ARTICLE
Auteur(s) : Aldona
Dunicz-Sokolowska1, Maria Długaszek2,
Krystyna Radomska2, Edyta Wlaźlak3, Grzegorz
Surkont3, Alfreda Graczyk2
1Polish Academy of Sciences, System Research
Institute, ul. Newelska 6, 01-447 Warsaw, Poland
2Institute of Optoelectronics, Military University of
Technology, ul. Kaliskiego 2, 01-489 Warsaw, Poland
3Department of Obstetrics and Gynecology Medical
University of Łódź, The Clinic of Operative Gynecology and
Gynecologic Oncology, Urogynecology Clinic, Madurowicz Hospital
ul.Wileńska 37, 94-029 Łódź, Poland
Diagnosis of the state of health based on chemical analysis of hair
elements appears to be a promising preliminary diagnostic procedure
[1-5] due to its non-invasive, painless and reliable method of
collecting diagnostic material (the research being based on
long-term > 2-3 months tendencies in the tested subject) [1, 4].
The interior of hair tissues ensures the chemical stability of
tested elements and their higher concentrations (i.e. than in
blood) [1, 2]. The isolation of the elements from co-existing
reactions inside the body lowers the error risk in assessing health
status [1-5].Population research on concentrations of bioelements
and toxic metals began in Poland in 1985 at the Institute of
Optoelectronics at the Military University of Technology in Warsaw.
The results (mean arithmetic and ranges) obtained from over 8000
subjects tested in 1986-1990 are shown in [3].The findings obtained
from over 20 000 healthy people from all over Poland tested in
1991-2004 are shown in [6, 7]. The results of this series of
analytical tests were obtained with the help of the computer
program STATISTICA 7.1 StatSoft PL [8, 9]. In Part I [6] the
results of our tests on a group of over 4000 formally healthy
children aged 1 to 10 years are presented. In Part II [7] the
results of tests on a group of 2850 formally healthy children aged
10-20 years are provided. The aim of Part III of our research was
to define referential concentration values of bioelements and toxic
metals in the hair of adults aged 20 to 40, on the basis of
biochemical analyses conducted from 1991 to 2006 on a group of 5733
formally healthy women and men. The period of life (20/25-40/50)
according to Tanner [11] called “life in all its fullness” may be
considered as a time of stabilization, presenting the
characteristics of relative equilibrium of anabolic and catabolic
processes. It is during this period that appear, at different
rates, involution processes which depend on genetic
predispositions, material conditions of life and life style
[11-15]. For women it is a period of maternity (pregnancy, feeding
and childcare) [11].
Materials and methods
The technology of hair collection and methodology of biochemical
analyses have been described previously [3, 6, 7]. This research
presents the statistical analyses of data on bioelements and toxic
metals in a group of formally healthy adults aged 20 to 40 (i.e.
adults with chronic diseases were excluded). Particular attention
was paid to collecting natural hair which was not subjected to any
chemical treatment (especially since dyeing hair is fashionable
among women in this age). Hair used in the analysis should not be
subjected to any chemical treatment which could disturb the
structure of the keratin sheath and thus facilitate penetration of
endogenous improprieties into the interior of the hair causing
false positive results and leading to mistakes in assaying. In
order to eliminate exogenous impurities, a hair washing procedure
has been elaborated. In all cases, 200 mg of hair cut at six
different points of the head, with a hair length of 3 to 4 cm
counting from the scalp, were used for testing. Hair was washed in
water with the addition of detergents free from metal ions and then
rinsed three times with water doubly distilled. After drying it up
it was subjected again to washing in a mixture of anhydrous
methanol and acetone in a Soxhlet apparatus. Subsequently, it was
washed for the second time and wet mineralized using a mixture of
nitric and perchloric acid, at a 1:3 ratio, the excess of acids
being vaporized afterwards; then, it was transferred to 25 mL
glass volumetric flask and made up to the 25 mL mark with
distilled water. The content of bioelements and toxic metals was
assayed by the AAS method (Atomic Absorption
Spectrophotometry): the PERKIN ELMER 2100 Vienna, Austria (in use
from 1992) and the AVANTA SIGMA GBC Scientific Equipment Pty Ltd.
Melbourne, Australia (in use from 2002). Bioelements: Ca, Mg, Cu,
Zn, Fe were essayed in flame, using standardization curves, whereas
heavy metals were essayed with AAS method using a graphite cuvette
HGA-700. The results obtained from chemical analysis were
elaborated with the help of the computer program STATISTICA version
7.1 StatSoft Inc., Tulsa, USA (in Poland StatSoft Sp. Z o. o.
Kraków) [8, 9].
Results and discussion
Statistical analyses were applied to hair bioelement concentrations
(Ca, Mg, Zn, Cu, Fe) and to heavy metals (Pb, Cd) obtained from
testing over 5730 sex-matched adults aged 20 to 40 (3780 women and
1953 men) (see the histogram of frequency in figure 1. The adults
originated from different regions of Poland; they were mainly
inhabitants of urban agglomerations, i.e. Warsaw and its
surroundings, Silesia, Kraków, Wrocław and Gdańsk. From the
frequency in each of the age groups, their random selection, an
almost equal number of subjects chosen from each part of Poland and
the fact that Poles belong to one homogenous race – we may conclude
(according to the central theorem) that the group tested is a good
representation of the healthy Polish population.
The results shown in the form of tables (table 1A and 1B)
and population tendency on diagrams (figure 2A-2G) were
obtained from the analysis of analytical data using descriptive
statistical methods [9, 10].
Our statistical calculations revealed that the error for
estimating mean concentrations of hair bioelements in the adult
population is less than 2%, assuming the level of confidence as
0.95. The above analysis confirms that the adults aged 20 to 40 are
characterized by a considerable dispersion of hair bioelement
concentrations (particularly by those of hair Ca in women),
resulting from a host of endogenous and exogenous factors
[12-15].
The main difficulty of statistical analysis in this case
consists in removing strongly extreme values from the data, as well
as in evaluating maximum concentrations around central measures and
defining tendencies of concentrations of tested elements in the
sex-matched age groups. Extreme outlying values distort tendencies
of the sets of data studied. However, compared to the study of the
group (children and youths) which had been carried out earlier [6,
7], our analysis of the adults aged 20 to 40 turned out to be
particularly difficult, due to large bioelement concentrations,
especially those of women’s hair Ca. For these reasons the most
outlying data were rejected and subjected to other analyses, in
order to establish the causes of such large element concentrations.
In biostatistics values outlaying more than +/-3 Standard
Deviations from the mean value are considered to be extreme values
[9, 10]. Thus, in the case of hair Ca in adults we can consider as
extremely large concentration the values which are higher than 1000
mcg/g d.h.m.
Statistical analyses make it possible to identify systematic
errors in the selection of data resulting in part from some women
subjects hiding the fact that their hair had been dyed or shampoo
colored. Long-term use of bad quality dyes could destroy the
structure of the keratin sheath that protects hair from the
penetration into its interior of endogenous impurities (air,
cosmetics) [1-4].
Since a large dispersion of data occurs which are difficult to
interpret and particularly hard to evaluate, the median was
accepted as being more resistant to outlying values than other
average values.
In the group of all tested adults, 33% of women had hair Ca
concentration higher than 500 mcg/g d.h.m., and 10% of them –
higher than 1000 mcg/g d.h.m. Eight percent of men had hair Ca
concentrations higher than 500 mcg/g d.h.m and 1% of them - higher
than 1000 mcg/g d.h.m. Very low Ca concentrations (less than 100
mcg/g d.h.m.) characterize 3% of women and 4% of men.
Hair Mg concentrations below 20 mcg/g d.h.m. are characteristic
for 9% of men and 7% of women. Concentrations higher than 40 mcg/g
d.h.m characterize 23% of women and 5% of men.
One percent of men have lower than 100 mcg/g d.h.m hair Zn
concentrations and so do 1% of women. Twenty-two percent of women
and 5% of men have hair Zn concentrations higher than 200 mcg/g
d.h.m.
Thirty percent of women and 18% of men have hair Cu
concentrations below 10 mcg/g d.h.m. Hair Cu concentrations higher
than 20 mcg/g d.h.m characterize 9% of women and 4% of men.
Ten percent of women and 5% of men are characterized by hair Fe
concentration of less than 10 mcg/g d.h.m. Fe concentrations
above 20 mcg/g d.h.m. characterize 14% of women and 6% of men
respectively.
Men have higher concentrations of toxic metals (figure 2F-G). Hair Pb
concentrations above 1 mcg/g d.h.m. characterize 18% of men and 15%
of women. Pb concentrations above 3 mcg/g d.h.m. characterize 0.5%
of women and over 5% of men. Nineteen percent of men and 22% of
women have hair Cd concentrations above 0.1 mcg/g d.h.m. Forty
percent of women and 18% of men have hair Cd concentrations below
0.1 mcg/g d.h.m. Over 3% of men have hair Cd concentrations above
0.3 mcg/g d.h.m. Average hair Cd concentrations in men amount to
approximately 0.2 mcg/g d.h.m. Women are characterized by a smaller
dispersion. Average hair Cd concentrations in women amount to
approximately 0.14 mcg/g d.h.m.
Tests on homogeneity of variances, significance of differences
of means, simultaneity of homogeneity of the groups were performed,
first on the sets of raw data, and afterwards once again, after
removing the excessively extreme values from the data (e.g. very
small and very large).
The analyses of variances (ANOVA/MANOVA) confirmed by the
Levene’s variance homogeneity tests confirm a high variation of
hair concentrations of the following sex-dependent elements studied
for: hair Ca < 1000 mcg/g d.h.m. with F = 3.493441 (p =
0.000000) and for hair Mg < 90 mcg/g d.h.m. with F = 4.0111613
(p = 0.000000); for hair Cu with F = 1.62046 (p = 0.007467); for
hair Fe with F = 1.92374 (p = 0.000372). Significant differences (p
= 0.000000) occur between variances women’s and men’s hair toxic
elements: hair Pb with F = 11.32356, and hair Cd with F = 3.40904.
The Levene’s test does not reveal significant differences between
hair Zn concentration variances in the hair of women and men (F =
1.22709, p = 0.152236). It could be proof of a similar distribution
of Zn in women’s and men’s hair.
The Brown-Forsythe test (ANOVA from the median) confirms a high
variation of concentrations of the following sex-dependent elements
with p = 0.000000, for: hair Ca < 1000 mcg/g d.h.m with F =
6.797729, and for hair Mg < 90 mcg/g d.h.m. with F = 3.721672.
Significant variances (p = 0.0001) also exist also between the
amounts of toxic elements in men and women: hair Pb with F =
6.336988 and hair Cd with F = 2.030435. The Brown-Forsythe test
does not reveal significant differences between variances of Zn, Cu
and Fe concentrations in women’s and men’s hair.
The ANOVA test confirms significant (p = 0.000000) differences
of variances of concentrations (for the assumed level of
significance α = 0.05) for hair Ca, Mg, Zn with F > 9.0, and for
hair Pb and Cd with F > 3.0 between sex-matched groups of
adults. The ANOVA test does not reveal significant differences
between variances of concentrations for women’s and men’s hair Cu
(F = 1.22455, p = 0.154623) and for hair Fe (F = 0.92822, p =
0.602020).
The Brown-Forsythe and the Levene tests for homogeneity of
variances confirm the significance of differences concentration
variances of all sex-matched bioelements tested in all age groups
with (p = 0.000000), with the exception of Fe, where the Levene
test indicates p = 0.004493, and the Browne-Forsythe test p =
0.049075 (for α = 0.05). In addition, the Hartley F-max, the
Cochran C and the Barlett Chi-sq sex-matched tests confirm
significance of variance differences on each level for Ca, Mg, Cu,
Pb, Cd (p = 0.000000; for Zn p = 0.000071) and do not confirm
significance for variances between the concentrations of Fe (p =
0.0894246).
Significant differences between element concentrations in the
hair of both sexes were tested by the post hoc tests. The following
tests were performed: tests on significance of differences of
arithmetic means, tests on group homogeneity with α assumption,
tests on simultaneous confidence intervals and tests on critical
ranges. The tests revealed considerable differences on different
levels of α. Minimal size of the groups and the power of tests were
examined.
All tests performed confirm the assumption that hair Ca
concentrations in women are higher than in men of the same age: the
Tukey, Scheffe and the Neuman-Keulus tests with p = 0.000000; the
NIR Fisher, Bonferroni, Duncan tests with p < 0.00001.
Significance (p = 0.000000) of differences between the groups of
women and men of the same age for hair Mg and Zn and larger amounts
of these hair elements in women on each level of significance α are
confirmed by the NIR Fisher, Bonferroni, Tukey, Duncan,
Newman-Keuls and the Scheffe tests.
All the tests on the significance of mean differences and the
tests on simultaneity of confidence intervals confirm (with p <
0.0001) higher Cu concentrations in women’s hair.
In the case of hair Fe, due to overlapping of fields of
variances, the post hoc tests do not confirm the significance of
differences of concentrations between men and women of the same age
(with p-values near 1.000000).
Tests of significance of mean differences and tests on
simultaneity of confidence intervals (with the exception of the
Sheffe test) confirm higher Pb and Cd concentrations in men’s
hair.
Analyses of correlation confirm the significance (α < 0.05)
of synergistic interactions between bioelements: Ca-Mg (r = +0.70),
Ca-Zn (r = +0.39), Ca-Cu (r = +0.14), Mg-Zn (r = +0.31), Mg-Cu (r =
+0.10), Zn-Cu (r = +0.11), Zn-Fe (r = +0.07). Significance (α <
0.05) of antagonistic interactions of bioelements with toxic metals
Ca-Pb (r = -0.13), Zn-Pb (r = -0.15), Mg-Pb (r = -0.15), Zn-Cd (r =
-0.02) was confirmed. Likewise, significance (with α < 0.05) of
synergistic interactions with toxic metals: Pb-Cd (r = +0.35),
Fe-Pb (r = +0.16), Fe-Cd (r = +0.08) was also determined.
Our analyses confirm significant differences in accumulations of
certain elements in women’s and men’s hair, which is in agreement
with other research [16, 17] and indicates a weak tendency of hair
Ca, Mg and Zn concentration levels to decrease with age (in the
tested range of 20 to 40 years) (figure 2A-C). However, due
to high level of unexplainable variances of bioelement
concentrations, this tendency is more noticeable between 20 and 50
years of age. Extremely low and extremely high hair Ca, Mg, Zn, Cu
and Fe concentrations indicate the necessity of further research in
order to diagnose this pathological state [1, 2, 4, 12-14, 18, 19].
Analysis (figure
2E) and the median tests indicate that average levels of
hair Fe concentrations are lower in women than in men. Undoubtedly
it is connected with loss of blood during menstruation, pregnancies
and births [11-13, 18].
Substantially greater concentrations of toxic metals in men’s
body may be explained by a higher percentage of smokers among them,
by jobs connected with harmful substances and by lower levels of
Ca, Mg and Zn which become antagonistic elements when interacting
with toxic metals [1-7, 12-15, 18-25].
Table 1A Concentration of bioelements and toxic metals
in the hair of Polish women aged 20-40.
|
Ca
|
Mg
|
Zn
|
Cu
|
Fe
|
Pb
|
Cd
|
|
Number tested
|
3579
|
3696
|
3762
|
3713
|
3709
|
2223
|
2041
|
|
Minimum-Maximum [mcg/g d.h.m.]
|
80.00 - 3250.00
|
7.00 - 300.00
|
48.00 - 440.00
|
3.20 - 150.00
|
3.00 - 160.00
|
0.01 - 19.70
|
0.01 - 1.90
|
|
Arithmetic mean [mcg/g d.h.m.]
|
507.00 - 632.60
|
39.20 - 43.20
|
178.00 - 196.00
|
11.60 - 12.14
|
15.40 - 16.30
|
0.780 - 1.020
|
0.110 - 0.141
|
|
Standard Error of Mean [mcg/g d.h.m.]
|
20.00 - 25.00
|
1.4 - 1.9
|
3.1 - 3.4
|
0.30 - 0.44
|
0.50 - 0.60
|
0.050 - 0.100
|
0.011 - 0.014
|
|
-95.00% Confidence Interval [mcg/g d.h.m.]
|
516.38 - 636.40
|
37.10 - 40.15
|
175.60 - 192.00
|
10.50 - 11.40
|
13.80 - 15.80
|
0.605 - 0.780
|
0.096 - 0.121
|
|
+95.00% Confidence Interval [mcg/g d.h.m.]
|
633.75 - 784.83
|
42.86 - 50.44
|
185.73 -208.10
|
11.80 -12.90
|
16.50 -17.30
|
0.889 -1.250
|
0.128 -0.216
|
|
Q25 [mcg/g d.h.m.]
|
300.00 - 380.00
|
25.00 - 27.10
|
159.00 - 173.70
|
8.50 - 9.00
|
11.00 - 12.00
|
0.300 - 0.400
|
0.050
|
|
Q50 = Median [mcg/g d.h.m.]
|
434.00 - 552.00
|
33.00 - 37.00
|
178.85 - 193
|
10.00 - 11.00
|
13.00 - 14.00
|
0.50 - 0.70
|
0.100
|
|
Q75 [mcg/g d.h.m.]
|
648.00 - 924.00
|
52.50 - 45.00
|
219.00 - 201.00
|
12.10 - 13.00
|
18.00 - 20.00
|
1.00 - 1.20
|
0.130 - 0.200
|
|
Standard Deviation [mcg/g d.h.m.]
|
300.00 - 330.00
|
19.00 - 25.00
|
38.00 - 41.00
|
5.20 - 5.60
|
6.3 - 7.8
|
0.560 - 0.900
|
0.12 - 0.20
|
Conclusions
1. Analyses of concentrations of hair elements in Polish adults
aged 20-40 years old have shown a high dispersion of results, more
sex than age dependent.
2. The values of the median along with their standard deviations
(either +/- 95% CI, or Q25-Q75) may be considered to be the most
reliable and stable measurements in defining reference values for
population studies (tables 1A, B) ).
3. Significant differences between women and men aged 20 to
40.were found in hair Ca, Mg, Zn (p = 0.0000000) and in hair Cu (p
= 0.0001). Mean hair Ca, Mg and Zn concentrations were confirmed to
be markedly higher in women than in men.
4. Lower hair Fe concentrations in women aged 20 to 40 connected
with women’s reproductive period were confirmed by some tests.
5. Higher (p = 0.0000) concentrations of toxic metals (hair Pb
and hair Cd) increasing with age were confirmed in men.
6. Significance (α < 0.05) of synergistic interactions
between bioelements: Ca-Mg (r = +0.70), Ca-Zn (r = +0.39), Ca-Cu (r
= +0.14), Mg-Zn (r = +0.31), Mg-Cu (r = +0.10), Zn-Cu (r = +0.11),
Zn-Fe (r = +0.07) was established.
7. Significance (α < 0.05) of antagonistic interactions with
toxic metals: Ca-Pb (r = -0.13), Zn-Pb (r = -0.15), Mg-Pb (r =
-0.15), Zn-Cd (r = -0.02) was confirmed.
8. Significance (α < 0.05000) of synergistic interactions
with toxic metals: Pb-Cd (r = +0.35), Fe-Pb (r = +0.16), Fe-Cd (r =
+0.08) was established.
9. Adults in whom hair bioelements and hair toxic metal
concentrations have shown values outlying from reference and who
have too many toxic elements and too few bioelements should undergo
further diagnostic tests since the results could be a sign of
disturbances that could lead to various diseases.
Table 1B Concentration of bioelements and toxic metals
in the hair of Polish men aged 20-40.
|
Ca
|
Mg
|
Zn
|
Cu
|
Fe
|
Pb
|
Cd
|
|
Number tested
|
1922
|
1926
|
1940
|
1915
|
1925
|
1249
|
1164
|
|
Minimum -Maximum [mcg/g d.h.m.]
|
51.60 - 2600.00
|
3.70 - 287.00
|
35.70 - 700.00
|
4.70 - 54.00
|
4.80 - 140.00
|
0.04 - 30.00
|
0.01 - 1.85
|
|
Arithmetic mean [mcg/g d.h.m.]
|
385.00 - 445.00
|
26.60 - 31.80
|
162.10 - 173.80
|
10.80 - 11.60
|
15.10 - 17.20
|
1.252 - 2.100
|
0.155 - 0.220
|
|
Standard Error of Mean [mcg/g d.h.m.]
|
21.00 - 25.00
|
1.4 - 1.9
|
3.1 - 3.4
|
0.30 - 0.60
|
0.60 - 0.90
|
0.160 - 0.350
|
0.001 - 0.004
|
|
-95% Confidence Interval [mcg/g d.h.m.]
|
318.70 - 381.10
|
25.50 - 30.25
|
151.00 - 167.50
|
9.20 - 11.60
|
14.20 - 15.70
|
1.010 - 1.480
|
0.120 - 0.200
|
|
+95% Confidence Interval [mcg/g d.h.m.]
|
426.96 - 497.56
|
32.00 - 40.35
|
170.00 - 205.66
|
11.50 - 13.20
|
16.50 - 19.20
|
1.700 - 2.700
|
0.180 - 0.360
|
|
Q25 [mcg/g d.h.m.]
|
220.00 - 256.00
|
19.00 - 22.30
|
135.00 - 156.00
|
8.00 - 9.00
|
11.70 - 12.00
|
0.520 - 0.750
|
0.05 - 0.10
|
|
Q50 = Median [mcg/g d.h.m.]
|
330.00 - 346.00
|
23.00 - 28.00
|
163.00 - 178.00
|
9.80 - 10.40
|
13.80 - 14.00
|
0.90 - 1.20
|
0.10 - 0.165
|
|
Q75 [mcg/g d.h.m.]
|
415.00 - 540.00
|
34.00 - 39.00
|
182.00 - 190.00
|
11.00 - 13.10
|
17.60 - 22.00
|
1.700 - 2.800
|
0.20 - 0.35
|
|
Standard Deviation [mcg/g d.h.m.]
|
178.00 - 210.00
|
16.00 - 17.00
|
32.00 - 36.00
|
2.5 - 5.6
|
6.80 - 7.20
|
1.7 - 2.1
|
0.14 - 0.30
|
Acknowledgements
We gratefully acknowledge Mrs. Marzena Duk for her constant
technical assistance in collecting research data and Dr. Richard
Sokolowski for his in-depth English proofreading.
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