Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl Accident Liquidators

Abstract

The present article is an attempt to reveal the connection (Pearson) between potassium (K) and sodium (Na), K and zinc (Zn) levels on the basis of analytical determination of elemental content in human scalp hair (atomic emission spectrometry in 954 Chernobyl accident liquidators and 947 healthy persons). The negative K-Zn correlation and also the increase in epidermic cells K and Na and reduction of calcium (Ca) and Zn can indirectly point, in the authors opinion, to the participation of membrane АТРаs (P-type) in the origin of metal-ligand homeostasis shifts and serve as oxidative and nitrosative stress discriminators.

Full text

Volume 1 • Issue 2 • 1000102
Vitam Trace Elem
ISSN: 2167-0390 VTE, an open access journal
Vitamins & Trace Elements
Petukhov et al., Vitam Trace Elem 2011, 1:2
http://dx.doi.org/10.4172/2167-0390.1000102
Research Article Open Access
Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl Accident
Liquidators
Petukhov VI1*, Dmitriev EV3, Kalvinsh I1, Baumane LKh1, Reste ED2, Zvagule T2, Skesters AP2 and Skalny AV4
1Latvian Institute of Organic Synthesis, Riga, Latvia
2Riga`s Stradin`s University, Riga, Latvia
3Institute of Numerical Mathematics, Russian Academy of Sciences, Moscow, Russia
4ANO “Centre of Biotic Medicine”, Moscow, Russia
Abstract
The present article is an attempt to reveal the connection (Pearson) between potassium (K) and sodium (Na), K
and zinc (Zn) levels on the basis of analytical determination of elemental content in human scalp hair (atomic emission
spectrometry in 954 Chernobyl accident liquidators and 947 healthy persons). The negative K-Zn correlation and also
the increase in epidermic cells K and Na and reduction of calcium (Ca) and Zn can indirectly point, in the authors
opinion, to the participation of membrane АТРаs (P-type) in the origin of metal-ligand homeostasis shifts and serve as
oxidative and nitrosative stress discriminators.
*Corresponding author: Valery Petukhov, LV-1045, Riga, Latvia Eksporta st., 14-
14, Russia, Tel: 8-10 (371)6 7381393; E-mail: vip-val@yandex.ru
Received December 05, 2011; Accepted January 30, 2012; Published February
06, 2012
Citation: Petukhov VI, Dmitriev EV, Kalvinsh I, Baumane LKh, Reste ED, et al.
(2011) Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl Accident
Liquidators. Vitam Trace Elem 1:102. doi:10.4172/2167-0390.1000102
Copyright: © 2011 Petukhov VI, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Introduction
Analysis of dynamic characteristics of metal-ligand homeostasis
(MLH) in dierent biosubstrates is an intriguing, virtually not studied
question of modern elementology. Such analysis, of course, cannot
be conned (though this information itself is undoubtedly valuable)
to only quantitative estimation of the content of one or another metal
in a biosubstrate. Data on the metal-ligand complexes themselves,
rst on metalloproteins, which are the main participants of intra- and
extracellular MLH events, are not less actual.
It is known that existence of metals in the organism in “free” form
(as ions) is virtually “forbidden” even for such essential metals as copper
(Cu), zinc (Zn), magnesium (Mg) etc., to say nothing of cadmium (Cd)
and mercury (Hg), which are yet considered in literature as “toxic” [1].
As it was shown in recent experiments on a model of yeast cells, copper
concentration in their plasma was less than 10-18 M, i.e. less than one
copper atom per cell [2].
At the same time, there are apparently no dierence in principle
between toxic eect of Zn2+ or Cu2+ on a cell and that of Cd2+ or
Hg2+. Most probably, the matter is some faults in binding metals by
specialized protein molecules (metallothioneins, metallochaperones)
and/or ineective removal of these ions from cells by assistance of
membrane ATPases.
e major role in binding (detoxication) of metals in the
organism belongs to metallothioneins (MT), low-molecular proteins
(6-7 kDa), where 20 of 60-68 amino-acid residues are cysteine. Since
1957, when a Cd, Zn-containing MT was found rst [3], new data on
MT participation in MLH appeared.
It was found that MT besides detoxication of heavy metals and/
or metals with variable valence (Fe, Cu) protect cell structures from
damaging eect of oxygen/nitrogen radicals [4,5], inuence activity of
nuclear transcription factor NF-kB, participate in regeneration of liver
and nervous cells [6,7], cause corrigent inuence on humoral and cell
immunity [8,9].
In cells, MT synthesis is induced (besides metals themselves and
oxygen/nitrogen radicals) by glucocorticoids, anti-inammatory
cytokines (TNFα, IL-1), α-interferon [10-13].
ere are four MT classes. Two of them, MT-1 and MT-2, are
expressed in almost all mammalian tissues. ey play a key role
in homeostasis of Zn2+, Cu2+, Cd2+ and Hg2+. e other two, MT-3
and MT-4, are tissue-specic for neural (CNS) and epithelial tissue,
respectively [14]. Zn2+ ions incorporate MT molecule relatively easy
and are claimed to be equally easy displaced by excess Cd2+ under
certain conditions.
e metal/thiols ratio in MT is not random. ere is about 1 Cu
atom per 2 cysteine residues in Cu-containing MT (Cu-MT), and 1
atom of Cd or Zn per 3 cysteine residues in Cd/Zn-containing MT
(Cd/Zn-MT). In Cu-MT, each copper atom is trigonally surrounded
by sulphur (S) atoms, while the corresponding complex in Cd/Zn-MT
has tetrahedral structure.
Structural regularity of the metalloprotein (MP) molecules allows
existence of linear bounds between metals included in MP. ese bonds
can serve like “markers” of MP, and can be detected by correlation
analysis (Pearson) of metal concentrations in a certain biosubstrate.
We guess that such approach could be ecient for studying dynamic
changes of MLH in normal and various pathological states. Using
this method, one could trace MLH changes in such a biosubstrate as
epidermis under conditions of oxidative and nitrosative stress, e.g.
in Chernobyl disaster liquidators. Moreover, the subject for direct
determination of metal concentrations could be not the epidermal
cells themselves, but their derivatives (hair). ough, in this case, we
should assume that metal concentrations in cells correspond to those
in hair. is assumption looks reasonable and can be veried in further
observations. Advantages of hair use for non-invasive and retrospective
observation on shis in MLH of epidermal cells are obvious and would
be especially appropriate for mass (population) investigations.
e deepness of retrospection in such observations can be easily
estimated on the basis of average speed of human hair growth ca. 0.2
mm/day: a sample of hair ca. 3 cm long reects events, happened in
epidermis during ve recent months.
At the same time, the choice of biosubstrate for elemental analysis
(hair) requires carefulness when interpreting the investigation results.

Citation: Petukhov VI, Dmitriev EV, Kalvinsh I, Baumane LKh, Reste ED, et al. (2011) Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl
Accident Liquidators. Vitam Trace Elem 1:102. doi:10.4172/2167-0390.1000102
Page 2 of 8
Volume 1 • Issue 2 • 1000102
Vitam Trace Elem
ISSN: 2167-0390 VTE, an open access journal
It is known that epithelium (with its derivatives), which takes
part in assimilation of chemical elements and their removal from the
organism, is an area, where regulatory mechanisms of MLH cannot
help manifesting themselves. erefore an increase in relative amount
of metals (or other chemical elements) in hair, if not being due to
external causes, can be explained by:
1) normal working of MLH regulatory system (e.g. accelerated
excretion of a metal in response of its excess intake from food);
2) a fault of regulatory mechanisms promoting metal retention
in the organism, that leads to development of metal-decient
states.
In its turn, at insucient intake of one or another metal from
outside (or from chelate complexes inside the organism), proper
work of the homeostasis regulatory system is directed to retention of
the metal inside (minimization of losses), that can be manifested in a
decrease of relative amount of the metal in such biosubstrate as hair.
Among oscillations in concentration of chemical elements (in a
rather wide range) registered in spectrometric analysis of hair, how
to distinguish those inherent in normal homeostatic regulation from
pathological shis, signs of elemental imbalance? Where are distinctive
criterions of these shis?
ese questions are yet to be answered. However already today
one should admit that it is hardly reasonable to extrapolate data of
hair analysis upon whole organism, i.e. to diagnose “total” elemental
imbalance on the basis of just quantitative determination of hair
mineral content (“more - less”).
Such extrapolation is probably applicable only in case of distinct
(general) deciency or excess of certain elements, when rough
disturbances of mineral content can be found in most tissues.
at is why a signicant advance (in theoretical and especially
practical sense) would be the very possibility to use results of hair
spectrometry for retrospective estimation of MLH events in epidermal
cells. In other words, in case of quite predictable identity of mineral
content of epidermis and its derivatives (hair), the latter could be a
reliable and aordable source of information about mineral status of
epidermal cells.
In this work we made an attempt to reveal pair associations between
concentration of sodium (Na) and potassium (K), as well as between
K and Zn as an indirect conrmation of participation of membrane
ATPases (P-type) in MLH changes, observed on the background of
oxidative and nitrosative stress.
For this purpose we used data on atomic-emission spectrometry of
hair from Chernobyl disaster liquidators and healthy persons.
Materials and Methods
Hair mineral content of 947 healthy persons, 2-86 years old (238
men, 709 women) and 954 Chernobyl accident liquidators, 37-82 years
old, living in Moscow, Russia (741 men, 213 women), was analysed by
atomic emission spectrometry with inductively coupled plasma (ICP-
AES) on an Optima 2000 DV (Perkin Elmer Inc., USA) instrument in
ANO “Centre for Biotic Medicine” (Moscow, Russia).
In addition, in order to reveal possible gender and age dependent
dierences, hair mineral content of 402 healthy residents of Riga,
Latvia, 2-86 years old (322 women, 80 men) was also investigated by
ICP-AES. All the persons were divided in 3 groups by age: Group 1
– 2-32 years old (n=154), Group 2 – 33-44 years old (n=127), Group
3 – 45-86 years old (n=121). Comparative analysis of the spectrometry
data was made for dierent genders and for two opposite age groups:
Group 1 and Group 3.
e hypothesis of normal data distribution was tested using
Jarque-Bera [15] and Kolmogorov-Smirnov [16] tests. In statistical
calculations of the spectrometry data we did not use ordinary
statistical methods (t-test), because the normality testing disproved
the hypothesis of normal data distribution with high probability for
all chemical elements except zinc (Zn) [17]. erefore, alternative
approaches (bootstrap method) were applied, which do not require
normal distribution of a priori data [18]. Correlation analysis was
made using standard computer application packages Microso Excel
and Matlab.
Results
Comparative analysis of gender and age dependent dierences in
mineral hair content of healthy persons has given the results presented
in (Tables 1,2).
Interval estimation of average hair content of such elements as
calcium (Ca), magnesium (Mg), manganese (Mn), phosphorus (P),
tin (Sn) was considerably dierent in healthy men comparing to
women. Mean values (M, µg/g) and limits of the condential intervals
(bootstrapping) for men are: Ca=749.1 [521.1-1125.4]; Mg=73.4 [59.9-
89.6]; Mn=0.73[0.55-0.97]; P=154.3 [146.3-161.8]; Sn=0.18[0.14-
0.21]; V=0.12[0.09-0.15]; for women: Ca=1537.5[1410.9-1666.4];
Mg=188.9 [169.6-209.1]; Mn=1.61 [1.33-1.96]; P=139.2 [134.7-143.9];
Sn=0.85[0.52-1.34]; V=0.07 [0.06-0.08]. Age dependent dierences
between two marginal age groups (Group 1 and Group 3) were found
only for Mg: in Group 1 Mg=141.9 [120.9-166.1] µg/g; in Group 3 Mg
=203.4 [168.9-243.4] µg/g.
e results indicate necessity of being careful, as mentioned above,
when generalizing estimation of mineral status by hair spectrometry on
the whole organism. For instance, the fact of higher Ca content in hair
of women comparing to men do not testify good state of Ca homeostasis
in women’s bone tissue, where 99% of this mineral is situated.
Comparative analysis of mean concentrations of 23 chemical
elements in hair of healthy persons and Chernobyl disaster liquidators
was made using interval estimation of the mean [18]. e results are
presented in (Table 3).
Sex Ca (μg/g)
[M]
Mg (μg/g)
[M]
Mn (μg/g)
[M]
P (μg/g)
[M]
Sn (μg/g)
[M]
V (μg/g)
[M]
Men
(n=80) 521,1<749,1<1125,4 59,9<73,4<89,6 0,55<0,73<0,97 146,3<154,3<161,8 0,14<0,18<0,21 0,09<0,12<0,15
Women
(n=322) 1410,9<1537,5<1666,4 169,6<188,9<209,1 1,33<1,61<1,96 134,7<139,2<143,9 0,52<0,85<1,34 0,06<0,07<0,08
Note: hereinafter in the tables the bold font designates values of average (M), usual - borders of condential intervals (bootstrap-method).
Table 1: Sex dependent differences in hair mineral content of healthy persons.

Citation: Petukhov VI, Dmitriev EV, Kalvinsh I, Baumane LKh, Reste ED, et al. (2011) Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl
Accident Liquidators. Vitam Trace Elem 1:102. doi:10.4172/2167-0390.1000102
Page 3 of 8
Volume 1 • Issue 2 • 1000102
Vitam Trace Elem
ISSN: 2167-0390 VTE, an open access journal
As follows from (Table 3), content of virtually all investigated
elements in hair of Chernobyl disaster liquidators signicantly diers
from control. It is notable that content of essential metals (Cu, Zn, Mg
etc.) in hair of liquidators is lower than in control group, but content
of so-called “toxic” metals (Cd, Pb, Sn) is higher. What is behind these
shis? Could one consider them as distinct faults of metal-ligand
homeostasis, or they are just a peculiar compensatory response of the
organism under conditions of oxidative/nitrosative stress, aimed at
saving vital elements inside and removing less important or potentially
toxic ones?
To try answering these questions, we have postulated the following
issues, based on published data and our own observations.
1. e main distinctive feature of biochemical processes in
Chernobyl liquidators is high activity of oxygen/nitrogen
radicals, or chronic oxidative/nitrosative stress, which directly
concerns events of metal-ligand homeostasis observed inside
and/or outside cells.
2. Key role in metal-ligand homeostasis belongs to metal-proteins
(MT, chaperons, metal-enzymes).
3. If assume that ratio of metals (and, consequently, their protein
ligands too) in hair is equal to that in epidermal cells (and
this assumption can be conrmed by further investigations),
therefore a real possibility appears to judge about events of
metal-ligand homeostasis in epidermis on the basis of changes
in mineral content of hair.
4. Linear correlations in pairs “metal-metal” or “metal-protein”
can serve as additional characteristics of their intracellular
interactions and/or as an indirect evidence of structural bonds
between metals (e.g. in MT molecule).
Some technical peculiarities of detection of the linear associations
between metals in hair spectrometry results should be explained [19].
It was established that a reduction of the subgroup size (to n = 100)
caused a multiple increase in the number of detected linear associations
as compared with the whole group (n = 1000). It points to the fact that
most of these correlations are casual and dependent on the subgroup
size. is circumstance determines the following rules of the correlation
analysis procedure.
To reveal the maximum number of signicant correlations, entire
assemblies of spectrometry data (in our observations n=947 and n=954)
was subjected to repeated “shue” (“shaking”). e “shue” was made
in accordance with specially designed formats (standards). For this
purpose, in the entire assembly we found the ratio of concentrations
in each possible pair of chemical elements for each observation. It
was essential that the sum of numerators was more than the sum of
denominators in each set of ratios for a given pair.
e set of such pairs, as determined by number of possible paired
combinations of m (where m is the number of analysed minerals), is a
set of formats for the ‘shue’ of the entire assembly. If m = 23 (as in this
case), the number of formats is 253.
In the present study we used the following 20 formats only: Mg/Pb,
Al/As, Cu/As, As/Cd, K/As, Zn/Li, Ca/Al, K/Mg, Fe/Cu, Fe/Cd, Si/Cr,
Cr/Co, Si/Ni, Si/Se, Na/Mn, P/Ni, Si/Co, P/Si, Na/Se, Ni/Cr.
In each obtained format, the ‘ranking’ (or ‘formatting’) of
individual data was performed according to value of elements ratio in
the given pair in descending order (from high to low). e optimum
size of sample for studying correlations is approx. 100 persons. In the
same order (from high to low) groups for the correlation analysis were
designed. For example, if the entire assembly is ca.1000 persons, than
Group 1 includes #1-#100, Group 2 - #101-#200, Group 3 - #201-#300,
etc.
e correlations between chemical elements, on which the shue
was formatted (and only within the given particular format), were not
allowed for, because, by experience, this method of formatting leads to
articial overstating of Pearson correlation coecient (r).
Aer calculating r in all groups and both entire assemblies (healthy
persons and disaster liquidators), a new combined assembly was
created involving only groups with r>0.2. Within this new assembly,
formed for a given pair, the spectrometry results were selected further
depending on r values.
For this purpose, for each case a so-called ‘occurrence coecient’
(OC) was found, which was equal to the number of repetitions of a
given observation in the entire assembly. At no repetitions OC=0, one
repetition corresponds to OC=1, two repetitions - OC=2 etc. For the
investigated relations, the near-to-zero r values were found among
those cases, where repetitions were absent (OC=0) or rarest. At the
same time, the maximum number of repetitions was accompanied by
maximum r values. Such an approach allowed relatively easy separation
of persons with absence or presence of a sought-for linear association
Age groups Mg [M] мкг/г
Group 1 (n=154)
men – 32 (20,8%);
women – 122 (79,2%)
120,9<141,9<166,1
Group 3 (n=121)
men – 26 (21,5%);
women – 95 (78,5%)
168,9<203,4<243,4
Table 2: Age dependent differences in hair mineral content of healthy persons.
Healthy persons (n=947)
M (μg/g)
Chernobyl disaster liquidators (n=954)
M (μg/g)
Al=8.1<8.77<9.5
As=0.07<0.09<0.13
Be=0.007<0.008<0.01
Ca=1176.8<1249<1318.9
Cd=0.04<0.05<0.06
Co=0.04<0.05<0.06
Cr=0.48<0.51<0.54
Cu=19.06<20.7<22.3
Fe=19.3<21.07<23.1
K=277.4<317.7<361.1
Li=0.03<0.04<0.05
Mg=125.5<134.3<143.2
Mn=1.02<1.17<1.3
Na=427.9<480.9<542.9
Ni=0.53<0.62<0.73
P=144.3<146.8<149.5
Pb=1.04<1.1<1.27
Se=0.62<0.91<1.3
Si=44.7<48.5<52.7
Sn=0.39<0.51<0.7
V=0.06<0.072<0.077
Ti=0.84<1.17<1.66
Zn=181.5<185.2<189.3
*Al=19.3<20.1<20.9 ↑
*As=0.38<0.40<0.43 ↑
Be=0.008<0.01<0.02
*Ca=623.4<654.8<685.2 ↓
*Cd=0.23<0.25<0.29 ↑
*Co=0.14<0.15<0.16 ↑
*Cr=0.85<0.9<0.92 ↑
*Cu=10.6<10.99<11.4 ↓
Fe=22.4<23.7<25.07 ↑
*K=365.8<394.8<422.4 ↑
*Li=0.053<0.06<0.062 ↑
*Mg=43.9<46.8<49.9 ↓
*Mn=0.74<0.8<0.86 ↓
*Na=757.5<822.3<892.4 ↑
*Ni=0.41<0.46<0.51 ↓
*P=150.8<153<155.4 ↑
*Pb=1.5<1.8<2.2 ↑
*Se=1.46<1.55<1.65 ↑
*Si=18.4<19.9<21.6 ↓
*Sn=0.91<0.96<1.0 ↑
*V=0.10<0.11<0.12 ↑
*Ti=0.49<0.54<0.59 ↓
*Zn=162.5<165.8<169.0 ↓
Note: *– signicant difference
Table 3: Interval estimation of mean content of chemical elements in hair of
Chernobyl disaster liquidators and healthy persons.

Citation: Petukhov VI, Dmitriev EV, Kalvinsh I, Baumane LKh, Reste ED, et al. (2011) Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl
Accident Liquidators. Vitam Trace Elem 1:102. doi:10.4172/2167-0390.1000102
Page 4 of 8
Volume 1 • Issue 2 • 1000102
Vitam Trace Elem
ISSN: 2167-0390 VTE, an open access journal
e obtained results have not just conrmed the existence of the
K-Zn correlation but also revealed the dierences in its manifestation
in the compared groups. us, the negative linear correlation between
K and Zn (r = -0.43; p<0.05) was only manifest in 18.1% of practically
healthy persons; in 36.3% it was negligible (r = -0.23; p<0.05), and in
45.6% it was altogether absent (r = 0.05).
Whereas the K-Zn correlation was negative and signicant (r -
from -0.41 to -0.62; p<0.05) in the absolute majority of the Chernobyl
disaster liquidators (88%), and in 12% of the liquidators it was not
revealed at all (r = - 0.03). Besides, the K-Zn correlation at the highest
|r| (in both the liquidators and the healthy subjects) was accompanied
by a signicantly higher level of K and Na and lower level of Zn than in
subjects showing no K-Zn correlation (See Table 4).
It is indicative that the |r| size at K-Zn correlation varies with the
increase of OC (Table 5,6), just like the average concentration values of
not only K, Na and Zn but some other metals as well.
e negative correlation between K and Zn means that a decrease of
intracellular zinc concentration must result in a proportional increase
of intracellular potassium concentration. is fact should be discussed
at greater length. Here, however, we can only conne ourselves to
assuming that the most probable cause of such relations between K and
Zn is an increase in production of nitric oxide (NO), which selectively
releases Zn from MT molecules. Besides, NO (or its derivatives and
between concentrations of chemical elements among the whole totality
of cases.
Alongside with the measurements of pair correlations between
metals, we were interested in the associations between the concentrations
of these metals in hair and the r values. Of special interest were possible
deviations of the results obtained in the Chernobyl disaster liquidators
from the norm. Such deviations could claim to be the distinctive
markers of the chronic oxidative/nitrosative stress provided that the
destructive action of oxygen/nitrogen reactive species extends to
metal-ligand homeostasis of epidermal cells.
When performing the correlation analysis, we were rst interested
in associations between concentrations of Zn, K and Na. It should
be noted that Zn is a part of MT molecule while a linear correlation
between K and Na concentrations in hair, according to our preliminary
data [19], is very constant, characterized by relatively high r values (0.6-
0.7), and does not depend on the sample size. At the same time, the
K-Na association depends on the biosubstrate type: it is present in hair
and absent in plasma. It suggests that this correlation reects, directly
or indirectly, the ne-tuned operation of membrane Na,K-ATPases,
which are constantly present in the cell and ensure the transmembrane
transport of metals.
It was interesting to nd out whether there were any correlations
between K and Zn and what kind of correlations could be observed in
the Chernobyl disaster liquidators and healthy persons.
Groups
Correlation (r)
and incidence of K-Zn
association
at the given r
Potassium (K)
μg/g
[M]
Sodium (Na)
μg/g
[M]
Zinc (Zn)
μg/g
[M]
Healthy
subjects
r = - 0,05 [45,6%]
(n=432) 92,2<125,3<165,8 183,8 <209 <243,2 194,3<200,1<205,7
r = - 0,43 [18,1%]
(p<0,05) (n=171) 729,0<894<1084,5 996,2<1233,9<1474,1 143,5<150,7<158,1
Chernobyl
disaster
liquidators
r = - 0,03 [12,0%]
(n=115) 102,5<150,5<208,3 199,4 <261,8< 328,0 172,3<183,5<194,5
r = - 0,62 [21,5%]
(p<0,05) (n=205) 502,9<578,1<660,5 982,7<1131,3 <1286,4 153,5<159,3<165,4
Note: The Table gives the extreme (max and min) r values only
Table 4: K-Zn correlation and K, Zn content in hair of Chernobyl disaster liquidators and healthy persons.
Number OC n r
Cu
μg/g
[M]
K
μg/g
[M]
Na
μg/g
[M]
Zn
μg/g
[M]
Ca
м
μg/g
[M]
Ca
f
μg/g
[M]
Mg
м
μg/g
[M]
Mg
f
μg/g
[M]
1-253 4-10 253 -0,01 21,0 127,0 197,8 204,3 915,3 1737,9 93,9 171,0
254-432 11 179 -0,17 23,1 122,9 224,8 194,2 938,5 1463,8 92,3 151,6
433-619 12 187 -0,20 21,5 210,8 356,8 189,4 719,5 1287,0 76,3 141,1
620-776 13 157 -0,24 21,5 346,8 557,0 176,8 900,2 1234,8 107,0 145,1
777-872 14 96 -0,31 16,8 522,9 943,2 164,0 1040,1 970,8 116,0 109,9
873-947 15-17 75 -0,44 15,0 1368,9 1605,9 133,6 637,0 915,2 72,9 138,6
Note: Caм and Mgм - Ca and Mg concentrations in men’s hair; Caf and Mgf - in women’s hair
Table 5: K-Zn correlation and concentration of metals in epidermic cells of healthy subjects depending on occurrence coefcient (OC).
Number OC n r
Cu
μg/g
[M]
K
μg/g
[M]
Na
μg/g
[M]
Zn
μg/g
[M]
Ca
м
μg/g
[M]
Ca
f
μg/g
[M]
Mg
м
μg/g
[M]
Mg
f
μg/g
[M]
1-115 6-11 115 -0,03 10,9 150,5 261,8 183,5 706,5 1418,8 47,1 119,6
116-229 12 154 -0,31 10,8 224,9 477,2 177,4 592,9 1081,7 38,2 89,1
270-503 13 234 -0,43 11,2 394,9 833,2 158,6 595,5 910,5 40,3 64,6
504-749 14 246 -0,43 10,9 464,8 1036,3 162,4 531,2 710,8 35,5 55,9
750-954 15-17 205 -0,62 11,0 578,1 1131,3 159,3 537,7 611,3 40,5 44,6
Note: Ca
м
and Mg
м
- Ca and Mg concentrations in men’s hair; Ca
f
and Mg
f
- in women’s hair
Table 6: K-Zn correlation and concentration of metals in the epidermic cells of Chernobyl disaster liquidators depending on occurrence coefcient (OC).

Citation: Petukhov VI, Dmitriev EV, Kalvinsh I, Baumane LKh, Reste ED, et al. (2011) Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl
Accident Liquidators. Vitam Trace Elem 1:102. doi:10.4172/2167-0390.1000102
Page 5 of 8
Volume 1 • Issue 2 • 1000102
Vitam Trace Elem
ISSN: 2167-0390 VTE, an open access journal
rst of all – peroxynitrite) apparently initiates transmembrane metal
transport processes (Zn – from the cell, K – into the cell), where an
active role is played by transport proteins (metallochaperones) and
membrane ATPases [20].
Assuming that the linear association between Na and K is due to the
coordinated work of membrane ATPases, there emerges a reasonable
interest in comparative analysis of K–Zn correlation in Chernobyl
disaster liquidators and healthy subjects because both Na,K-ATPase
and the membrane pump for Zn belong to the same type (P-type) of
ATPases, although they represent dierent subgroups of those.
In our view, the negative nature of association between Na and K
concentrations in both assemblies (in the presence of K-Zn association)
can testify an increase in activity of P-type ATPases regarding
dierently-directed transmembrane transport of K+ and Zn2+ under the
conditions of oxidative/nitrosative stress.
In this connection, it would be appropriate to refer to the results
of the redox status research in Chernobyl disaster liquidators received
earlier [21] (See Table 7).
As Table 6 shows, the analysis of the redox status of Chernobyl
disaster liquidators evidences an appreciable and stretched in time
prooxidant shi (chronic oxidative stress) which cannot be liquidated
completely even aer antioxidant therapy.
At the same time, accepting the assumption that the negative K-Zn
association can indirectly point to an increased intracellular production
of oxygen and nitrogen radicals, one must explain the fact that a part
of healthy subjects (18.1%, see Table 4) in this respect varies little from
disaster liquidators. It is also unclear why in both investigated groups,
regardless of gender (Table 5 and 6), the Ca concentration in epidermic
cells falls while |rK-Zn| increases?
e close relation between NO and intracellular calcium
homeostasis is well known: via activation of soluble guanylyl cyclase
(sGC), cyclic guanosine monophosphate (cGMP), cGMP-dependent
protein kinase (PKG-I) and Ca2+ATPase [22]. One of the nal eects of
NO-dependent induction of this signalling path (at least for muscular
cells) is increased excretion of Ca from the cell.
Our comparative analysis of Ca concentration values in people
with and without detected negative K-Zn correlation separately for
men and women (let us remind that hair Ca content according to our
data was dependent on gender) gave the following results.
Regardless of gender, in groups with rK-Zn= -0,43 (healthy persons)
and rK-Zn= -0.62 (disaster liquidators) the hair Ca content was
signicantly lower than in the group with rK-Zn= -0.05 (healthy persons)
and rK-Zn= -0.03(disaster liquidators).
us, in healthy men, the mean Ca content in the group with rK-
Zn = -0.05 was 934.7 [745-1137.5] µg/g, while in the group with rK-Zn
= -0.43 it was just 856.9 [633.9-1107.1] (in square brackets – interval
estimation of the mean by the bootstrap method). In healthy women
with rK-Zn = -0.05 this parameter was equal to 1627.8 [1486.8-1776.5]
µg/g while in those with rK-Zn = -0.43 – just to 946.8 [787.1-1128.5] µg/g.
In the group of Chernobyl disaster liquidators with coecient rK-Zn =
-0.03, equal for men and women, the Ca level in men was 706.5 [622.9-
803.4] µg/g, in women – 1418.8 [1059.5-1815.8] µg/g. while in those
with rK-Zn = -0.62 this parameter in male liquidators was equal to 537,7
[506,1-570,4] μg/g; in female liquidators it was 611,3 [508,1-793,3] μg/g
(See Table 8).
e obtained results agree with our suggestion that the negative
association between K and Zn together with signicant decrease of Ca
level can indicate activation of intracellular radical reactions with NO
participation.
One can try to reveal interrelation between Ca, K and Zn
concentrations without correlation analysis. For this, in both entire
assemblies (separately for men and women) one needs to compare
mean levels of K, Zn at patently low level of Ca (lower than the lower
limit of interval bootstrap estimation of the mean) with mean K,
Zn in the rest of persons within the subsample. e results of such
comparison are shown in (Tables 9,10).
As (Tables 9,10) show, the low Ca level (groups I) was accompanied
by increased K concentration and decreased Zn concentration (as
compared with Groups II), with condence being proved by interval
estimation of the mean (bootstrapping) in most cases.
Analyses/Units Years 1998 – 1999 Year 2007
Lipid peroxides and hydroperoxides [LOO. , LOOH] (conv.units) 210,0 ± 30,7 [normal value < 80 ] 121,7 ± 10,84 [normal value < 80 ]
Blood plasma oxydizability (conv. units) 450, 0 ± 44,8 [normal value < 200] 440,6 ±51,82 [normal value < 200]
Lipid peroxidation processes ratio (conv. units) 9, 94 ± 1,01 [normal value < 4,0 ] 6,67 ± 0,71 [normal value < 4,0 ]
Selenium content in blood plasma (µg/L) 56,1 ± 3,3 (min.17,0 µg/L) [normal value. 80
- 120]
79,8 ± 3,94 (min.43,0µg/L) [normal value 80
- 120]
Glutathione Peroxidase in blood plasma (IU/L) 380,0 ± 19,4 [normal value 450 - 600] 398,0 ± 21,[normal value 450 - 600]
Glutathione Peroxidase in blood (IU/gHb) 26,1 ± 1,98 [normal value 35 - 50] 38,25 ± 1,84 [normal value 35 - 50]
Table 7: Redox status of Chernobyl disaster liquidators.
Groups Correlation
( r )
Ca (μg/g)
[M]
men women
Healthy
subjects
r = - 0,05
(n = 432)
745,0 <934,7< 1137,5
1486,8<1627,8<1776,5
r = - 0,43 (p<0,05)
(n = 171)
639,9< 856,9< 1107,1
787,1 <946,8<1128,5
Chernobyl
disaster
liquidators
r = - 0,03
(n = 115)
622,9 <706,5 < 803,4
1059,5<1418,8<1815,8
r = - 0,62 (p<0,05)
(n = 205)
506,1<537,7< 570,4
508,1<611,3< 739,3
Note: The Table gives the extreme (max and min) r values only
Table 8: K-Zn correlation and Ca concentration in epidermic cells.

Citation: Petukhov VI, Dmitriev EV, Kalvinsh I, Baumane LKh, Reste ED, et al. (2011) Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl
Accident Liquidators. Vitam Trace Elem 1:102. doi:10.4172/2167-0390.1000102
Page 6 of 8
Volume 1 • Issue 2 • 1000102
Vitam Trace Elem
ISSN: 2167-0390 VTE, an open access journal
Discussion
Undoubtedly, any conclusions that could be drawn on the basis
of this investigation are largely provisional. However, the long-felt
requirement in additional (apart from quantitative analysis) information
about the events really happening with metal-ligand complexes seems
inarguable. ese events, named metal-ligand homeostasis, are unlikely
to be correctly interpreted without understanding the role of ligands
in realization of the known biological eects which are traditionally
attributed to metals only.
Analysis of linear correlations between chemical elements (on the
basis of spectrometry results) is to help in getting such information
about MT, which are the most prevalent protein ligands for cadmium,
copper and zinc. In this connection it seems expedient (together with
determination of metals) to make quantitative estimation of MT or
their apoforms, thioneins, in one or another biosubstrate (which can
be the subject of further investigations).
It was found that a MT molecule includes two domains, α and β,
which have considerable, dierences [23]. One of these dierences
is unequal terminal amino acid residues in metal-binding clusters.
In the α domain, designed mostly for ‘toxic’ metals (particularly for
Cd), the terminal residues are carboxyls (carboxyl-terminal domain),
in the β domain – amino groups (amino-terminal domain). is β
domain chiey binds essential metals (Zn, Cu), and it is the one from
which nitroxide selectively releases Zn2+ through nitrosylation of thiols
(formation of S-nitrosothiols), leaving Cd-containing α domain intact
[24].
Other examples of functional interrelation between MT and NO
are also known. It was found that they have some common transmitters
(TNFα, IL-1, lipopolysaccharides) for induction of their intracellular
synthesis, and the MT molecule can serve as a trap for aggressive
nitrogen radicals. In addition, according to some researchers, Zn2+
released from MT can inhibit inducible NO-synthase (iNOS) and thus
prevent NO hyperproduction in cells [24].
Zn2+ release largely depends on the redox status of the cell where
prooxidant shis (e.g. accumulation of oxidized glutathione – GSSG)
facilitate Zn2+ release from MT molecules while reduced glutathione
(GSH) without presence of GSSG inhibits this process [25,26]. us,
Zn bond with β domain becomes unstable under conditions of both
oxidative and nitrosative stress [27,28].
is circumstance directly concerns Chernobyl disaster liquidators,
whose redox status was found [21] to demonstrate distinct prooxidant
shis: 3-5 fold increase of plasma chemiluminescence above norm, a
signicant decrease in activity of erythrocytic Se-dependent glutathione
peroxidase (GSH-Px-I).
Nitrosylation of thiols by nitroxide in β domains can release not
only Zn2+ but also Cu2+ from Cu-containing MT (Cu-MT). In this
case, if Cu2+ binds to apoform of Cu,Zn-superoxide-dismutase (apo-
ZnSOD), then Cu-MT can play the role of metallochaperone for one of
the key antioxidant enzymes in the cell [29].
Aer breaking from bonds of MT by means of nitrogen or oxygen
radicals, essential metals (Zn, Cu) have a choice: either to remain in
the epidermal cell in order to help it overcome oxidative/nitrosative
stress (e.g. as a part of antioxidant enzymes and/or as a suppressor
of NO hyperproduction), or to leave the cell. e latter choice looks
apparently less benecial for epidermis and its derivatives. However,
this is the choice which allows the organism to save essential metals,
whose necessity obviously rises at activation of radical processes.
An absolutely dierent situation exists for ‘toxic’ metals,
particularly Cd, in epidermis and its derivatives. Being eciently
secured from the release-eect of nitrogen/oxygen radicals in MT’s α
domain, these metals not only can remain in the cell but, according to
our observations (See Table 3), can also accumulate in the epidermal
derivative (hair). What does such accumulation mean for the whole
organism?
It means that all the body surface (in a man – ca.2m2), almost
completely covered by hair (except for palms and soles), becomes
the area where the organism removes excess of heavy and/or ‘toxic’
metals by pushing them into permanently desquamating epidermis
and growing hair. Such a reaction seems ecient and evolutionary
reasoned, and it can be surely claimed a compensatory/adaptive one
not only against heavy metal poisonings, but also in case of chronic
disturbances of the organism’s redox status – prooxidant shi, which
happens in Chernobyl disaster liquidators.
is idea is further conrmed by the data on the signicant
increase of Zn, Cu level in blood of disaster liquidators as compared
to norm [21]. It is notable that the considerable increase of Cu
concentration in blood of liquidators was accompanied by normal
level of ceruloplasmin. Unfortunately, the aforesaid authors did not
determine the MT concentration in plasma; therefore, it is dicult to
interpret the data.
Metal
Healthy subjects (n=947)
Women (n=709) Men (n=238)
Group I (n=437) Group II (n=272) Group I (n=149) Group II (n=89)
Ca (μg/g) 675.7<707.5<739 2494.6<2615.7<2746.6 318.5<335.4<351.3 888.1<1096.5<1408.2
K (μg/g) 268.6<336.3<412.6 171.6<226.2<293.2 290.5<380.2<475.2 191.1<324.7<494.7
Zn (μg/g) 171.3<176.2<181.1 196.4<205.6<215.1 154.5<162<168.8 190.2<205.3<220.3
Table 9: Comparative analysis of hair K, Zn content in groups of healthy subjects with different Ca level.
Metal
Chernobyl disaster liquidators (n=954)
Women (n=213) Men (n=741)
Group I (n=134) Group II (n=79) Group I (n=453) Group II (n=288)
Ca (μg/g) 477.6<505<739 1457.6<1634.7<1852.2 394.8<403.1<411.9 814.7<851.9<893.3
K (μg/g) 285.6<357.2<439.3 159.9<216<281 424<468.8<512.6 291.9<346.3<404.1
Zn (μg/g) 156.7<164.2<171.6 170.9<183.5<197.8 148.7<152.6<156.8 176.3<182.6<189.4
Note to Tables 9, 10: Group I – persons whose individual Ca levels do not exceed the lower limit of interval bootstrap estimation of the mean; Group II – the rest of the
given subsample. Bold font – mean values, regular font – limits of condence intervals
Table 10: Comparative analysis of hair K, Zn content in groups of Chernobyl disaster liquidators with different Ca level

Citation: Petukhov VI, Dmitriev EV, Kalvinsh I, Baumane LKh, Reste ED, et al. (2011) Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl
Accident Liquidators. Vitam Trace Elem 1:102. doi:10.4172/2167-0390.1000102
Page 7 of 8
Volume 1 • Issue 2 • 1000102
Vitam Trace Elem
ISSN: 2167-0390 VTE, an open access journal
Particular mechanisms of metal transfer through cell membrane
are insuciently studied. A key role in this process belongs to rather
large set of ATPases, which consists of ve subsets (I, II, III, IV and
V). e P-type of these enzymes (rstly PIb and PII) is most interesting
for us because the P-type ATPases provide transmembrane transport
of such ions as H+, Na+, K+, Cu+, Zn2+, Ca2+, Mg2+, Cd2+ etc [30,31].
Energy necessary for such transport, which runs against concentration
gradient, comes from ATP hydrolysis. It is assumed that nitrosylation
of S-containing proteins in the structure of KATP channels under
conditions of nitrosative stress leads to activation of these channels
[20].
It seems no mere chance to us that the existence of interrelation
between K+ and Zn2+ ion counter-ows (which can be seen in the
detected negative correlation K - Zn in Chernobyl liquidators and
partly in healthy subjects) is accompanied by a signicant decrease
of Ca concentration in epidermal cells. In our opinion, such a
combination (being a kind of discriminator) can point to activation of
redox processes involving nitrogen/oxygen radicals in epidermis.
Unfortunately, the authors had no data about the doses of
radioactive elements (J131, Cs137, Sr90) received by the Chernobyl
accident liquidators. erefore, it was not deemed possible to evaluate
the dierent eects of these radioactive atoms on the change of metal-
ligand homeostasis.
Conclusions
1. e epidermis derivative (hair) represents a convenient object
for noninvasive and retrospective supervision of events in
metal-ligand homeostasis epidermic cells, suitable for mass
(population) investigations. However, when conducting a
quantitative analysis of hair’s mineral structure, one should
avoid generalizing estimations by virtue of special, still
understudied role of epithelium in receipt and deducing of
metals. Moreover, one cannot exclude that in most cases
changes of metal concentration in epidermis can be of a re-
distribution nature, mismatching their true content in the
organism.
2. e correlation analysis of spectrometry data with the
subsequent selection of signicant correlations opens new
opportunities for studying metal-ligand homeostasis and
its changes connected with the redox status in organism (in
particular, with oxidative and nitrosative stress).
3. e results of the present work allow oer the following
parameters as kind of discriminators of an oxidizing and
nitrosative stress: a) increase in epidermis concentration values
K and Na alongside with reduction Ca and Zn, b) the presence
of signicant negative K-Zn correlations according to hair
spectrometry analyses.
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Citation: Petukhov VI, Dmitriev EV, Kalvinsh I, Baumane LKh, Reste ED, et al. (2011) Metal-Ligand Homeostasis in Epidermic Cells of Chernobyl
Accident Liquidators. Vitam Trace Elem 1:102. doi:10.4172/2167-0390.1000102
Page 8 of 8
Volume 1 • Issue 2 • 1000102
Vitam Trace Elem
ISSN: 2167-0390 VTE, an open access journal
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