d. Cosmet.
sci., 56, 1-16 (January/February
2005)
Characterization
of the lipid compositionat the proximal
root reDionsof humanhair
YOSHINORI
MASUKAWA,
HIROFUMI
NARITA,
and
GENJI IMOKAWA, TochigiResearch
Laboratories,
Kao Corporation,
2606 Akabane,Ichikai,Haga, Tochigi,321-3497Japan.
Accepted
for publication
December
3, 2004.
Synopsis
The hair lipid composition
collectedfrom44 Japanese
femalesbetween1 and81 yearsof agewasexamined
for eightlipids includinghydrocarbons
(HCs), squalene
(SQ),waxesters(WEs), triglycerides
(TGs), fatty
acids(FAs), cholesterol(CH), ceramides(CERs), and 18-methyl eicosanoic
acid (MEA). In this study, the
5-cm length from the proximalroot end of hair fibers,which had neverbeenexposedto any chemical
treatment,wasusedafter 5-min incubationwith hexanefollowing shampooing.Hair lipids were extracted
with solventand subsequent
alkali-solventand werethen analyzedby a combinationof chromatography.
Although the averagecontentsof the lipids showedgreat fluctuationsamongindividuals,there were
significant
correlations
betweenthelevelsof eachlipid, whichallowedfor theclassification
of thehairlipids
into four groups:groupA: SQ, WEs, TGs, and FAs (designated
as endogenous
lipids basedupon their
sebumorigin);groupB: CH andCERs(designated
asendogenous
lipids);groupC: HC (unknownorigin);
and groupD: MEA (the otherendogenous
lipid). A principalcomponentanalysisfor eight lipidsrevealed
that the hair lipid composition
wascharacterized
by a predominant
negativecorrelation
betweeneachlipid
for groupsA and B. This negativecorrelationsuggests
that the endogenous
lipids in groupB serveas a
barrieragainstthe penetrationof predominantlysebum-derived
exogenous
lipids (groupA). Endogenous
lipidsconsisting
of CH andCERs(groupB) andMEA (groupD) shouldbe designated
asintrinsicinternal
lipids of humanhair.
INTRODUCTION
A fine structure,calledthe cell membranecomplex(CMC), existsbetweencorticalor
cuticle cellsof human hair aswell aswool (1,2). The CMC is visualizedby transmission
electronmicroscopy
asa tram-linestructureincludinga denselystainedB-layersandwichedbetweenlightly stainedp-layerson both sides.The main components
of the
B-layerand the p-layershavebeenthoughtto consistof proteinsand lipids, respectively
(1,3). The factthat lipid extractsfrom woolor from humanhair form liposomes
suggests
that lipidsmight comprisea lipid bilayerat the [3-1ayers
of the CMC (4,5). A characteristiclipid, 18-methyleicosanoic
acid(MEA), is chemicallyboundto the surfaceof
Addressall correspondence
to Genji Imokawa.
2
JOURNAL OF COSMETIC SCIENCE
cuticlecellsby a thio-esterlinkage(6). Many studieshavesuggested
that hair lipids can
contributeto physicochemical
phenomenasuchasdiffusion,cell cohesion,
andmechanical strength(3,7-10), althoughlipidsoccurat a muchlowercontent(1-9% dry weight)
than proteins(>90%).
Many analyticalstudieson the lipids of humanhair havebeenperformed(6,7,11-20).
Hair fibershavenot only surfacelipidsoriginatingfrom sebum(11,14), but alsolipids
within the hair (11-13). Curry and Golding reported that hair yields its lipids to
successive
Soxhletextraction,suggestingthe existenceof lipids within hair (11). Using
hair fibers treated with successiveSoxhlet extraction, Sakamoto et •l. (12) first demon-
strated the existenceof lipids within them suchas fatty acids (FAs) and wax esters
(WEs).Theydesignated
themas"internallipids"of humanhair (12-14). Subsequently,
hydrocarbons
(HCs) (15), squalene(SQ) (7,15,20), wax esters(WEs) (15,17,20), triglycerides(TGs) (15,17), FAs (7,15-18,20), cholesterol(CH) (7,15-17,20), cholesterolsul-
fate (CS) (16), MEA chemicallyboundto hair fibers(6,16), and ceramides(CERs)(19)
havebeenfoundas hair lipids. However,in all previousstudiesdescribed
above,hair
sampleswerenot from the proximalroot endsor werenot clearlydescribed.Sincehair
lipids are graduallylost or alteredas hair fibersgrow, the proximalroot endsof hair
fibersor the nearestregionsshouldbe usedto characterize
the precisehair lipid composition.In addition,all previousstudieshavenot focusedon comprehensive
hair lipid
composition,includingits individualvariation,althougha great deal of knowledgeon
the intercellularlipid compositionof the stratumcorneumhasaccumulated(e.g., 2123). The aim of the presentstudyis to clarifythe precisehair lipid compositionat the
proximalroot regionsof hair fibersby a combinationof chromatography.
MATERIALS
AND
METHODS
CHEMICALS
HPLC grade chloroform,methanol, hexane,and acetonitrilewere purchasedfrom
Kanto-Kagaku(Tokyo,Japan).Biochemistry
gradeo-phtalaldehyde
(OPA) and 2-mercaptoethanol
(2-ME) werefromWako (Tokyo,Japan),9-anthryldiazomethane
(ADAM)
wasfrom Funakoshi(Tokyo,Japan),andTMS-HT wasfrom GL Science
(Tokyo,Japan).
All other chemicalswere analyticalgrade.
REFERENCES
FOR
LIPID
ANALYSIS
Tetracosane
for HCs waspurchased
from Kanto-Kagaku.Squalenefor SQ, tripalmitin
for TGs, tricosanoic
acidfor an internalstandard(IS) of MEA, D-sphingosine,DL-threodihydrosphingosine,
DL-erythro-dihydrosphingosine,
and N-palmitoyl-DL-dihydro-
sphingosine
for CERswerepurchased
fromSigma(St.Louis,MO). Cetylpalmitatefor
WEs, palmiticacidfor FAs,and cholesterol
for CH werepurchased
from Tokyo-Kasei
(Tokyo,Japan).All references
wereusedwithout furtherpurification.
MATERIALS
Hair fiberswereobtainedfrom 44 healthyJapanese
femalevolunteers
whoseagesranged
HAIR
LIPID
COMPOSITION
3
from 1 to 81 years.About 200 fibersof 5-cm length takenfrom the proximalroot end
perindividual(ca.100 mg) werecollected.
The volunteers
hadnot beenexposed
to any
chemicaltreatmentssuchaspermingor bleachingfor at leastsix months.Hair fibers
werewashedwith plainshampoo
twiceandwerethenincubated
with hexanefor 5 min
prior to extractionof hair lipids.
EXTRACTION
OF HAIR
LIPIDS
Each hair bundle washedwith shampooand hexanewas cut into small piecesusing
scissors.
They wereimmersedinto a seriesof CHC13/CH3OH2:1, 1:1, 1:2 (v/v) and
CHC13/CH•OH/water
18:9:1(v/v/v)for24 hr each,at roomtemperature.
Themixture
of solvents
wasfilteredthrougha solvent-resistant
0.5-1amMillipore filter. The filtered
solutionwastakento dryness,andwasthen usedfor analysisof extractablelipids.
Delipidizedhairfibersweresaponified
by heatingfor2 hr at 60øCin 1N KOH in 90%
CH•OH (16). AfterwaterandCHCI• wereadded,themixturewasshaken
in a separatoryfunnel.TheCHCI• phase
wastransferred
to a flask,andtheupperphase
including
hair residuewasacidifiedby the additionof 6N HC1. The acidifiedupperphasewas
shaken
againwith CHC13.Thecombined
CHCI.•phasewaswashed
twicewith saturated
NaC1aqueous
solution.The washedCHC13phasewasthenfilteredthrougha solventresistant0.5-1amMillipore filter. The filteredsolutionwastakento dryness,and was
thenusedfor analysis
of integrallipids,whicharedefinedasthe hair lipidsthat canbe
extracted
by alkalisaponification
followingsolventextraction
(16).
IDENTIFICATION
OF CERs WITH
GAS CHROMATOGRAPHY/MASS
SPECTROMETRY
(GC/MS)
Thisprocedure
wasperformed
with slightmodifications
of methods
previously
reported
(19). CERswereisolatedfromthe extractedlipidsby preparative
TLC (silicagel 60,
Merck,Darmstadt,Germany).The isolatedCERsweresilylatedwith TMS-HT at 60øC
for 20 min. Massspectra
wereobtainedby GC/MSwith a Hewlett-Packard
5988A mass
spectrometer
coupledto a Hewlett-Packard
5890 SeriesGC and a Hewlett-Packard
59970C workstation (Hewlett-Packard, Palo Alto, CA). An Ultra ALLOY-I(HT)
30-m
x 0.25-mm x 0.15-1ammetalcapillarycolumn(Frontier-Labo,
Fukushima,
Japan)was
usedwith a temperature
programof 150øCto 360øCat 5øC/min,whichwasheldfor 5
min. Aliquotsof the sialylated
CER solutions
wereinjectedvia splitless
modeat 370øC.
Helium wasusedasa carriergasat a flow rate of 1.16 ml/min. The massspectrometer
scannedfrom 50 to 1000 amu in 0.78 sec, with an electron ionization of 70 eV. The
sourceand the transferline temperatures
werekept at 300øCand 320øC,respectively.
DETERMINATION OF HCs, SQ, WEs, TGs, AND FAs WITH HIGH-PERFORMANCE THIN-LAYER
CHROMATOGRAPHY
(HPTLC)/DENSITOMETRY
Theseprocedures
wereperformedwith slightmodifications
of methodspreviouslyreported(16,17).Thus,for separation,
HPTLC plates(silicagel 60, 20 x 10 cm,Merck)
wereused.Aliquotsof theextractable
lipid solutions
wereapplied2 cm fromthe bottom
edge of the plate, which was developedfirst to 3 cm with CHCI•/CH3OH/water
(100:10:0.5) twice, secondto 12 cm with hexane/acetic
acid (80:10), and third to 15 cm
with petroleumether.After drying,theplatewassprayed
with 10% coppersulfateand
4
JOURNAL OF COSMETIC SCIENCE
8% phosphoric
acidsolutionand wascharredby heatingat 180øCfor 5 min. Eachspot
wasdensitometricallydeterminedwith a ShimadzuCS-900 photodensitometer
(Kyoto,
Japan) and was comparedto a calibrationreferencecurve. Duplicate analyseswere
performedfor eachmeasurement.
DETERMINATION OF MEA WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
This procedure
wasperformedwith slightmodifications
of methodspreviouslyreported
(6). Thus, tricosanoicacid asthe IS of MEA and ADAM wereaddedto the integral lipid
solution(CHC13/CH3OH1:1) andthe solutionwasthenderivatized
for 1 hr at room
temperature.Aliquotsof the derivatizedintegrallipid solutionwere injectedinto an
HPLC apparatus
equippedwith a fluorescence
detector(Hitachi L-6000 series,Hitachi,
Ibaragi,Japan)and a 12.5-cm x 4.0-mm Superspher
60 RP-8e (Merck, Darmstadt,
Germany)kept at 40øC.The derivatives
weredetectedat EX.365 nm andEM.412 nm.
Two solventsof (A) acetonitrile/water85:15 and (B) acetonitrilewere usedfor separation at a flow rate of 1.5 ml/min with a gradientelutionprogram,which includedfirst
(A) 100%, second(B) 2.5%/min to (B) 100% (40 min), andfinally holdingof(B) 100%
for 20 min. The contentof MEA wascalibratedbasedon an equivalentmolar sensitivity
of MEA to the IS. Duplicateanalyseswereperformedfor eachmeasurement.
DETERMINATION
OF CH WITH
GC
This procedure
wasperformedwith slightmodifications
of previouslyreportedmethods
(7). Thus,analyses
wereperformedon a Hewlett-Packard5890 seriesGC equippedwith
a flame ionizationdetector,an Ultra #2 25-m x 0.25-mm x 0.33-pm fusedsilica
capillarycolumn(Hewlett-Packard),
anda Hewlett-Packard
3396 SeriesII integrator.
Aliquotsof the lipid solutionwereinjectedvia splitmodein a ratioof 1:50at 340øC.
The temperature
of the detectorwasmaintainedat 340øC.Helium wasusedat a flow
rate of 1.0 ml/min. The columntemperaturewasprogrammedfrom 200øC to 320øC at
20øC/min, which was held for 10 min. The content of CH was determined using a
calibrationcurve.Duplicateanalyses
wereperformedfor eachmeasurement.
DETERMINATION
OF CERs WITH
HPLC
Procedures
for alkaline hydrolysisof CERs for the isolationof sphingoidswere performed,basedon a slight modificationof a methodreportedfor CERs of the stratum
corneum(24). Thus,the lipids weresaponified
by heatingfor 18 hr at 65øCin 10 ml
I N KOH in CH3OH/hexane5:1. Ten micrograms
of threo-dihydrosphongosine
was
then addedas the IS. By meansof liquid-liquidextraction,CHCI• extractswereobtained.Thesphingoids
extracted
wereisolated
bypreparative
TLC (silicagel 60) because
hydrolyzed
fattyacidsinterruptedthe reactionbetweensphingoidandOPA. The sphingoldsisolatedwerederivatizedat roomtemperature
for 30 min with an addedsolution
of OPA methanol,a 2-ME/methanol solution,and a sodium tetraborateaqueoussolu-
tion. Aliquotsof the derivatives
wereinjectedinto the sameHPLC equipmentusedfor
theMEA analysis
with a 15-cmx 4.6-mm L-ColumnODS (Kagakuhin-Kensa-Kyoukai,
Tokyo,Japan)keptat 40øC.The derivatives
weredetectedat EX.344 nm andEM.433
nm. Two solvents
of (A) CH•OH/water85:15and(B) CH•OH wereusedfor separation
HAIR
LIPID
COMPOSITION
5
at a flow rate of 1.0 ml/min with a gradient elution program, which included first
holding (A) 100% for 15 min, second(B) 5%/min to (B) 100% (20 min), and finally
holdingof (B) 100% for 15 min. The contentof sphingoidswascalibratedbasedon area
ratiosof the peaksof interestto the IS (threo-dihydrosphingosine),
resultingin CER
content.Duplicate analyseswere performedfor eachmeasurement.
STATISTICAL
ANALYSES
Correlationanalyses
amongthe level of lipids and a principalcomponentanalysis(PCA)
with a correlation matrix for hair lipid compositionwere performed using Excel
Tahenryo-Kaisekiversion3.0 software(Esumi,Tokyo,Japan).
RESULTS
MOLECULAR
SPECIES
OF CERs IN
HUMAN
HAIR
A total ion GC/MS chromatogram
of silylatedCERs in extractablelipids is shownin
Figure1, andthosein integrallipidsexhibitedalmostthe samepeakpattern.PeaksA-F
in Figure 1 had distinctivefragments:(A) m/z: 313, 370, (B) m/z: 299, 313,458,
(C) m/z = 313,398, (D) m/z = 313,426, (E) m/z = 313,454, and (F) m/z = 313,480.
It hasbeenestablished
that a massspectrumof silylatedCERs can be characterized
by
a C2-C3 fragmentationof its sphingoidsamidified with its N-acyl moiety (19,25).
Therefore,fragmentsofm/z: 311 or m/z: 313 and m/z: [M-311] or m/z = [M-313]
providesufficientinformationabouttheir sphingoidssuchassphingenine(sphingosine)
or sphinganine(dihydrosphingosine)
and abouttheir N-acyl moieties.Accordingly,the
peakswereidentifiedasFollows:
(A) 2-N-palmitoylamino-octadecane-l,3-diol,
(B) 2-Not-hydroxypalmiroylamino-ocradecane1,3-diol, (C) 2-N-stearoylamino-octadecane1,3-
A
0
B
.._0
A
F
½
•o
'
15
'
30
Ti•
'
25
F
•o
Figure 1. A GC/MS total ion chromatogram
of silylatedCERsfrom extractablelipids.A, B, C, D, E, and
F show their
molecular
structure.
6
JOURNAL OF COSMETIC SCIENCE
diol, (D) 2-N-eicosanoylamino-octadecane-l,3-diol, (E) 2-N-docosanoylaminooctadecane-l,3-diol,and (F) 2-N-tetracosanoylamino-octadecane-l,3-diol.
DETERMINATION
OF THE
HAIR
LIPIDS
BY CHROMATOGRAPHY
The optimal conditionsfor extractionof hair lipids, excludingcontaminatedlipids, were
examinedfor the relationshipbetweenhexaneincubationtime after twice shampooing
hair fibersand the extractedlipid contents.When the squareroot of time wasplotted
againstthe relativeextractedlipid content,a linear plotting wasobtainedduring 5 to
30 rain of incubationwith hexane(Figure2). In general,the followingequationis used
for the diffusionof a soluteinto a cylindricalfiber of infinite length (26):
Ct/Coo
= 4(Dt/(q-rr2))
1/2
where C•/Coorepresentsrelative soluteuptake at a certain time (t), D is the diffusion
coefficient,and r is the radiusof the cylindricalfiber. Accordingto this equation,if
diffusionof the soluteoccurs,the relativesoluteuptakeplottedagainstthe squareroot
of time should become linear becauseof the constant D, q-r,and r. The observedlinear
line (Figure2) impliesthe existenceof diffusionof the lipids from the hair insideto the
outsideduring the incubation.Therefore,in our study,a 5-rain incubationwith hexane
wasperformedprior to the extractionof hair lipids to deletecontaminatedlipids.
0.4
30 min
0.3
5 min
10min
20min
•
•
2min
+
•
•
•
1min
+.......................
0.2
0.1
0.0
0
1
2
3
4
Root square of hexaneincubation time
5
6
min •a
Figure 2. Effectof hexaneincubationtime aftertwiceshampooing
of hairfiberson extractedlipid contents.
Eachpoint represents
the mean + SD calculatedin five hair samplesfor eachhexaneincubationtime.
Relativeextractedlipid contentwasdefinedasa ratioof lipid contentsextractedat a certaintime (t) to that
of total lipids exhaustively
extracted.
HAIR
LIPID
COMPOSITION
7
HCs, SQ, WEs, TGs, and FAs in the extractablelipids were determinedby HPTLCdensitometryaccordingto the conventional
method(16,17). A representative
chromatogram is shown in Figure 3. MEA in the integral lipids was determined by HPLC
followingits derivatizationwith ADAM accordingto a previouslyreportedmethod(6),
and a representative
chromatogram
is shownin Figure4. CH in the lipids wasdetermined by GC, basedon a previouslyreportedmethod (7), and a representativechromatogramis depictedin Figure 5. Sphingoidsgeneratedby hydrolysisof CERs in the
lipids were determinedby HPLC followingtheir derivatizationwith OPA (Figure 6).
The major and minor sphingoidswere identifiedas erythro(C•8-)dihydrosphingosine
(peakC in Figure6), and (C•8-)sphingosine
(peakA in Figure6) by comparison
with
authentic standards.Peak D in Figure 6 was identified as erythro-C2o-dihydrosphingosine
baseduponcomparison
with GC/MS datafor acetylatedandsilylatedsphin12 cm
,
HCs
9 cm
SQ
WEs
I
TGs
!
6 cm
FAs
3 cm
0 cm
II
A
Ill III Ill Illlllllllllllllllllllllll
B
C
D
E
F
Figure 3. A HPTLC chromatogram
of lipid references
and extractablelipids.LaneA: tetracosane.
LaneB:
squalene.Lane C: cetyl palrnitate.Lane D: tripalrnitin. Lane E: palrnitic acid. Lane F: extractablelipids.
8
JOURNAL OF COSMETIC SCIENCE
A
B
10
C
2o
30
D
40
50
Time/min
Figure 4. A HPLC chromatogram
of fatty acid-ADAMderivatives
in integrallipids.A: palmiticacid.
B: stearic acid. C: MEA.
D: tricosanoic
acid as an internal
standard.
golds(data not shown).The absenceof peaksat ca. 11 rain in Figure 6 indicatesthat
human hair containslittle CERs with phytosphingosine.
Both CH and CERs were
calculatedas the sum of the extractablelipids and the integralones,sinceit is unclear
whetherthey shouldbe chemicallydistinguishedfrom eachother or not in the caseof
CH and CERs different from MEA, which is clear in its chemicalstate (6).
STATISTICAL
ANALYSIS
OF THE
LEVELS
OF THE
HAIR
LIPIDS
The variationof the hair lipidsamongindividualsis shownin Table I. An averageof the
level of eachlipid wasin the orderof (FAs) > (WEs) > (HCs)>(CH) > (SQ) > (TGs) >
(MEA) •. (CERs).The majorhair lipidscanbeassigned
asFAsandWEs sincetheir sum
accountedfor morethan 85% of the total lipids (average23.5 mg/g hair). The percentageof coefficientof variationwasin the orderof (TGs) > (SQ) > (WEs) •. (FAs) • (HC)
> (total lipid) > (CH) > (CERs) > (MEA), thusindicatingthat their levelsof TGs, SQ,
WEs, FAs, and HCs amongindividualsfluctuatemorethan thoseof the total lipids.
Relationships
betweenthe levelsof eachlipid were analyzed(Table II). There was a
significantpositivecorrelation(p < 0.01) amongthe levelsof SQ, WEs, andFAs,oneof
which is shownin Figure 7A. A significantpositivecorrelation(p < 0.01) betweenthe
levelsof CH and CERswasalsoobserved(Figure7B). Further, therewasa significant
negativecorrelationbetweenthe levelsof SQ and CH (p < 0.01), SQ and CERs (p <
0.05), WEs andCH (p < 0.01), WEs andCERs(p < 0.01), TGs andCH (p < 0.01), TGs
and CERs (p < 0.05), FAs and CH (p < 0.01), and FAs and CERs (p < 0.01) (Table II).
A representative
negativecorrelationbetweenWEs and CH is shownin Figure 7C.
While HCs were weakly correlatedonly with CERs (p < 0.05), MEA exhibited no
significantcorrelationwith any other lipids.
HAIR LIPID COMPOSITION
9
A
1'o
fs
Time/min
Figure 5. A GC chromatogram
of cholesterol
in extractablelipids.A: cholesterol.
A principalcomponent
analysis
(PCA)forthelevelsofeightlipidswasperformed.
Factor
loading,eigenvalue,andcontributionobtainedby the PCA areshownin Table III. The
first two components
accountedfor 60.4% of the total variation,with 42.3% in the first
component(Z1) and 18.1% in the secondcomponent(Z2). The valuesin Z1 increased
in accordance with the increase in the levels of CH and CERs and the decreasein the
levelsof SQ, WEs, FAs,andTGs. The valuesin Z2 mainlyincreased
in concertwith the
increase
in the levelof HCs. Therefore,Z1 wasinterpretedto imply a negativecorre-
lationbetweenonelipid consisting
of SQ, WEs, FAs,and TGs and anotherlipid
consisting
of CH and CERs(TableII). Z2 wasalsointerpretedto imply a definite
contributionto the level of HCs. A two-dimensional
projectionof the PCA, which
impliesa total variationfor the hair lipid composition
of 44 Japanese
females,is shown
in Figure8. The individualswho werecharacterized
by the lowerlevelsof SQ, WEs,
FAs,andTGs,andbythehigherlevelsofCH andCERs,weresituated
at a positivevalue
of Z1 (Figure8). On theotherhand,individuals
whowerecharacterized
by thehigher
levelsof SQ, WEs, FAs,andTGs, andby the lowerlevelsof CH andCERs,weresituated
at a negativevalue of Z1 (Figure 8).
BaseduponFigure8, the individuals
weresymbolized
according
to theirage(Figure9).
Young individuals(ages1-10) tendedto locateat the right-handside, and older
10
JOURNAL OF COSMETIC SCIENCE
B
IIIIIIIIIIIIII
I0
IIIIIIIIllllllllll
tl')
•)
(S)
II
It')
I
(S)
Time/rain
Figure 6. A HPLC chromatogram
of sphingoid-OPAderivatives
in CERsisolatedfrom extractable
lipids.
A: (C•8-)sphingosine. B: threo-(C18-) dihydrosphingosine as an internal standard. C: erythro(C•s-)dihydrosphingosine.
D: erythro-C2o-dihydrosphingosine.
Table
I
Variation of Hair Lipids Among Individuals
Statistical values
HCs
SQ
WEs
TGs
FAs
Maximum (mg/g hair)
Minimum (mg/g hair)
8.9
<0.2
3.5
<0.2
17.1
0.3
4.6
<0.2
56.2
2.4
Mean(mg/ghair)•
2.4
0.7
4.9
0.5
Variance•
3.7
0.9
17.7
0.9
SD (mg/ghair)•
CV%
1.9
81
1.0
131
4.2
85
0.9
176
15.4
166
12.9
84
CH
CERs
MEA
2.8
0.4
0.49
0.04
0.47
0.22
1.3
0.29
0.30
0.5
0.02
0.00
0.7
52
0.13
45
0.07
22
Total
63.7
5.6
23.5
231
15.2
65
• <0.2 mg/ghairwascalculated
as= 0.
Abbreviations:
HCs: hydrocarbons;
SQ: squalene;
WEs: waxesters;TGs: triglycerides;
FAs:fatty acids;CH:
cholesterol;CERs: ceramides;MEA: 18-methyl eicosanoic
acid.
individuals(ages11-40) tendedto distributeat the left-handsidein the PCA, showing
total variationof hair lipids as shownin Figure 9.
DISCUSSION
Sincehair lipids are graduallylost ashair fibersgrow, the hair lipid compositionat the
distal region of hair fibers may not reflect the "precisehair lipid composition."The
HAIR
LIPID
COMPOSITION
Table
11
II
Correlation
Coefficients
for theLevelsof EachLipidAmongIndividuals
•
Correlation
Lipid
HCs
SQ
SQ
-0.1815
WEs
TGs
FAs
-0.2232
0.0410
0.0463
0.7386**
0.2421
0.4879**
CH
CERs
MEA
-0.1404
-0.3378*
0.1640
-0.5353**
-0.3260*
-0.1565
WEs
coefficient
TGs
FAs
CH
CERs
----
---
----
-0.6479**
-0.5019'*
-0.1171
0.7716
0.2172
--0.1130
....
-0.2123
0.3942**
-0.6133'*
-0.4516'*
-0.1240
0.0718
-0.4422**
-0.3263*
-0.2219
• <0.2 mg/ghairwascalculated
as: 0.
**p < 0.01; *p < 0.05.
Abbreviations:
as described in Table I.
phrase"precisehair lipid composition"meansthe hair lipid compositionthat is not
changedby anyexternalfactorssuchasweathering(shampooing,
heat,light, and soon),
perming,or coloring.In all paststudies,hair sampleswerenot from the proximalroot
endsor were not preciselyidentifiedfor sitesof the hair shaftcollected(6,7,11-20). In
this study,only the 5-cm lengthfrom the proximalroot endof hair fiberswasused,and
the hair lipids without contaminated
lipidswereobtainedby extractionwith CH3CI/
CH3OH/waterandwith 1 N KOH in 90% CH3OH following5-minhexaneincubation
after shampooing.
In our comprehensive
analysisof hair lipids at the proximalroot
regionsof hair fibersfrom 44 Japanese
females,the averagecontentof total hair lipids
was23.5 mg/g hair, comprising2.4, 0.7, 4.9, 0.5, 14.4, 1.3, 0.29, and 0.30 mg/g hair
for HCs, SQ, WEs, TGs, FAs,CH, CERs,andMEA, respectively
(TableI). The levelof
eachlipid showeda great fluctuationamongindividuals(Table I), suggestingthat the
hair lipid composition
of humanhair variesconsiderably
amongindividuals.
The molecularspecies
of CERsidentifiedin Japanese
females(Figure1) werealmostthe
sameasthoseidentifiedin Caucasian
males,asreportedby HussleretM. (19). Thus,hair
CERsbelongedto class2 or class5 accordingto the ceramideclassification
reportedfor
the stratumcorneum(27). However,the contentof hair CERsdeterminedin our study
(average0.29 mg/g hair) wasmuchhigher than that reportedby Hussleret•/. (average
0.1 mg/g hair) (19). The differencebetween the content of hair CERs in those two
studiesmight be due to differentsampleoriginsasdescribedabove,althoughit might
alsoresultfrom otherfactorssuchasdifferentracesand/orgenderof the subjects,and/or
hair diameter/volumeper cm length.
The resultsfromthe relationships
amongthe levelsof lipidsindicatethat thehairlipids
may be tentativelyclassifiedinto the followingfour groups:groupA (SQ, WEs, TGs,
and FAs);group B (CH and CERs);group C (HCs); and group D (MEA). While each
lipid level in groupA or eachlipid level in group B is positivelycorrelatedwith each
other,eachlipid levelin groupA is negativelycorrelated
with eachlipid levelin group
B (Figure 7, Table II). Sincethe level of HCs or MEA did not correlatein most cases
with eachlevelin otherlipids,HC andMEA wereclassified
into the othergroups(group
C and groupD). The PCA showingthe total variationof hair lipids among44 individualsindicatesthat the hair lipid compositioncan be especiallycharacterizedby a
negativecorrelationof eachlipid levelbetweengroupA and groupB, whichpredomi-
12
JOURNAL OF COSMETIC SCIENCE
2O
A
R=0.7386 P<0.01
ß
I
I
I
$Q m•/• hair
0,6
B
R=0.7716 P<0.01
ß
ß
0.4
ß
ß•t•..'"
,+*
•ß ß ....*'0
ß
ß
ß
ß ß
0.2
ß .,,"
ß
:
ß
ß
I
0,0
CH
i
mg/g hair
•..CR=-0.6133
P<0.01
ß
ß
ß
•'"'.,p.%.
ß
0
ß
!
!
5
10
!
15
2O
WEs mg/g hair
Figure7. Therelationship
between
thelevels
oflipidsfrom44Japanese
females.
Eachpointrepresents
the
levelof thelipidsforeachindividual.
A: SQandWEs.B: CH andCERs.C: WEsandCH.
HAIR
LIPID
COMPOSITION
Table
13
III
FactorLoading,Eigenvalue,Contribution,and AccumulatedContributionfor PrincipalComponents
of
Eight Lipids
Component
Lipid
First
Factor
loading
Second
Third
Fourth
Sixth
HCs
-0.022
0.728
0.155
-0.469
0.388
SQ
•-0.411
-0.310
-0.201
0.065
-0.364
0.539
WEs
TGs
-0.425
-0.253
-0.293
0.003
-0.183
0.558
0.040
0.737
-0.476
0.159
FAs
-0.381
-0.405
-0.124
CH
CERs
MEA
0.500
0.421
0.118
-0.089
-0.120
-0.636
0.070
0.171
0.620
Eigenvalue
0.160
-0.133
-0.353
0.346
3.386
Contribution (%)
1.444
-0.130
Fifth
1.196
-0.325
0.134
0.600
-0.170
0.003
-0.081
0.345
0.132
0.508
-0.199
Seventh Eighth
0.209
0.116
-0.455
-0.245
0.443
0.012
0.412
0.194
0.111
-0.403
0.613
-0.052
0.400
0.715
-0.140
-0.170
0.709
0.539
0.415
0.187
0.122
42.3
18.1
15.0
8.9
6.7
5.2
2.3
1.5
42.3
60.4
75.3
84.2
90.9
96.1
98.5
100.0
Accumulated
contribution (%)
Abbreviations:
as described in Table I.
o
o
o
o
o
o
o •
o o
o
8 o
o0 o o o
o
o
o
o
-2
o
o o
08
o
o
OoOO
0
o
oo
o
I
-4
o
o
-2
I
I
0
2
4
Z1
Figure 8. Two-dimensional
projectionof 44 Japanese
femalesby the first two components
(Z1 and Z2) of
PCA for the levelsof eightlipids.Eachpoint(C)) represents
the valuesof Z1 andZ2 for eachindividual.
nantlyaccounted
for 42.3 % of the total variation(TableIII, Figure8). Individualswhose
hair fiberscontainlower lipid levelsin groupA and higher lipid levelsin groupB are
distributedat the right-handsideof the projectionin Figure 8. In contrast,individuals
whosehair fiberscontainhigher lipid levelsin group A and lower lipid levelsin group
B aredistributedat the left-handsideof Figure8. Distributionof the youngerandolder
individualsaccordingto their agesin the PCA, asshownFigure 9, suggeststhat the hair
lipid compositionmay changewith increasingage.
Sincelipids in group A (SQ, WEs, TGs, and FAs) are componentssimilar to thoseof
sebum(28), it is likely that the lipids in group A result from sebumattributable to
14
JOURNAL OF COSMETIC SCIENCE
ß
-2
-4
-2
0
2
4
Z1
Figure 9. Two-dimensional
projectionof 44 Japanese
femalesby the first two components
(Z1 andZ2) of
PCA for the levelsof eight lipids. Eachpoint represents
the valuesof Z1 and Z2 for eachindividual.
Symbols:
¸: ages1 to 10. [•: ages11 to 20. /•: ages21 to 30. O: ages31 to40. ß: ages41 to 50. ': ages
51 to 60. •': ages61 to 70. x: ages71 to 81.
sebaceous
glands.Thus, the lipids in group A may originatefrom sebumpenetrating
into the inside of hair fiberswithin hair follicleswhere sebaceous
glandsare included
histologically.Koch eta/. (15) designatedhair lipids within hair fibers as "internal
sebum"in their paper.Although someFAs may originatefrom hair matrix cells,the
existenceof higher levels of unsaturatedand branchedFAs in hair lipids (7,18,28)
suggeststhat most FAs result from decompositionof TGs or WEs as sebum.On the
other hand, lipids in group B (CH and CERs) are thought to result from intrinsic
constitutivelipids biosynthesized
in hair matrix cells,basedupon the fact that they are
not synthesizedin sebaceous
glands(28). HCs (classifiedinto group C) consistingof
carbonnumber 22--44 has been reportedin the analysisof hair lipids (29), but their
originremainsunclear.It hasbeenproposed
by a studyusingMapleSyrupUrine Disease
patients(6) that MEA (classifiedinto group D) may resultfrom an intermediarymetabolite of isoleucinewithin hair matrix cells. Therefore,accordingto their origins,
groupA canbe designatedasexogenous
lipids andgroupsB andD asendogenous
lipids.
Changesin hair lipid compositionwith special referenceto exogenouslipids with
increasingage (Figure 9) could be explainedin terms of increasedsebumsecretion
during adolescence
(30). The level of MEA did not correlatewith both lipids in group
B (CH and CERs), althoughthesethree lipids are endogenous.
This suggeststhat the
productionof eitherCH or CERsmay interactwith eachotherduring the biosynthetic
process,while MEA may be regulatedby anothermetabolism.
The reasonthat there wasa negativecorrelationbetweeneachlevel of exogenous
lipids
(groupA) andendogenous
ones(groupB) might be dueto the contributionof the CMC.
Sinceendogenous
lipids in group B are the main components
of cell membranelipids
that form CMC (4,17), they can lead to the formationof stableand strongCMC.
Therefore,stableandstrongCMC may resultin enhancingthe barrierfunction(31,32),
HAIR
LIPID
COMPOSITION
15
which is attributableto preventingexternalmaterialsfrom entering into hair fibers as
is the casein skin. Consequently,
higher levelsof endogenous
lipids in group B may
resultin diminishedlevelsof exogenous
onesdue to their difficulty in penetration,while
lower levelsof endogenous
lipids in groupB may resultin increasedexogenous
onesin
hair fibers.
Available studiesdealing with lipids within human hair have hitherto usedthe term
"internallipids"for lipids within hair fibers(6,7,11-20). This term for lipids penetrating from a sebaceous
gland into hair fibersmay be not misdirectedbecause
theselipids
areinternalized.However,baseduponthe fact that they arenot intrinsicinternallipids,
they shouldbe designatedas exogenous
lipids (group A), as revealedin this study. On
the other hand, endogenous
lipids consistingof CH and CERs (group B) and MEA
(groupD) shouldbe designatedas intrinsicinternallipids of humanhair. While some
FAs originatefrom hair matrix cells,most FAs may occuras a resultof the decompositionof TGs or WEs assebumcomponents.
This is corroborated
by the fact that there
arehigherlevelsof unsaturated
and branchedFAs in hair lipids (7,18,28) and that there
is the positivecorrelationbetweenFAs and the other exogenous
lipids. Therefore,it is
likely that a lipid classof FAs within hair fibersoccursmainly as exogenouslipids.
CONCLUSION
In summary,the lipid compositionat the proximalroot regionsof human hair hasbeen
characterized
for the first time. The hair lipids canbe classifiedinto four groups:group
A (SQ, WEs, TGs, and FAs); group B (CH and CERs); group C (HCs); and group D
(MEA). Sincegroup A or groupsB and D originatefrom sebumpenetratingthe hair
shaft or from constitutivehair lipids biosynthesizedin hair matrix cells, they were
designatedexogenouslipids or endogenousones,respectively.The hair lipid composition among individualswas also characterizedby a predominantnegativecorrelation
betweenlipids for groupsA and B. This negativecorrelationsuggeststhat the endogenouslipids in group B serveas a barrieragainstthe penetrationof exogenous
lipids
(groupA). Although many researchers
havehitherto usedthe term "internallipids" for
all hair lipids including exogenous
lipids (groupA in our study),only endogenous
lipids
consistingof CH and CERs (group B) and MEA (group D) shouldbe designatedas
intrinsic internal lipids of human hair.
ACKNOWLEDGMENTS
We expressour cordialgratitude to Dr. Yoshiaki Fujikura and Dr. Katsumi Kita for
their discussions
and encouragement
of this study.Our sincerethanksare alsodue to
Mr. ToshihikoSakaiandMs. Kumi Suginofor their assistance
in hair samplecollections.
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