CLIN. CHEM. 36/3, 477-480 (1990)
Distribution of Trace Elements in the Lipid and Nonlipid Matter of Hair
KhudreM. Attar, M. A. Abdel-AaI, and P. Debayle
We studied the effect of lipid removal on the concentrations
of 13 trace elements measured in human hair. We used a
pooled specimen of hair from a barber shop, initiallywashed
with de-ionized water, with ultrasonic cleaning, then analyzed
for Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Si, Sr, and Zn with
use of an inductively coupled plasma emission spectrometer.
The lipid was removed by Soxhlet extraction with ethanol,
and the hair was re-analyzed. We found several elements
present in a relatively large proportion (>20%) in the lipid
fraction, mainly Na, K, Ca, Mg, Ni, and Sr. We suggest that
removal of part or all of the lipids from hair by using
detergents or other lipid-removing solvents for washing may
account for the variability in data on elements in hair reported
by different laboratories, and that those elements largely
present in the lipid fraction are the result of environmental
exposure, whereas those retained in the hair fiber after lipid
removal can be attributed to nutritional and clinical aspects.
We believe that such determination of the distribution of
elements may help validate the use of hair in assessing trace
elements in the body.
AdditIonal Keyphrases: inductively coupled plasma emission spectrometry
variation, source of
Soxhlet extraction
There is increasing evidence that hair can be considered
a minor excretory organ, supporting its use as a biopsy
material representing the body (1-3). The ease with which
it can be collected, its stability on storage, and its high
concentrations of trace elements as compared with other
body tissues and fluids (4) all make hair a suitable specimen for epidemiological studies and potentially for diagnostic considerations. These advantages, however, are
counteracted by the lack of agreement in results obtained
by laboratorians involved in hair analysis (5).
The predominant
reason for such disagreement is differences in pre-analysis treatment of hair samples, specifically the way it is washed. Because dust and cosmetic
treatment may contribute most of the element concentrations in hair, samples must be washed before analysis.
However, differences in washing procedures cause results
to differ (6, 7). The importance of a standardized washing
procedure-including
the type of washing solution, the
duration of washing, and the number of successive washes-has been widely recognized and has lead to the development of the standard practice recommended by the International Atomic Energy Agency for assessment of environmental pollutants (8). Even so, Salmela et al. (7) have
disputed this recommendation because they found that, for
every element determined, there was a point below which
the concentration could not be reduced by further washing,
and that this point was a function of the type of hair, the
washing solvent, and the duration and number of succes-
Central Analytical and Materials Characterization Laboratories, Research Institute, King Fahd University of Petroleum and
Minerals, Dharan 31261, Saudi Arabia.
Received October 13, 1989; accepted December 18, 1989.
sive washes. They therefore recommended an additional
prerequisite in the pre-analysis treatment of human hair:
that washing should be prolonged, or the number of washes
increased, until further washes do not change the measured concentration of the elements to be determined. This
seems a plausible criterion, analogous to the accepted
practice of drying solid samples until no further change in
weight is observed. It might well explain the variability in
hair elements reported by workers in different laboratories
who used the same washing solutions, but it would not
explain why different washing solutions give different
results.
We believe that an important factor contributing to the
problem and overlooked by many investigators is the hair
sample itself. Analytically, hair has been regarded hitherto
as a homogeneous sample within which the elements are
homogeneously dispersed. Such is not the case. Hair is
actually a heterogeneous entity with well-defined separate
parts, a complex system consisting of several morphological
components. Dry human hair is approximately 90% to 95%
protein. The remaining constituents are lipids, pigments,
and trace elements. Endogenous trace elements probably
constitute an integral part of the fiber structure, in salt
linkages or coordination complexes with the side chains of
the protein (9). However, they can also exist as part of the
lipid matter or in association with the pigments, in which
case removal of all or part of the lipid matter would
undoubtedly affect results for hair analyses.
For example, if the elements were dispersed homogeneously in both the lipid and nonlipid matter of hair, then
removal of part or all the lipid would not change the
relative measured concentrations of the elements. However, if the elements were associated only with the protein
matter, then removal of the lipid would increase the measured concentrations of the elements in the hair. Also, if an
element to be determined existed only in the lipid layer,
then lipid removal would reduce its measured concentration in the hair to zero. The possibility also exists that
different elements have different distribution patterns
within the hair fiber; i.e., they are heterogeneously distributed between the lipid and nonlipid matter, depending on
specific functional or excretory routes. Consequently, the
use of washing solutions with different lipid-removing
capacities could lead to erroneous
results in the final
analysis.
To investigate such possibilities, we extracted the lipid
matter from hair and observed the changes, if any, in the
concentrations of the following elements in the extracted
hair: Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Si, Sr, and Zn.
We wanted to elucidate the distribution pattern of these
elements in the lipid and nonlipid matter of human hair.
Materials and Methods
Sampling:
The pooled specimen of hair clippings from 49
men (ages 18-47 years) was collected from a barber shop
during a week. It was freed from extraneous debris by
confining one portion at a time between two stainless-steel
seives and blowing it with oil-free laboratory air for 1 mm.
CLINICAL CHEMISTRY, Vol. 36, No. 3, 1990 477
The cleaned portions were then cut into 1- to 2-cm pieces
with stainless-steel scissors, mixed thoroughly with a Teflon rod, and stored in five separate sample bags.
Because the pooled hair was a heterogeneous sample, we
made it into a composite by mixing and remixing randomly
in a common bag and redividing into separate composites.
To select an analytical sample, we combined portions from
each composite, mixed, and removed the sample for analysis. This sampling procedure was performed on a clean-air
benchtop with use of plastic gloves.
Washing and d7ying the hair: The solvent used to wash
the hair before analysis had to have minimum lipidremoving ability, yet effectively remove dust without influencing the concentration of endogenous elements. We used
ultrasonic cleaning with de-ionized distilled water, which
satisfies such criteria (3), and we prolonged the washing
until further washing did not change the concentrations of
the elements. We transferred the samples into plastic
beakers and washed by ultrasonic cleaning for 5 mm per
washing, stopping the ultrasonic treatment for the last
minute to let most of the hair settle down and drain the
excesswater. We evaluated the effect of 2,4,6,8, 10, and 12
successive washings. Samples were then dried at 60#{176}C
for
24 h and stored in a desiccator for later determination of
the elements by inductively coupled plasma (ICP) spectrometry.
Extraction and determination
of lipid: Ethanol, a solvent
that can swell hair, removes more lipid than do nonswelling solvents such as diethyl ether, benzene, or chloroform (9). To extract lipids from hair, we treated about 1-2
g of hair by Soxhiet extraction with 100 mL of “HPLC”
grade ethanol (Fluka AG, Chemische Fabrik, Buchs CH9470, Switzerland) for 7 h. The ethanol extract was evaporated to 10 mL in a rotary evaporator on a water bath
maintained at 85 #{176}C
under reduced pressure, then transferred onto a pre-weighed aluminum dish and placed in a
desiccator, which was purged with nitrogen gas to remove
the last traces of solvent. We dried the sample in the dish at
60 #{176}C
for 24 h and obtained the weight of the lipid. The
extracted hair was analyzed for the elements.
Preparation
of hair samples and determination
of the
elements by ICP: About 0.5 to 1.0 g of dry hair was packed
into a pre-cleaned and pre-weighed Vycor crucible (capacity
30 mL, Coming brand; Fisher Scientific Co., Springfield, NJ
07081). The hair was charred in the flame of a Terrill burner
(Fisher Scientific Co.). (This operation was conducted in the
hood owing to liberation of hydrogen sulfide gas.) The
charred hair was then dry-ashed in a muffle furnace, at
550-600 #{176}C
for 5 h. We added 5 mL of a 50 mLIL solution of
HC1 (“Baker-Analyzed” reagent; J.T. Baker Chemical Co.,
Phillipsburg, NJ 08865) to the cooled ash and heated the
crucible on a hotplate until the ash was completely dissolved. We then diluted the solution to 10 mL with a 50
mJJL solution of HC1 in a Class A volumetric flask.
The elements were determined with an ICP spectrometer
(Model 3580, vacuum version; Applied Research Laboratories, Ecublens, Switzerland) equipped to measure 48 elements simultaneously and fitted with a nozzle purged with
argon gas to allow observation of emissions in the vacuum
ultraviolet region. Table 1 summarizes
the instrument
settings.
Calibration standards were prepared by serial dilution of
standard spectrometric solutions (National Institute of
Standards and Technology, Gaithersburg, MD) with dilute
(50 mL/L) HC1.
478
CLINICAL
CHEMISTRY,
Vol. 36, No. 3, 1990
Table 1. Instrument SettIngs for ICP Analysis of Hair
Solutions
Forward power
1200 W
Observation height
Plasma gas flow
15mm
12.8 Llmin
Carriergas flow
Nozzle argon flow
Sample uptake rate
0.8 L/min
1.5 Llmin
2
mL/min
Results
Washing of hair:
elements
remaining
ionized
Figure
1 shows the percentages of
after successive washings with deDifferent elements required different num-
water.
bers of successive washings until no further change in
concentration was observed, ranging from two successive
washings for Cu and Zn toll for Fe, as shown in the figure.
We therefore washed the hair samples 12 successive times
with de-ionized water to ensure removal of extraneous
contamination before multi-element analysis by ICP spectrometry.
Lipid content of hair: The mean amount of lipid removed
from the hair after 7 h of Soxhlet extraction with ethanol
corresponded to 4.65% (SD 0.21%) of the weight of the dry
hair. This figure is the average of five replicate determinations.
Concentrations
of elements in hair samples: Table 2
presents data on the concentrations of the elements in the
C,
#{163}
so
0
i
I.
4
I
10
12
Number of SuccessiveWashings
Number
of SurcessfveWashings
Fig. 1. Percentages of elements remainingafter successivewash-
ingswith de-ionizedwater for Cu, Fe, Ca, Cr, Mg, Zn, P, Ni, Sr, Si,
Mn, K, and Na
Each point representsthe averageof threedeterminations
Table 2. Concentrations of Elements In Hair before
Washing, after Washing, and after Extraction of Lipid
Concentration, pg/g, mean
Element
Ca
Beforewashing
2210(25)
(and SD)
AfterwashIng
Afterextraction
1140 (29)
1380 (44)
0.95 (0.11)
21.4 (0.6)
35.5 (1.1)
13.7 (1.7)
0.83 (0.03)
20.1 (0.2)
32.4 (1.5)
<4.0(1.3)
Cr
Cu
Fe
K
1.81 (0.21)
23.0 (2.5)
58.1 (1.2)
191 (6)
Mg
518 (10)
Mn
Na
1.08 (0.02)
962 (8)
Ni
P
170 (2)
156 (2)
157 (2)
Si
Sr
Zn
452 (7)
31.3 (0.5)
181 (7)
222 (7)
21.1 (0.7)
178 (3)
201 (7)
17.0 (0.4)
179 (3)
248(6)
197(6)
0.51 (0.03)
6.43 (2.72)
3.91 (0.05)
0.48 (0.03)
2.48 (0.46)
2.24 (0.06)
2.70 (0.25)
Values are for five replicate measurements.
hair before washing, after 12 successive washings with
de-ionized water, and after extraction of lipid. All values for
the extracted
hair, except for potassium,
exceeded the
detection limit of the ICP we used. The absence of potassium was confirmed
by scanning the potassium
channel.
Evaluation
and nonlipid
of concentrations of the elements in the lipid
matter of hair: Let
L (%) = the percentage of lipid extracted (4.65 ± 0.21),
y (pg)
=
the amount of element present in 1 g of washed
hair before lipid extraction,
x (pg)
the amount of element left in 1 g of hair after
lipid extraction,
a (pg)
=
the amount of element in the lipid matter
extracted from 1 g of washed hair, and
b (zg)
=
the amount of an element in the nonlipid matter of 1 g of washed hair.
Then the following equations will be valid:
=
y=a+b
x
=
Discussion
It is necessary to describe the morphology
and mention
some pertinent properties of the hair fiber before commenting on the distribution of the elements in the lipid and
nonlipid matter of hair.
Three distinct types of cells are generally apparent in
crosssections of human hair fibers. The outermost region of
cells, which forms a thick protective coating enclosing the
hair fiber, is called the “cuticle.” It consists of flat overlapping cells (scales), with each of the cells surrounded
by a
membrane called the “epicuticle,” which is thought to
contain a lipid layer. The cuticle envelops the “cortex.”
Cortical cells comprise the major part of the fiber mass and
contain keratin-based
fibrous proteins and intercellular
binding materials. The cortex also contains the pigment
granules, dispersed throughout.
A third type of cells, usually found in hair fibers of large diameter and called the
“medulla,”
is located along the fiber axis of the hair fiber. It
constitutes a very small proportion of the fiber mass.
Extraction of hair with hot ethanol can remove a portion
of or the entire epicuticle membrane and release the lipid
material. The isoelectric pH of a hair fiber is about 3.67 (9).
Elements in the lipid fraction: We found (Table 3) that
Na, K, Ca, and Mg constitute a major proportion of the
elements found in the lipid fraction.
Because the isoelectric pH of hair fibers is 3.67, at any
higher value the surface of the fiber bears a negative
charge and can attract cations such as Na4 and K4 during
frequent washing with soaps, detergents, or shampoos. It
can also attract Ca2 and Mg2 cations during rinsing,
especially if the domestic tapwater available for washing is
“hard.” Such cations can diffuse through the epicuticle
membrane and so are difficult to rinse out (10). However,
they are eventually released with the lipid fraction after
the epicuticle membrane is destroyed with hot ethanol.
(1)
(2)
b {l + [L/(100-L)]}
L/(l00-L)
being the extra fraction of extracted hair taken,
to achieve a weight of 1 g.
The percentage distribution of the elements in the lipid
and nonlipid fractions of hair can then be calculated as
follows:
Percent of an element in lipid fraction
Table 3 gives the results so calculated for the percentage of
each element measured in the lipid and nonlipid matter of
hair, along with the 95% confidence interval for five replicate measurements, as determined from the variance.
=
Percent of an element in nonlipid fraction
100 aly
=
100 bly
(4)
Values for y and x can be substituted from the second and
third columns of Table 2, into equations 1 and 2, from
which a and b are evaluated and used to calculate results
for equations 3 and 4.
The standard deviation
for the calculated
percentage
results in equations 3 and 4 can be evaluated by considering the percentages as a function of three variables y, x, and
L. Because the values as well as the variances of the
variables are known from Table 2, one can apply the rule of
partial derivatives to determine the variance of the result.
Table 3. Percentages of Total Element Concentrations
DIstributed between the Lipid and Nonlipld Fractions
of Hair
Percent of total
Element
Ca
Cr
Cu
Fe
In lipid
21.1
16.7
10.4
13.0
In nonlipid
78.9
83.3
89.6
87.0
K
Mg
Mn
Na
Ni
>72.2
24.2
10.3
63.2
20.9
<27.8
75.8
89.7
36.8
P
Si
4.2
13.7
Sr
Zn
22.9
4.4
Confidence
Intervala
±4.4
±14.2
±3.6
±5.8
±11.9
±3.7
±9.6
±21.1
79.1
95.8
86.3
±11.2
±2.0
±4.5
77.1
95.6
±4.5
±2.5
95% confidence interval calculated as (±
IS/\r0) of the derived percent-
age values, where t = Student’s f-test value, s = calculated standard
deviationsof the derived percentages, and n = 5 replicate measurements.
CLINICAL CHEMISTRY, Vol. 36, No. 3, 1990 479
Some Cu and Ni, which, excreted in sweat, play an important regulatory role in hot climates (11), also appeared in
the lipid fraction.
Elements in the nonlipid fraction: Except for Na and K,
the major proportion of all the elements was contained in
the nonlipid fraction. Cu, Cr, Fe, Mg, Mn, and Zn, all of
which act as enzyme cofactors and are always associated
with proteins in the cells, constituted a major part. Also the
presence of Ca and P in the nonlipid fraction can be related
to their important role in the hardening and mineralization
of keratm (12).
We believe that most of the elements that could have
contacted hair via external routes such as environmental
contamination, application of cosmetic preparations, or
excretion of sweat have been deposited on the hair fiber and
mixed with the sebaceous excretion, becoming resistant to
rinsing with water. However, they were released in the
lipid fraction of the hair, which mainly is sebum. The other
elements, which could be related to nutritional and clinical
aspects, were still present in the hair fiber after removal of
lipid.
Use of different washing solutions: Washing
hair fibers
with solvents having various lipid-removing capacities can
alter the distribution of the elements in the lipid fraction.
Consequently, any comparison of results of hair analysis
from laboratories using different solvents will be meaningless for the elements that are found in the lipid fraction, as
evidenced by the data presented in Table 2. The need for a
standard washing solution and procedure has already been
recognized (7, 8). However, for elements that predominate
in the nonlipid matter of hair, e.g., Cu, Cr, Fe, Mn, and Zn,
data from laboratories using even different washing solutions might retain their validity, because the lipid content
of hair is low (4.65% for our samples). This has been
verified in the many publications on these elements.
Use of standard washing solution: Conclusions and judgments derived from results of hair analysis related to
nutritional, environmental, epidemiological, and clinical
studies can also be obscured for some of the elements when
their distribution between the lipid and nonlipid matter is
not known, even if a standard washing solution and procedure are used. For example, if the Ca and P concentrations
in hair were to be related to their mineralization of keratins, from Table 3 we see that only 78.9% of the total Ca
found after washing with water would have to be used for
data analysis rather than the total itself, whereas for P the
total value could be used, because most of the P is in the
nonlipid fraction. Also, if the concentration of Ni in hair
were to be related to sweat excretion, only that portion
associated with the lipid fraction (20.9% of the total Ni
found), which is equivalent to 0.56 .tg of Ni per gram of
480 CLINICAL CHEMISTRY, Vol. 36, No. 3, 1990
hair, should be used for data analysis rather than the total
value, 2.70 tzg/g, from Table 2.
Failure to realize that some of the elements in hair can
be found in the lipid layer and that hair is not really a
homogeneous
sample has contributed
significantly to the
lack of agreement of results from various laboratories. This
lack of agreement is reflected in the reference values for
concentrations of elements in hair (13), where it was
possible to arrive, for instance, at reference values for Cu,
Cr, Mn, Fe, and Zn, but not for Ni, which we found to be
partly in the lipid fraction. Also the ranges of the values for
those elements in hair were large as compared with their
ranges in (e.g.) urine and serum.
Unlike serum or urine, which can give an indication of
the status of the body at a specific time or on a daily basis,
trace-element concentrations in hair represent timeweighted average-exposure values, which are useful for
epidemiological and nutritional studies as well as for environmental and forensic investigations.
This work is part of KFUPMIRI project no. 15010, supported by
the Research Institute of King Fahd University of Petroleum and
Minerals.
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