in the cell membrane
complex. Genetics seem to play an important role in the properties of these membranes,
as genetically similar hair samples from father and daughter and identical twins showed
identical patterns, though hair from fraternal twins did not.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
This research was funded by the Natural Sciences and Engineering Research Council of
Canada (NSERC), the National Research Council Canada (NRC), the Canada Foundation
for Innovation (CFI) and the Ontario Ministry of Economic Development and Innovation.
MCR is the recipient of an Early Researcher Award of the Province of Ontario. The
funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors:
Natural Sciences and Engineering Research Council of Canada (NSERC).
National Research Council Canada (NRC).
Canada Foundation for Innovation (CFI).
Ontario Ministry of Economic Development and Innovation.

Competing Interests
The authors declare there are no competing interests.

Yang et al. (2014), PeerJ, DOI 10.7717/peerj.619 15/19

 Author Contributions
• Fei-Chi Yang, Yuchen Zhang and Maikel C. Rheinstädter conceived and designed
the experiments, performed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables,
reviewed drafts of the paper.

Human Ethics
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
Hamilton Integrated Research Ethics Board (HIREB) under approval number 14-474-T.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/
10.7717/peerj.619#supplemental-information.

REFERENCES
Armstrong CL, Marquardt D, Dies H, Kučerka N, Yamani Z, Harroun TA, Katsaras J, Shi A-C,
Rheinstädter MC. 2013. The observation of highly ordered domains in membranes with
cholesterol. PLOS ONE 8:e66162 DOI 10.1371/journal.pone.0066162.
Astbury WT, Sisson WA. 1935. X-ray studies of the structure of hair, wool, and related fibres. III.
The configuration of the keratin molecule and its orientation in the biological cell. Proceedings
of the Royal Society of London. Series A, Mathematical and Physical Sciences 150:533–551
DOI 10.1098/rspa.1935.0121.
Astbury WT, Street A. 1932. X-ray studies of the structure of hair, wool, and related fibres. I.
General. Philosophical Transactions of the Royal Society of London. Series A, Containing Papers of
a Mathematical or Physical Character 230:75–101 DOI 10.1098/rsta.1932.0003.
Astbury WT, Woods HJ. 1934. X-ray studies of the structure of hair, wool, and related fibres.
II. The molecular structure and elastic properties of hair keratin. Philosophical Transactions of
the Royal Society of London. Series A, Containing Papers of a Mathematical or Physical Character
232:333–394 DOI 10.1098/rsta.1934.0010.
Barrett MA, Zheng S, Roshankar G, Alsop RJ, Belanger RKR, Huynh C, Kučerka N,
Rheinstädter MC. 2012. Interaction of aspirin (acetylsalicylic acid) with lipid membranes.
PLoS ONE 7:e34357 DOI 10.1371/journal.pone.0034357.
Barrett MA, Zheng S, Toppozini LA, Alsop RJ, Dies H, Wang A, Jago N, Moore M,
Rheinstädter MC. 2013. Solubility of cholesterol in lipid membranes and the formation of
immiscible cholesterol plaques at high cholesterol concentrations. Soft Matter 9:9342–9351
DOI 10.1039/c3sm50700a.
Berg BO, Rosenberg SH, Asbury AK. 1972. Giant axonal neuropathy. Pediatrics 49:894–899.
Briki F, Busson B, Kreplak L, Dumas P, Doucet J. 2000. Exploring a biological tissue from atomic
to macroscopic scale using synchrotron radiation: example of hair. Cellular and Molecular
Biology 46:1005–1016.
Briki F, Busson B, Salicru B, Estève F, Doucet J. 1999. Breast-cancer diagnosis using hair. Nature
400:226–226 DOI 10.1038/22244.

Yang et al. (2014), PeerJ, DOI 10.7717/peerj.619 16/19

 Busson B, Engstrom P, Doucet J. 1999. Existence of various structural zones in keratinous
tissues revealed by X-ray microdiffraction. Journal of Synchrotron Radiation 6:1021–1030
DOI 10.1107/S0909049599004537.
Cohen C, Parry DA. 1994. Alpha-helical coiled coils: more facts and better predictions. Science
263:488–489 DOI 10.1126/science.8290957.
Creasy RK, Resnik R, Iams JD, Lockwood CJ, Greene MF (eds.) 2013. Creasy and Resnik’s
maternal-fetal medicine: principles and practice. WB Saunders.
Crewther WG, Dowling LM, Steinert PM, Parry DAD. 1983. Structure of intermediate filaments.
International Journal of Biological Macromolecules 5:267–274
DOI 10.1016/0141-8130(83)90040-5.
Crick FHC. 1952. Is α-keratin a coiled coil? Nature 170:882–883 DOI 10.1038/170882b0.
Franbourg A, Hallegot P, Baltenneck F, Toutaina C, Leroy F. 2003. Current research on ethnic
hair. Journal of the American Academy of Dermatology 48:S115–S119
DOI 10.1067/mjd.2003.277.
Fraser RD, MacRae TP, Parry DA, Suzuki E. 1986. Intermediate filaments in alpha-keratins.
Proceedings of the National Academy of Sciences of the United States of America 83:1179–1183
DOI 10.1073/pnas.83.5.1179.
Fraser RD, MacRae TP, Rogers GE. 1962. Molecular organization in alpha-keratin. Nature
193:1052–1055 DOI 10.1038/1931052a0.
Fraser RDB, MacRae TP, Sparrow LG, Parry DAD. 1988. Disulphide bonding in α-keratin. Inter-
national Journal of Biological Macromolecules 10:106–112 DOI 10.1016/0141-8130(88)90017-7.
Furt F, Simon-Plas F, Mongrand S. 2011. Murphy AS, Schulz B, Peer W, eds. The plant
plasma membrane, Plant cell monographs, vol. 19. Berlin, Heidelberg: Springer-Verlag, 57–85
DOI 10.1007/978-3-642-13431-9 1.
Harroun TA, Heller WT, Weiss TM, Yang L, Huang HW. 1999. Experimental evidence for
hydrophobic matching and membrane-mediated interactions in lipid bilayers containing
gramicidin. Biophysical Journal 76:937–945 DOI 10.1016/S0006-3495(99)77257-7.
Howell A, Grossmann JG, Cheung KC, Kanbi L, D Gareth RE, Hasnain SS. 2000. Can hair be
used to screen for breast cancer? Journal of Medical Genetics 37:297–298
DOI 10.1136/jmg.37.4.297.
Iida A, Noma T. 1993. Synchrotron X-ray muprobe and its application to human hair analysis.
Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials
and Atoms 82:129–138 DOI 10.1016/0168-583X(93)95092-J.
James V. 2001. The importance of good images in using hair to screen for breast cancer. Journal of
Medical Genetics 38:e16 DOI 10.1136/jmg.38.5.e16.
James VJ, Amemiya Y. 1998. Intermediate filament packing in α-keratin of echidna quill. Textile
Research Journal 68:167–170 DOI 10.1177/004051759806800303.
James V, Kearsley J, Irving T, Amemiya Y, Cookson D. 1999. Using hair to screen for breast
cancer. Nature 398:33–34 DOI 10.1038/17949.
Kajiura Y, Watanabe S, Itou T, Nakamura K, Iida A, Inoue K, Yagi N, Shinohara Y, Amemiya Y.
2006. Structural analysis of human hair single fibres by scanning microbeam saxs. Journal of
Structural Biology 155:438–444 DOI 10.1016/j.jsb.2006.04.008.
Katsaras J, Raghunathan VA, Dufourc EJ, Dufourcq J. 1995. Evidence for a two-dimensional
molecular lattice in subgel phase dppc bilayers. Biochemistry 34:4684–4688
DOI 10.1021/bi00014a023.

Yang et al. (2014), PeerJ, DOI 10.7717/peerj.619 17/19

 Kreplak L, Briki F, Duvault Y, Doucet J, Merigoux C, Leroy F, Lévêque JL, Miller L, Carr GL,
Williams GP, Dumas P. 2001a. Profiling lipids across Caucasian and Afro-American hair
transverse cuts, using synchrotron infrared microspectrometry. International Journal of
Cosmetic Science 23:369–374 DOI 10.1046/j.0412-5463.2001.00118.x.
Kreplak L, Mérigoux C, Briki F, Flot D, Doucet J. 2001b. Investigation of human hair
cuticle structure by microdiffraction: direct observation of cell membrane complex
swelling. Biochimica et Biophysica Acta (BBA)—Protein Structure and Molecular Enzymology
1547:268–274 DOI 10.1016/S0167-4838(01)00195-9.
Kučerka N, Liu Y, Chu N, Petrache HI, Tristram-Nagle S, Nagle JF. 2005. Structure of fully
hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented
multilamellar arrays and from unilamellar vesicles. Biophysical Journal 88:2626–2637
DOI 10.1529/biophysj.104.056606.
Kučerka N, Tristram-Nagle S, Nagle JF. 2006. Closer look at structure of fully hydrated fluid phase
dppc bilayers. Biophysical Journal 90:L83–L85 DOI 10.1529/biophysj.106.086017.
Lupas AN, Gruber M. 2005. The structure of α-helical coiled coils. Advances in Protein Chemistry
70:37–38.
Mercer EH. 1953. The heterogeneity of the keratin fibers. Textile Research Journal 23:388–397
DOI 10.1177/004051755302300603.
Mills TT, Huang J, Feigenson GW, Nagle JF. 2009. Effects of cholesterol and unsaturated dopc
lipid on chain packing of saturated gel-phase dppc bilayers. General Physiology and Biophysics
28:126–139 DOI 10.4149/gpb 2009 02 126.
Mills TT, Toombes GES, Tristram-Nagle S, Smilgies D-M, Feigenson GW, Nagle JF. 2008. Order
parameters and areas in fluid-phase oriented lipid membranes using wide angle X-ray
scattering. Biophysical Journal 95:669–681 DOI 10.1529/biophysj.107.127845.
Nussbaum RL, McInnes RR, Willard HF, Hamosh A. 2007. Principles of molecular
disease: lessons from the hemoglobinopathies. In: Thompson & Thompson genetics in
medicine, vol. 6, 181–202.
Ohta N, Oka T, Inoue K, Yagi N, Kato S, Hatta I. 2005. Structural analysis of cell membrane
complex of a hair fibre by micro-beam X-ray diffraction. Journal of Applied Crystallography
38:274–279 DOI 10.1107/S002188980403403X.
Pan J, Mills TT, Tristram-Nagle S, Nagle JF. 2008. Cholesterol perturbs lipid bilayers
nonuniversally. Physical Review Letters 100:198103 DOI 10.1103/PhysRevLett.100.198103.
Pauling L, Corey RB. 1950. Two hydrogen-bonded spiral configurations of the polypeptide chain.
Journal of the American Chemical Society 72:5349–5349 DOI 10.1021/ja01167a545.
Pauling L, Corey RB. 1951. The structure of hair, muscle, and related proteins.
Proceedings of the National Academy of Sciences of the United States of America 37:261–271
DOI 10.1073/pnas.37.5.261.
Petrache HI, Gouliaev N, Tristram-Nagle S, Zhang R, Suter RM, Nagle JF. 1998. Interbilayer
interactions from high-resolution X-ray scattering. Physical Review E 57:7014–7024
DOI 10.1103/PhysRevE.57.7014.
Pinto N, Yang F-C, Negishi A, Rheinstädter MC, Gillis TE, Fudge DS. 2014. Self-assembly
enhances the strength of fibers made from vimentin intermediate filament proteins.
Biomacromolecules 15:574–581 DOI 10.1021/bm401600a.
Poinapen D, Toppozini L, Dies H, Brown DCW, Rheinstädter MC. 2013. Static magnetic
fields enhance lipid order in native plant plasma membrane. Soft Matter 9:6804–6813
DOI 10.1039/c3sm50355k.

Yang et al. (2014), PeerJ, DOI 10.7717/peerj.619 18/19

 Rafik MEr, Doucet J, Briki F. 2004. The intermediate filament architecture as determined
by X-ray diffraction modeling of hard α-keratin. Biophysical Journal 86:3893–3904
DOI 10.1529/biophysj.103.034694.
Randebrook RJ. 1964. Neue erkenntnisse über den morphologischen aufbau des menschlichen
haares. Journal of the Society of Cosmetic Chemists 15:691–706.
Rappolt M, Rapp G. 1996. Simultaneous small- and wide-angle X-ray diffraction during the
main transition of dimyristoylphosphatidylethanolamine. Berichte der Bunsengesellschaft and
Physikalische Chemie 7:1153–1162 DOI 10.1002/bbpc.19961000710.
Rheinstädter MC, Das J, Flenner EJ, Brüning B, Seydel T, Kosztin I. 2008. Motional coherence in
fluid phospholipid membranes. Physical Review Letters 101:248106
DOI 10.1103/PhysRevLett.101.248106.
Rheinstädter MC, Ollinger C, Fragneto G, Demmel F, Salditt T. 2004. Collective dynamics of
lipid membranes studied by inelastic neutron scattering. Physical Review Letters 93:108107
DOI 10.1103/PhysRevLett.93.108107.
Rheinstädter MC, Seydel T, Salditt T. 2007. Nanosecond molecular relaxations in lipid bilayers
studied by high energy resolution neutron scattering and in-situ diffraction. Physical Review E
75:011907 DOI 10.1103/PhysRevE.75.011907.
Robbins CR. 2012. Chemical and physical behavior of human hair. 5th ed. New York: Springer.
Rogers GE. 1959. Electron microscopy of wool. Journal of Ultrastructure Research 2:309–330
DOI 10.1016/S0022-5320(59)80004-6.
Schneggenburger P, Beerlink A, Weinhausen B, Salditt T, Diederichsen U. 2011. Peptide model
helices in lipid membranes: insertion, positioning, and lipid response on aggregation studied
by X-ray scattering. European Biophysics Journal 40:417–436 DOI 10.1007/s00249-010-0645-4.
Swift JA, Smith JR. 2001. Microscopical investigations on the epicuticle of mammalian keratin
fibres. Journal of Microscopy 204:203–211 DOI 10.1046/j.1365-2818.2001.00957.x.
Tristram-Nagle S, Liu Y, Legleiter J, Nagle JF. 2002. Structure of gel phase dmpc determined by
X-ray diffraction. Biophysical Journal 83:3324–3335 DOI 10.1016/S0006-3495(02)75333-2.
Ward WH, Lundgren HP. 1954. The formation, composition, and properties of the keratins.
Advances in Protein Chemistry 9:243–297.
Welti R, Rintoul DA, Goodsaid-Zalduondo F, Felder S, Silbert DF. 1981. Gel-phase phospholipid
in the plasma membrane of sterol-depleted mouse lm cells. The Journal of Biological Chemistry
256:7528–7535.
Wilk KE, James VJ, Amemiya Y. 1995. The intermediate filament structure of human hair.
Biochimica et Biophysica Acta (BBA)-General Subjects 1245:392–396
DOI 10.1016/0304-4165(95)00111-5.
Yang F-C, Peters RD, Dies H, Rheinstädter MC. 2014. Hierarchical, self-similar structure in native
squid pen. Soft Matter 10:5541–5549 DOI 10.1039/C4SM00301B.

Yang et al. (2014), PeerJ, DOI 10.7717/peerj.619 19/19