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A study of association between fingernail elements and osteoporosis by laser

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A study of association between fingernail elements and osteoporosis by
laser-induced breakdown spectroscopy
Maryam Bahreini, Zahra Hosseinimakarem, and Seyed Hassan Tavassoli

Citation: J. Appl. Phys. 112, 054701 (2012); doi: 10.1063/1.4747934

View online: http://dx.doi.org/10.1063/1.4747934
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Published by the American Institute of Physics.

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 JOURNAL OF APPLIED PHYSICS 112, 054701 (2012)

A study of association between fingernail elements and osteoporosis

by laser-induced breakdown spectroscopy
Maryam Bahreini,a) Zahra Hosseinimakarem, and Seyed Hassan Tavassoli
Laser and Plasma Research Institute, Shahid Beheshti University, G. C., Evin, Tehran 1983963113, Iran

(Received 30 November 2011; accepted 20 July 2012; published online 6 September 2012)
Laser induced breakdown spectroscopy (LIBS) is used to investigate the possible effect of
osteoporosis on the elemental composition of fingernails. Also, the ability to classify healthy,
osteopenic, and osteoporotic subjects based on their fingernail spectra has been examined. 46
atomic and ionic emission lines belonging to 13 elements, which are dominated by calcium and
magnesium, have been identified. Measurements are carried out on fingernail clippings of 99
subjects including 27 healthy, 47 osteopenic, and 25 osteoporotic subjects. The Pearson
correlations between spectral intensities of different elements of fingernail and age and bone
mineral densities (BMDs) in nail samples are calculated. Correlations between line intensities of
some elements such as sodium and potassium, calcium and iron, magnesium and silicon and also
between some fingernail elements, BMD, and age are observed. Although some of these
correlations are weak, some information about mineral metabolism can be deduced from them.
Discrimination between nail samples of healthy, osteopenic, and osteoporotic subjects is shown to
be somehow possible by a discriminant function analysis using 46 atomic emission lines of the
LIBS spectra as input variables. The results of this study provide some evidences for association
between osteoporosis and elemental composition of fingernails measured by LIBS. V C 2012

American Institute of Physics. [http://dx.doi.org/10.1063/1.4747934]

I. INTRODUCTION between toenail selenium concentration and bladder cancer

risk in women, but such a relation was not observed in
Analysis of human tissues can be an ideal method for
men.12 However, there are some literatures that could not
screening a population for identification of diseases. There is
find any relation between nail and disease. In a prospective
conflicting epidemiologic data supporting this novel idea or
study, association between level of arsenic, copper, chro-
not. Patriarca et al. claimed that determination of trace ele-
mium, iron, or zinc of toenail and breast cancer risk were
ments in body fluids and tissues is essential for prevention,
studied and results did not provide evidence for an important
diagnosis, and treatment of some related diseases.1 Among
effect of these five trace elements on breast cancer risk.13
human tissues, nail is a favorable biomarker for clinical
Osteoporosis is a skeletal disease and it can probably
investigations because it is easy to collect, store, and trans-
influence the nail mineral content. The possible correlation
port. The component of nail can contain information about
between osteoporosis and elemental composition of finger-
metabolic events that occurred during the time of its forma-
nail offers that analyzing of mineral content of nail can be
tion.2,3 Accepting the idea that the trace element composition
served as a diagnostic tool for osteoporosis. There are differ-
of nail can be changed by several pathological, physiologi-
ent methods for elemental analysis of biological matrix
cal, and environmental factors,4–7 it is possible that some dis-
including neutron activation analysis (NAA),14 atomic
ease influences the trace element content of nail and nails
absorption spectrophotometry (AAS),15 inductively coupled
serve as an accessible biopsy material. Garland et al.
plasma mass spectrometry (ICP-MS),16 x-ray fluorescence
assessed the reproducibility over a 6-year period of 16 trace
(XRF),17 and proton particle-induced x-ray emission (proton
elements measured in toenails and suggested that a single
PIXE).18 Vecht-Hart et al. concluded that Ca and Mg meas-
measurement of these biomarkers reflects long-term expo-
urements in nail clippings by NAA cannot be used for
sure.8 Nail clipping test for diagnosis of cystic fibrosis has
screening purposes in the prevention of osteoporosis.19 Oth-
been done and concluded that this method may be useful in
erwise, Ohgitani et al. could find relation between nail cal-
cases in which a sweat test cannot be performed or gives bor-
cium and magnesium and bone mineral density (BMD) and
derline values, or when a patient is far from a diagnostic cen-
claimed that nail mineral content may be utilized as one of
ter.9 In a similar work, Leonard and Morris could find
the indicators of bone mineral metabolism.15 Towler et al.
relation between sodium, calcium, and magnesium level of
proved that change in the protein phase of human nail meas-
nail, and cystic fibrosis of pancreas.10 In another work, they
ured by Raman spectroscopy is correlated with relevant
found an increase in sodium and calcium and also a decrease
changes in bone proteins.20–22
in magnesium concentration of fingernail in a kwashiorkor
A recently proposed technique for analysis of biological
group.11 Michaud et al. observed a strong inverse relation
samples is laser induced breakdown spectroscopy (LIBS).
This technique is based on emission of excited atoms, ions,
a)
E-mail address: M_Bahreini@sbu.ac.ir. and molecules in plasma produced by focusing a high power

0021-8979/2012/112(5)/054701/9/$30.00 112, 054701-1 C 2012 American Institute of Physics

V
054701-2 Bahreini, Hosseinimakarem, and Tavassoli J. Appl. Phys. 112, 054701 (2012)

laser pulse on sample surface.23,24 Several advantages of BMD measurements and 19 of them have done extra meas-
LIBS including little or no sample preparation; minimally urements of forearm BMD. A panel of the World Health Or-
invasive; fast analysis time and very easy to use,25 make it ganization (WHO) recommended that the diagnosis of
very feasible for analysis of biological samples such as osteoporosis be made when the T score on bone-mineral-
teeth,26,27 hairs,28 cells,29 and bacteria.30–35 As an applica- density measurement by dual-energy x-ray absorptiometry is
tion of LIBS for elemental analysis of nails, Haruna et al. 2.5 or lower. They also suggested that the term “osteopenia,”
used it for calcium detection.36 Also, Hamzaoui et al.. have or “low bone mass,” be applied when T scores are from "1.0
used LIBS as a potential method for simultaneous measure- to "2.5.43–45 Here, 99 subjects are categorized into three
ment of the Ca, Na, and K in normal and pathological groups: 27 healthy subjects with age of 48.85 6 10.575; 47
nails.37 This technique has been used in a previous work in osteopenia patients with the age of 54.72 6 9.134 years, and
analysis of human fingernails and has been found some rela- 25 osteoporosis patients with age of 59.40 6 13.074 years
tion between elemental composition of nails, gender, and (mean 6 SD). Healthy means that the person has no identifi-
age.38 able risk of fracture; osteopenia means that the patient may
Up to now, the possible correlations between nail miner- be developing a tendency to fracture, and osteoporosis means
als and bone mineral density have investigated rarely. At the that the patient has a high spontaneous fracture probability.
present time, osteoporosis diagnosis, based on BMD meas- Fingernail clippings were taken from free edge of nail plate
urements is done by dual energy x-ray absorptiometry which by a stainless steel nail scissors and kept separately in non-
is so complicated and expensive. In the present study, laser reactive plastic envelopes. Then, all of them were stored at
induced breakdown spectroscopy is used for elemental anal- room temperature until analysis. In order to eliminate any
ysis of fingernail of healthy and osteoporotic subjects. In surface contaminations, specimens were washed by soaking
order to find evidence of associations between elemental in acetone for 5 min followed by soaking in distilled water
composition of fingernails and osteoporosis, the Pearson cor- for the same time. Some of them were dirtier and were
relations between fingernail element intensities, age, and washed more. Then they were dried at room temperature.
BMD in 99 nail samples are calculated. To investigate the
possible classification of high-risk groups of osteoporosis by B. Experiment
LIBS, the subjects are categorized in three groups of healthy,
The LIBS experimental setup used to analyze fingernails
osteopenia, and osteoporosis, based on their BMD measure-
is shown in Fig. 1. Typically, a laser pulse is focused on the
ments and a statistical multivariate method named discrimi-
nail surface. The intense electric field of the laser pulse cre-
nant function analysis (DFA) is used to discriminate and
ates a micro-plasma on the samples and light emitted out of
classify samples in different groups.
the plasma is analyzed by a spectrometer. We use a
Q-switched Nd:YAG laser operating at wavelength of
II. SUBJECTS AND METHODS
1064 nm, pulse energy of 25 mJ/pulse, repetition rate of
A. Subjects 5 Hz, and pulse duration of 6 ns. The energy of laser pulses
can be tuned by using a combination of rotator and Glan–
The subjects participated in this study were randomly
Taylor prism. Laser pulse is focused on the nail samples by a
chosen among population of a bone densitometry center in
lens with focal length of 8 cm. Plasma emission is collected
the city of Tehran. They have been told of the purpose of the
by two lenses with focal lengths of 4 cm and 1.5 cm and is
study and then, all subjects having given informed consent
guided by an optical fiber to an Echelle spectrograph with
for participation to this work. The sampling of fingernails
the resolving power of 1700. The spectrograph is equipped
included administration of questionnaires containing individ-
by an intensified charge-coupled device (ICCD) camera for
ual information such as personal information, medical his-
time resolved detection. A part of laser beam is sent to a pho-
tory, drug consumptions, and diets. Some useful information
todiode by means of a beam splitter and then a signal is send
are obtained from their medical file including important risk
to a delay generator. ICCD is triggered by delay generator,
factors for osteoporosis like: Age, gender, race, bone struc-
800 ns after the plasma initiation. Each spectrum recorded in
ture and body weight, status of menopause, prior history of
gate width of 20 ls.
fracture, and family history.39 99 subjects participated in this
study and most of them are females (94 female and 5 male),
because this disease is more epidemic in women.40 All sub-
jects are Caucasian. Research has shown that Caucasian and
Asian women are more likely to develop osteoporosis.41
Subjects are aging from 22 to 89 years old with the mean of
54.30 and standard deviation of 11.206 years old. Among
these subjects, 69 person are postmenopausal women (mean-
¼ 56.57, SD ¼ 9.111). Menopause is characterized by the
loss of estrogen production by the ovaries and the loss of
estrogens accelerates bone loss.42
BMD measurements were done using a dual-energy
x-ray absorptiometry (DEXA; Hologic Explorer) bone densi-
tometer. All subjects have done lumbar spine and femur FIG. 1. Schematic diagram of the experimental setup for LIBS measurements.
054701-3 Bahreini, Hosseinimakarem, and Tavassoli J. Appl. Phys. 112, 054701 (2012)

Five LIBS experiments were done on each nail sample III. RESULTS AND DISCUSSION
and the spectrum of each experiment is obtained by accumu-
A. LIBS spectrum of human fingernail
lation of 5 laser shots. The samples are moved in a perpen-
dicular plane respect to the incident laser beam by means of Characteristic LIBS spectra of human fingernail samples
a motorized xyz stage so that every laser shot hits a new tar- from a healthy, an osteopenic, and an osteoporotic subject
get position to improve the repeatability of the results. A spe- are presented in Fig. 2. Five different points were selected on
cialized sample holder has been designed in which the
situation of all nail with respect to laser pulse is the same
and laser pulses are focused on a flat surface of nails. All
nail samples were fixed on this holder in the order that dur-
ing experiment, the healthy-patient status of samples was
unidentified.

C. Statistical methods
In the statistical analysis, we calculated the Pearson cor-
relation coefficients which are calculated by dividing the co-
variance of the two variables by the product of their standard
deviation. In classification part of this paper, we performed
DFA. DFA is a multivariate analysis of variance which uses
independent variables and forms linear combinations to pre-
dict and quantify group memberships between distinct
groups. In our study, for example, all the spectra acquired
from fingernails of healthy subjects should ideally be identi-
cal, and thus constitute a group. Fingernail spectra of osteo-
porotic subjects would constitute different groups. DFA uses
set of independent variables from each spectrum to predict
the group membership of that particular spectrum. This is
processed in three basic steps: construction of discriminant
functions, test of significance, and classification.
In the first step, a set of orthogonal discriminant func-
tions is constructed from the data sets from all the groups. In
order to do this, a canonical correlation analysis produces a
set of canonical discriminant functions which are essentially
the eigenvectors of the data expressed in a basis that maxi-
mizes the difference between groups. N " 1 discriminant
functions are constructed for discrimination between N
groups. The first canonical discriminant function (denoted
DF1) is constructed such that it accounts for more of the var-
iance between groups than the second canonical discriminant
function (DF2) and so on. In second step, a test for signifi-
cance in the discriminant functions is performed. This is
done by checking the differences in the means of the groups.
Wilks’ lambda is used in (F) test of mean differences in
DFA, which varies from 0 to 1, with 0 meaning group means
differ (thus, the more the variable differentiates the groups),
and 1 meaning all group means are the same. The F test of
Wilks’ lambda shows which contributions of variables are
significant. In the last step, classification of variables is
undertaken. The group membership of each subject is pre-
dicted based on the calculated discrimination score. Further
detailed information on the statistical procedures can be
found in Refs. 46–49.
All the independent variable vectors from all groups
were then analyzed simultaneously by a commercial DFA
program (SPSS Inc., SPSS v17.0) to construct the canonical
discriminant functions which were in turn used to calculate
FIG. 2. Characteristic LIBS spectra of human fingernail samples from (a) a
for each data spectrum a discriminant function score for that healthy with some atomic emissions identified, (b) an osteopenic, and (c) an
particular function. osteoporotic subject.
054701-4 Bahreini, Hosseinimakarem, and Tavassoli J. Appl. Phys. 112, 054701 (2012)

TABLE I. Identification and intensity of important atomic and ionic lines elements like Cu, Cr, Se, Si, Sr, Ti, K, Ni, Pb, V, Co, Cd,
used in the spectral fingerprint of fingernails. Mn, As, Sb, Sn, and Hg can be found in trace levels.7,16,50
The basis of our spectroscopic identification is formed
Line identification Wavelength (nm) Fractional spectral power
by measurement of the intensities of the 46 emission lines
C(I) 247.85 0.0239 from 13 elements mentioned above. Every line intensity is
Mg(II) 279.55 0.0355 obtained by integrating the area under the peak minus back-
Mg(I) 285.21 0.0032 ground. In each spectrum, the intensities of all 46 observed
Si(I) 288.15 0.0027 lines are summed which is called the total spectral power
Ca(II) 393.36 0.1810
and then the intensity of each spectral line is divided by this
Ca(II) 396.84 0.1333
total spectral power to obtain the fractional spectral power.
Ca(II) 315.88 0.0108
Ca(I) 422.67 0.0492
These fractional spectral powers are used as variables in the
Ca(I) 612.22 0.0074 statistical method. This evaluation is used previously in bio-
Ca(I) 643.90 0.0038 logical application of LIBS.33,38,51
Ca(I) 442.54 0.0060 Therteen elements in fingernail spectrum have been
AI(I) 394.40 0.0065 identified including calcium, magnesium, silicon, sodium,
AI(I) 396.15 0.0072 potassium, titanium, strontium, iron, aluminium, carbon,
H(I) 656.27 þ 656.28 0.2359 nitrogen, hydrogen, and oxygen. Table I lists some specific
Na(I) 588.99 þ 589.59 0.0166 lines used in this work, the wavelength of the emission line,
N(I) 742.36 0.0095 and the fractional spectral power of these lines which are the
N(I) 744.22 0.0178
average of 99 nail samples. It should be mentioned that four
O(I) 777.19 þ 777.41 þ 777.53 0.0540
elements of O, H, N, and C are common in air and nail and
K(I) 766.48 0.0021
K(I) 769.89 0.0015
thus, we cannot simply distinguish between them in air and
nail.

each nail plate and five laser pulses hit to each point. Thus,
each spectrum is the average of about 25 laser shots.
These spectra can be used as fingerprint of nails. Similar
LIBS spectra of fingernail have been observed by some
research groups.29,37,38 As depicted in the graphs, fingernails
from three different health status present similar spectra. As
a result, the differences between their LIBS spectra consist
of subtle variations in the line intensities which are not
obvious in a visual examination. The dominant emission
lines in the fingernail spectrum were atomic lines of inor-
ganic elements, as have been addressed on the graph. It is
known that the dominant inorganic elements in nail composi-
tion are Ca, Mg, P, Na, K, Fe, Zn, and A1 while other
TABLE II. The Pearson correlation coefficients between age and three BMD measurements.
B. Relationship between fingernail element
intensities, age, and BMDs
The relations between spectral intensities of 9 fingernail
elements including: silicon, sodium, strontium, calcium,
magnesium, aluminum, potassium, titanium, iron, and age
and three BMD measurements were examined in 99 study
participants. Here, we use the sum of all line intensities
belonging to each element.
1. Correlations between age and BMDs
The results of correlation between age and BMDs are
shown in Table II. The significant correlations have been

Age Menopause status (year) Femur Lumbar spine Forearm

a a a
Age Pearson correlation 1 0.336 "0.326 "0.31l "0.547b
Sig. (2-tailed) 0.005 0.001 0.002 0.015
N 99 69 99 99 19
Menopause status (year) Pearson correlation 1 "0.255b "0.266b "0.612b
Sig. (2-tailed) 0.034 0.027 0.026
N 69 69 69 13

Femur Pearson correlation 1 0.593a 0.713a

Sig. (2-tailed) 0.000 0.001
N 99 99 19
Lumbar spine Pearson correlation 1 0.837a
Sig. (2-tailed) 0.000
N 99 19
Forearm Pearson correlation 1
Sig. 2-tailed)
N 19

a
Correlation is significant at the 0.01 level (2-tailed).
b
Correlation is significant at the 0.05 level (2-tailed).
054701-5 Bahreini, Hosseinimakarem, and Tavassoli J. Appl. Phys. 112, 054701 (2012)

TABLE III. The Pearson correlation coefficients between spectral intensities of fingernail elements.

Si Na Ca Mg A1 K Ti Fe Sr
a a a a a
Si Pearson correlation 1 0.058 "0.146 0.401 0.535 0.282 0.470 0.765 0.039
Sig. (2-tailed) 0.566 0.148 0.000 0.000 0.005 0.000 0.000 0.703
N 99 99 99 99 99 99 99 99 99
Na Pearson correlation 1 0.163 "0.321a 0.238b 0.838a "0.118 "0.155 0.044
Sig. (2-tailed) 0.108 0.001 0.018 0.000 0.247 0.127 0.663
N 99 99 99 99 99 99 99 99

Ca Pearson correlation 1 "0.061 0.044 0.156 0.329a "0.503a "0.096

Sig. (2-tailed) 0.549 0.666 0.122 0.001 0.000 0.345
N 99 99 99 99 99 99 99
Mg Pearson correlation 1 0.027 "0.25lb 0.249b 0.577a "0.123
Sig. (2-tailed) 0.795 0.012 0.013 0.000 0.226
N 99 99 99 99 99 99
AI Pearson correlation 1 0.427a 0.216b 0.321a 0.029
Sig. (2-tailed) 0.000 0.032 0.001 0.773
N 99 99 99 99 99
K Pearson correlation 1 "0.125 "0.008 0.096
Sig. (2-tailed) 0.219 0.934 0.342
N 99 99 99 99
Ti Pearson correlation 1 0.544a "0.098
Sig. (2-tailed) 0.000 0.335
N 99 99 99
Fe Pearson correlation 1 "0.042
Sig. (2-tailed) 0.682
N 99 99
Sr Pearson correlation 1
Sig. (2-tailed)
N 99

a
Correlation is significant at the 0.01 level (2-tailed).
b
Correlation is significant at the 0.05 level (2-tailed).

flagged in the table. Obviously, three BMDs have positively
significant correlations with each other. Correlations
between age and femur and lumbar spine and Forearm
BMDs are negatively significant (r ¼ "0.326; p ¼ 0.001,
r ¼ "0.311; p ¼ 0.002, and r ¼ " 0.547; p ¼ 0.015, respec-
tively). These correlations become stronger in 69 post-
menopausal women (age and femur BMD: r ¼ "0.335, age
and lumbar spine BMD: r ¼ "0.461, age and forearm
BMD: r ¼ "0.743; at p < 0.005). Also, there are significant
correlations between menopause status (year) and
three BMDs. Menopause status (year) means the number
of years passed from menopause. The negative relation
between age and BMDs is mentioned in many publica-
tions52,53 and concludes that menopausal bone loss
occurs in addition to non-menopausal or age-related bone
loss.54

TABLE IV. The Pearson correlation coefficients between spectral intensities of fingernail elements and three BMD measurements.

C Si Na Sr Ca Mg A1 K Ti Fe

Femur Pearson correlation "0.127 "0.187 0.190 "0.106 0.227a "0.034 "0.113 0.105 "0.083 "0.128
Sig. (2-tailed) 0.209 0.063 0.060 0.295 0.024 0.740 0.266 0.303 0.416 0.206
N 99 99 99 99 99 99 99 99 99 99
Lumbar spine Pearson correlation "0.045 "0.051 0.077 "0.167 0.070 0.046 "0.057 0.039 0.019 0.025
Sig. (2-tailed) 0.657 0.615 0.448 0.099 0.490 0.649 0.572 0.702 0.853 0.806
N 99 99 99 99 99 99 99 99 99 99

Forearm Pearson correlation "0.137 0.105 0.195 "0.060 0.152 "0.158 0.472a 0.184 "0.162 0.064
Sig. (2-tailed) 0.576 0.669 0.423 0.807 0.536 0.518 0.042 0.451 0.508 0.794
N 19 19 19 19 19 19 19 19 19 19

a
Correlation is significant at the 0.05 level (2-tailed).
054701-6 Bahreini, Hosseinimakarem, and Tavassoli J. Appl. Phys. 112, 054701 (2012)

TABLE V. The Pearson correlation coefficients between spectral intensities of fingernail elements and age in different groups of healthy, osteopenic, and
osteoporotic subjects.

Si Na Ca Mg A1 K Ti Fe Sr

Age Pearson correlation 0.128 "0.161 0.086 0.487a 0.043 "0.189 0.174 0.169 "0.129
Healthy Sig. (2-tailed) 0.525 0.423 0.671 0.010 0.831 0.345 0.384 0.399 0.522
N 27 27 27 27 27 27 27 27 27
Age Pearson correlation "0.015 "0.059 0.347a 0.010 0.087 "0.051 "0.048 "0.121 0.249
Osteopenic Sig. (2-tailed) 0.920 0.692 0.017 0.948 0.562 0.735 0.749 0.419 0.091
N 47 47 47 47 47 47 47 47 47
Age Pearson correlation 0.282 0.160 0.238 0.171 0.380 0.281 "0.065 0.183 "0.580b
Osteoporotic Sig. (2-tailed) 0.171 0.445 0.252 0.415 0.061 0.173 0.758 0.381 0.002
N 25 25 25 25 25 25 25 25 25

a
Correlation is significant at the 0.05 level (2-tailed).
b
Correlation is significant at the 0.01 level (2-tailed).

2. Correlations between fingernail elements be due to the fact that body elements would be depleted
through both nail and urine.59
In this part, we investigated the correlation between
fingernail element intensities. The results are shown in
3. Correlations between fingernail elements and
Table III.
BMDs
The strongest correlation is between sodium and potas-
sium of fingernails (r ¼ 0.838; p < 0.01). A similar result Results of correlations between fingernail elements and
obtained in the previous work on 45 fingernail samples38 and BMDs are shown in Table IV. Bones consist of two major
also by Nowak.55 Such a correlation is also observed in ingredients: bone cells (comprising of osteoclasts, osteoblasts,
groups of 27 healthy, 47 osteopenic, and 25 osteoporotic and osteocytes) and minerals (including calcium).60 Calcium
subjects separately (r ¼ 0.942, r ¼ 0.748, and r ¼ 0.756; is the most important mineral in bone metabolism and it is a
p < 0.01, respectively) and correlation in healthy subjects is natural medication for treatment of osteoporosis. Low Ca
the strongest one. We also calculated these correlations in 69 intake is considered to be a modifiable risk factor for osteopo-
postmenopausal women among participants where this posi- rosis.61 So, any disorder of calcium concentration in finger-
tive correlation still exists (r ¼ 0.829; p < 0.01). Similar cor- nails could probably tell us something about the condition of
relation also observed by Liu et al. in serum of 290 bones. As it can be seen in Table IV, there is a significant
postmenopausal women from ages 45 to 65 in the Xi’an
urban area at p < 0.01 level.56
There is a significant negative correlation between Ca
and Fe of fingernails (r ¼ "0.503; p < 0.01). The same corre-
lation is observed in healthy (r ¼ "0.476; p ¼ 0.012) and
osteopenia (r ¼ "0.565; p < 0.01) groups separately. This
correlation exists in osteoporotic group (r ¼ "0.380;
p ¼ 0.061) but it is not significant. This correlation is also
significant in group of premenopausal (r ¼ "0.757; p < 0.01)
and is weaker in postmenopausal group (r ¼ "0.375;
p ¼ 0.002) while 2% of premenopausal and 22% of post-
menopausal people have osteoporosis. Liu et al. also have
reported a negative correlation between Ca and Fe in serum
of 281 postmenopausal women (r ¼ "0.156; p < 0.01) while
43.77% of them had morbidity rate of osteoporosis.56
A significant positive correlation is observed between
Mg and Si of fingernails (r ¼ 0.401; p < 0.01). This correla-
tion in the osteoporotic group is the strongest (r ¼ 0.523;
p ¼ 0.007). Magnesium is essential for the formation of col-
lagen as well as bone mineralization. Bone growth induced
by silicon is mainly due to an increase in collagen. Charnot
and P!erès57 suggested that silica controls the metabolism of
calcium and magnesium. Also, a stronger positive correla-
tion was observed in the previous work (r ¼ 0.65).38 The
FIG. 3. Discriminant function analysis plot showing the first two discrimi-
same correlation has been reported in urine sample by Ber- nant function scores of LIBS spectra obtained from fingernails of three dif-
lyne et al.58 The similar results between nail and urine may ferent health status (normal, osteopenia, and osteoporosis).
054701-7 Bahreini, Hosseinimakarem, and Tavassoli J. Appl. Phys. 112, 054701 (2012)

TABLE VI. Results of classification between healthy and osteopenic groups.

Predicted group membership

Category Healthy Osteopenia Total Correctly classified

Original Count Healthy 25 2 27

Osteopenia 4 43 47
% Healthy 92.6 7.4 100.0
Osteopenia 8.5 91.5 100.0 91.9

Cross-validated Count Healthy 15 12 27

Osteopenia 18 29 47
% Healthy 55.6 44.4 100.0
Osteopenia 38.3 61.7 100.0 59.5

positive correlation between Ca intensity and femur BMD
(r ¼ 0.227; p ¼ 0.024). This correlation is mainly due to
healthy subjects because the correlation between Ca and
BMD is significant in healthy subjects (r ¼ 0.421; p ¼ 0.029)
but not in patient groups. This shows that Ca metabolism is
regular in healthy subjects but disturbs in patients.
There is a correlation between Ca and Femur BMD in
group of 25 premenopausal women (r ¼ 0.538; p ¼ 0.006), but
not in postmenopausal women. The drop in estrogen that
occurs during menopause triggers the production of the inflam-
matory mediator interleukin-6, which stimulates the growth of
additional osteoclasts (cells that break bone down), increases
bone loss.42 The lack of significant correlation between Ca and
Femur BMD in postmenopausal women can be due to the
irregularity of Ca metabolism as a result of estrogen drop.
There is a weak but significant negative correlation
between Na intensity and lumbar spine BMD (r ¼ "0.377;
p ¼ 0.009) in 47 nail samples of osteopenic subjects. Several
reviews examining the impact of Na on bone health have
been published. The kidney plays a key role in maintaining
homeostasis of several mineral ions, particularly Ca, Na, and
K.62 Nordin et al. concluded that Na intake plays a very im-
portant role in determining the rate of obligatory Ca loss,
and must be a risk factor for osteoporosis.63 The negative
correlation between Na and lumbar spine BMD shows that
an increase in Na level in fingernail (which can be due to
increase in Na intake) leads to a decrease in the lumbar spine
BMD and emphasize on conclusion of Nordin et al. that Na
intake can be considered as a risk factor for osteoporosis.
4. Correlations between fingernail elements and age
Table V shows the correlation between fingernail ele-
ments and age in different groups. As we can see in Table V,
there is one significant positive correlation between age and
Mg intensity (r ¼ 0.487; p ¼ 0.01) only in 27 nail samples of
healthy group. Ohgitani et al. have found that fingernail Mg
concentration increases significantly with age in both men
(r ¼ 0.209; p ¼ 0.0145) and women (r ¼ 0.280; p < 0.0001).15
Therefore, we can state that Mg intensity of fingernails should
normally increase by increasing age.
In Table V, there is one weak but significant positive cor-
relation between age and Ca intensity (r ¼ 0.347; p ¼ 0.017)
in 47 osteopenic subjects. Recently, Suliburska concluded that
the concentration of minerals in hair of women is associated
with age and women aged 31–40 years had significantly lower
calcium levels in hair than women aged 41–50 years.64 In
another work, Baranowska et al. also observed positive corre-
lations between the content of Ca and age in hair samples.17
These results mean that by increasing age, the calcium levels
in hair increase. As hair and nails are often stated to have
much in common in relation to their origin, anatomical struc-
tures, and common involvement in many diseases,65 similar
result for hair and nail may be expected. But, we do not know
why this correlation exists only in osteopenic group.
As it is shown in Table V, there is a significant negative
correlation between Sr and age in 25 subject of osteoporotic
group (r ¼ "0.580; p ¼ 0.002). Chojnacka et al. investigated
the effect of age on the composition of hair and found that

TABLE VII. Results of classification between healthy and osteoporotic groups.

Predicted group memebership

Category Healthy Osteoporosis Total Correctly classified

Original Count Healthy 23 4 27

Osterporosis 2 23 25
% Healthy 85.2 14.8 100.0
Osterporosis 8.0 92.0 100.0 88.5
Cross-validated Count Healthy 15 12 27
Osterporosis 15 10 25
% Healthy 55.6 44.4 100.0
Osterporosis 60.0 40.0 100.0 48.1
054701-8 Bahreini, Hosseinimakarem, and Tavassoli
TABLE VIII. Results of classification between osteopenic and osteoporotic groups.
Category
Original Count Osteopenia
Osteoporosis
% Osteopenia
Osteoporosis
Cross-validated Count Osteopenia
Osteoporosis
% Osteopenia
Osteoporosis
the composition of hair varies with age and people from the
oldest group (45–65 years) in their analysis had lower level
of Sr in their hair in comparison to lower age groups.66 As it
is mentioned before, similar results for hair and nail are
expected and as we stated before, the mean age in this group
is 59.40 and is the biggest. So, we can conclude that by
increasing age, the Sr content of fingernail decreases and this
correlation in older ages is more obvious.
C. Classification
In the univariate analysis, some significant and insignifi-
cant correlations between BMDs and some elements were
found. In order to obtain a diagnostic technique, a multivari-
ate analysis with all elements of nail is performed by using
DFA statistical methods. Thus, all the independent variables
of 99 fingernail samples are simultaneously analyzed. 46 in-
dependent variables of line intensities completely described
each spectrum are written as a 1 $ 46 vector.
In this analysis, a discrimination between three groups
of 27 healthy, 47 osteopenia, and 25 osteoporotic subjects is
done. Also, a discrimination between each two groups is
done. A plot of three discriminant function scores belonging
to classification of three groups is shown in Fig. 3. In this fig-
ure, first two canonical discriminant functions are used for
classification. The first function accounts for the most dis-
crimination (58.4% of the variance) and the second one pro-
vides the second most (41.6% of the variance). As it is
shown in the graph, function 1 can better discriminate
between three groups while function 2 put considerable dis-
tance between osteoporotic (more risky) group and two
others. In this analysis, 85.9% of original grouped cases are
correctly classified which show that a discrimination
between three groups based on fingernail elements is possi-
ble. However, the validation results show that 38.4% of
cross-validated grouped cases are correctly classified. In
cross validation analysis, one sample is assumed to be
unknown and separated from other samples. Then, a discrim-
inant function is derived from all other samples and the
membership of unknown sample is determined by the func-
tion. The low cross-validation of this classification shows
that we should be careful in using LIBS spectra of fingernail
accompanied with DFA as a diagnostic method for osteopo-
rosis and further studies should be done to evaluate whether
this method is actually useful or not.
J. Appl. Phys. 112, 054701 (2012)
Predicted group membership
Osteopenia Osteoporosis Total Correctly classified
45 2 47
2 23 25
95.7 4.3 100.0
8.0 92.0 100.0 94.4
29 18 47
10 15 25
61.7 38.3 100.0
40.0 60.0 100.0 61.1
In the next step, a classification between each two groups
performed and results of this analysis are shown in Tables VI–
VIII. As it is shown in Table VI, 91.9% of original grouped
and 59.5% of cross-validated grouped cases of healthy and
osteopenic subjects are correctly classified. In Table VII, we
can see that in discrimination between healthy and osteoporotic
subjects, 88.5% of original grouped cases and 48.1% of cross-
validated grouped cases are correctly classified. Discrimination
between two groups of osteopenic and osteoporotic subjects
shows that 94.4% of original grouped cases and 61.1% of
cross-validated grouped cases are correctly classified as shown
in Table VIII. It can be seen that the discrimination between
osteopenic and other two groups are more efficient which may
be due to this fact that changes in elemental composition of
fingernail in osteopenic subjects are more noticeable.
IV. CONCLUSION
To investigate whether it is possible to identify high-risk
groups of osteoporosis by means of nail analysis, the LIBS
spectra of fingernails of 99 subjects including healthy, osteo-
penic, and osteoporotic subjects have been analyzed.
Although fingernails of three different health statuses present
similar spectral lines, the small differences in their intensities
may contain some information about disease. In order to find
evidence of associations between elemental composition of
fingernails and osteoporosis, the Pearson correlations
between spectral intensities of different elements of finger-
nail and age and BMDs have been calculated. Correlations
between line intensities of some elements such as sodium
and potassium, calcium and iron, magnesium and silicon are
significant. Also, significant correlations between some fin-
gernail elements, BMD, and age are observed. Although
some of these correlations are weak but say something about
mineral metabolism in fingernail, but the real physiological
mechanism is so complicated to describe.
DFA, a statistical multivariate method, has been used for
classification of subjects into healthy and patient groups. The
results show that the classification between nail samples of
healthy, osteopenic, and osteoporotic subjects is attainable.
Although more than 85.9% of original grouped cases is cor-
rectly classified, the validation results are not promising. As
many physiological factors can effect elemental composition
of nail, detailed studies in specific subgroups of subjects are
required. Furthermore, the variation of LIBS spectra based on
054701-9 Bahreini, Hosseinimakarem, and Tavassoli J. Appl. Phys. 112, 054701 (2012)

27
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