3.1.

Human Fingernail Spectrum

Samples were randomly collected from people of Tehran. Subjects were categorized into two sex groups and five age groups. Table 1 represents the distribution of nail samples among different age and sex groups. Aspects of gender, age, nutrition, and disease were assessed by questionnaires. All samples were cleaned with alcohol, acetone, and distilled water to remove the external contaminants and then dried before the experiment. We could identify 13 key elemental species and a molecule by measuring the samples. A representative LIBS spectrum of nail is shown in Fig. 2. This spectrum is an average of three spectra. Each of these spectra is formed by accumulation of 10 pulses ablating different locations of the sample. The sample is moved perpendicular to the incident laser beam in the vertical direction so that every laser shot hits a new target position. The emission lines are atomic and ionic lines from organic and inorganic elements, which have been labeled on the graph. It is known that the dominant elements in nail composition are Ca, Mg, P, Na, K, Fe, Zn, and A1 while other elements like Cu, Cr, Se, Ni, Pb, V, Co, Cd, Mn, As, Sb, Sn, and Hg can be found in trace or subtrace levels.¹⁸ In 1971, Harrison ⁸ detected up to 30 elements in human fingernail samples by spark source mass spectrometry including Na, Mg, Al, Si, P, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Ge, As, Se, Rb, Sr, Zr, Mo, Ag, Cd, Sn, Sb, Cs, Ba, La, Ce, Pb, and Bi. However, the elements detected with the present system are: aluminum (Al), carbon (C), calcium (Ca), iron (Fe), hydrogen (H), potassium (K), magnesium (Mg), nitrogen (N), sodium (Na), oxygen (O), silicon (Si), strontium (Sr), and titanium (Ti) as well as CN. Table 2 shows 63 emission lines related to these 14 species. The line signals are obtained by subtracting background emission from the peak area under the spectral lines. Here, an imaginary line between two either sides of each spectral line is considered and the area under this line is subtracted as the background. Also, the corresponding Wilks’ lambda for each emission line is presented in Table 2. The Wilks’ lambda will be discussed in Sec. 3.2 Although the calcium content is only 0.1% in human nail,¹⁹ the LIBS spectrum of nail is dominated by calcium, about 65% of the total spectral power. The total spectral power can be calculated by summing the intensities of all spectral lines in each spectrum.

Fig. 2

Typical LIBS spectrum of the human nail.

Fig. 6

Box plot of concentration of (a) sodium and (b) potassium, and representation of elevated levels in cases of hyperthyroidism.

Table 1

Distribution of nail samples among different age groups and genders.

Table 2

Identification of 63 atomic and ionic emission lines in human fingernail spectrum along with the Wilks’ lambda for sex and age group discriminations.

Here, we discuss concentration levels of elements in two sex groups. In order to do so, all emission lines of the same species are added up and then normalized to the total spectral power. This value shows the fractional spectral power of each element. Table 3 represents the average of the fractional spectral power of each species among 45 nail samples. The comparison between concentrations of elements of two sex groups is shown in Fig. 3. The x axis represents different sex groups and the z axis represents the average fractional spectral power of each element in every group. The average concentrations of Al, CN, Fe, H, K, Mg, N, Na, O, and Si are more elevated in men while concentrations of Ca and Ti are higher in the female group. The elevated concentration of Na and K in males in comparison with females was also reported by Vance ²⁰ They analyzed 117 subjects by instrumental neutron activation analysis. However, they reported that Ca concentration had no effect on either age or sex, which is in contradiction with our analysis. Concentrations of C and Sr are equal within the experimental error in both groups. Dittmar ²¹ reported that carbon content in male and female subjects was not different, which is in accordance with our analysis. In 1988, Takagi detected 18 elements in human nails by argon inductively coupled plasma emission spectroscopy.¹⁸ They reported that Ca, Mg, Na, K, and Fe generally exist at higher levels in males compared to females, which is in accordance with our results except for the Ca concentration. The average concentrations of each species in different age groups are displayed in Fig. 4. As the age increases, the overall carbon level in the fingernails increases, although there are slight fluctuations. This was also observed by Dittmar ²¹ They performed analysis on 225 individuals (93 males and 132 females). Takagi reported that Al concentration decreased with age, which is not exactly in accordance with our results.¹⁸ However, they performed testing on 512 samples (278 men and 134 women) from different countries of Japan, India, U.S.A, Canada, and Poland.

Fig. 3

Fractional spectral power of elements in nails of men and women. Y-axis has a logarithmic scale.

Fig. 4

Fractional spectral power of elements in nails in different age groups. Y-axis has a logarithmic scale.

Table 3

Fractional spectral powers for each species in human fingernails (average from 45 samples).

It should be mentioned that a number of samples in the present work and their distribution is not enough for making a statistically meaningful analysis. Also, the number of samples in the present work differs from the number of samples in other authors’ research^{18, 20, 21} The limited number of samples tested in the present work (45 samples) and also the number of samples in the original works, ranging from 117 to 512, do not allow one to draw definite conclusions on systematic difference in elements’ concentrations between different groups.

3.2.

Discriminant Function Analysis

Discriminant function analysis (DFA) is a data reduction analysis technique that is used to determine which variables discriminate between different groups. DFA uses a set of independent variables to separate cases based on groups one defines; the grouping variables are the dependent variables that are categorized as the variables of sex and age group in our analysis. Given a set of independent variables, DFA attempts to find linear combinations of those variables that best separate the groups of cases. These combinations are called discriminant functions. A canonical correlation analysis produces the discriminant functions and their canonical roots that are the eigenvectors and eigenvalues of the data. N-1 discriminant functions are constructed for discrimination among N groups. Canonical discriminant analysis constructs discriminant functions that separate the categories as much as possible. The first function separates the groups the most. The second function provides the second most separation. The procedure continues adding functions in this way until the maximum number of functions is reached. All of these constructed functions are uncorrelated.²² In this work, subjects are categorized in two sex groups and five age groups.

The null hypothesis states that the groups or categories under consideration are statistically the same or exhibit the same behavior without any significant difference. To reject the null hypothesis, a significance test is carried out by checking the differences in the mean values of the groups. The groups are significantly different if the difference of their mean values is greater than the variance of all samples constituting one normal group. Therefore, if the test is significant, the groups are discriminated according to the corresponding canonical discriminant function scores. By constructing discriminant functions, DFA can find small similarities in the variances of dependant variables.²² By giving each dependant variable a score, it can classify them. When the scores are plotted, we can observe the validity of our predictions.

Here, two approaches are used. In the first approach, the sum of all of the emission lines belonging to a species is used for discrimination.²³ In the second, all emission lines are individually inserted into DFA software (SPSS v16). The results of the first approach do not show a significant discrimination between groups, i.e., 73.3% for sex discrimination and 80% for age group discrimination. Therefore, the second approach is utilized in which 63 individual emission lines of 13 elements and one molecule are input into the software. Figure 5 shows a graph of the discriminant function scores for each subject. In Fig. 5, the first function accounts for the most discrimination (79.1% of the variance) and the second one provides the second most (20.9% of the variance). For the age groups, four discriminant functions are constructed, which account for 59.2, 21.6, 13.7, and 5.5% of the variance, respectively. The graphical output of the DFA analysis depicts only the scores from the first two functions.

Fig. 5

DFA plot showing the first two function scores that resulted from LIBS spectra obtained from nail samples. (a) Two groups of men and women. (b) Five age groups.

3.3.

Correlation Between Concentrations of Elements

Pearson correlations between concentrations of elements are shown in detail in Table 4. Only the significant correlations are displayed in Table 4. The Pearson correlation is obtained by dividing the covariance of the two variables by the product of their standard deviation. Concentrations of sodium and potassium are positively correlated in 45 nail samples at the level of 0.88. Nowak also observed a significant positive correlation between sodium and potassium in fingernails.²⁴ Calcium and magnesium concentrations are negatively correlated at the level of 0.49. This may be due to the fact that Ca and Mg compete for absorption on the gut surface.²⁵ Authors in Ref. 26 refer to this fact that magnesium inhibits the calcification. This fact may explain the negative correlation between Ca and Mg. Magnesium correlates with silicon at the level of 0.65. Berlyne showed that the urinary silicon was highly correlated with the urine magnesium concentration.²⁷ Silicon is bound with magnesium cations when excreted from the body mainly in the form of urine.²⁸ Nails can be considered as another depletion mechanism of elements in the body.²⁹ So, Si and Mg are simultaneously excreted by the nails. There is no significant correlation between pairs of elements like sodium-iron and sodium-silicon. In addition to that, aluminum, magnesium, and titanium do not significantly correlate.

Table 4

Pearson correlation between concentrations of elements in 45 nail samples.

3.4.

Hyperthyroidism

In this section a case report of hyperthyroidism based on the LIBS spectra of fingernails is presented. According to the questionnaires, case numbers 2, 3, 5, and 16 suffer from hyperthyroidism and case number 43 suffers from high blood pressure. Hyperthyroidism, an illness of the thyroid, is associated with an overproduction of the thyroid hormone. The symptoms of hyperthyroidism are palpitations, heat intolerance, nervousness, insomnia, breathlessness, increased bowel movements, light or absent menstrual periods, fatigue and fast heart rate, which are caused by the effects of too much thyroid hormone on tissues of the body.³⁰ By looking at the box plot depicting sodium (Na) concentration in Fig. 6a, it is shown that the Na concentrations of case numbers 3, 5, and 16, who are siblings, are outliers. An outlier is an observation that is numerically far from the rest of the data.³¹ If the groups of males and females are separately considered [Fig. 6a], case number 16 who is a hyperthyroidism patient is located in the normal distribution, while the sodium concentration of case number 43 in the female group is an outlier. By observing the box plot for potassium level in Fig. 6b, we can see that case numbers 2, 3, 5, 15, 16, and 43 have outlying levels of potassium in their fingernails. As it is mentioned, the cases 2, 3, 5, and 16 suffer from hyperthyroidism while case number 43 is a 47 year old woman who has high blood pressure. However, case number 15 does not suffer from any disease. If the sex groups are separately observed [Fig. 6b], all male subjects suffering from hyperthyroidism (2, 5, and 16) are diagnosed as healthy, which is a false negative diagnosis. Generally, it is observed that there is an agreement between the elevated levels of potassium and sodium in the fingernails and blood test results indicating hyperthyroidism and high blood pressure. Potassium deficiency is one of the symptoms of hyperthyroidism.³² On the other hand, the high level of an element in hair and nail may indicate depletion of that element in the body.²⁹ Thus, the elevated level of potassium in nails of hyperthyroidism patients can be due to the potassium depletion. In general, it should be mentioned that only individual cases were investigated for hyperthyroidism, and a more statistical approach is demanded to ascertain whether our results are of a more general nature.