In . J. Mol. Sci. 2013, 14, 12170-12185; doi:10.3390/ijms140612170
In e na ional Jou nal o
Molecula Sciences
ISSN 1422-0067
www.mdpi.com/jou nal/ijms
A icle
Se um Me allo hioneins in Childhood Tumou s—A Po en ial
P ognos ic Ma ke
Ja mila K useo a 1, Da id Hynek 2,3, Voj ech Adam 2,3, Rene Kizek 2,3, Richa d P usa 4,
Jan H abe a 1 and Tomas Eckschlage 1,*
1 Depa men o Paedia ic Haema ology and Oncology, 2nd Medical Facul y and
Uni e si y Hospi al Mo ol, V U alu 84, P ague CZ 150 06, Czech Republic;
E-Mails: ja mila.k useo a@ nmo ol.cz (J.K.); [email p o ec ed] (J.H.)
2 Depa men o Chemis y and Biochemis y, Facul y o Ag onomy, Mendel Uni e si y in B no,
Zemedelska 1, B no CZ 613 00, Czech Republic; E-Mails: [email p o ec ed] (D.H.);
[email p o ec ed] (V.A.); [email p o ec ed] (R.K.)
3 Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Technicka 3058/10,
B no CZ 616 00, Czech Republic
4 Depa men o Medical Chemis y and Clinical Biochemis y, 2nd Medical Facul y and
Uni e si y Hospi al Mo ol, V U alu 84, P ague CZ 150 06, Czech Republic;
E-Mail: [email p o ec ed]
* Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed];
Tel.: +42-0-224-436-450; Fax: +42-0-224-436-420.
Recei ed: 10 Ap il 2013; in e ised o m: 22 May 2013 / Accep ed: 30 May 2013 /
Published: 6 June 2013
Abs ac : Me allo hioneins (MT) a e low molecula weigh , cys eine- ich p o eins
main aining me al ions homeos asis. They play a ole in ca cinogenesis and may also cause
chemo esis ance. The aim o he s udy was o explo e he impo ance o MT se um le els
in child en su e ing om malignan umou s. This p ospec i e s udy in ol es examina ion
o 865 samples om 172 pa ien s wi h malignan umou s ea ed om 2008 o 2011 a
Uni e si y Hospi al Mo ol. MT se um le els we e de e mined using di e en ial pulse
ol amme y–B dicka eac ion. Mean MT le el was 2.7 ± 0.5 μM. The e was no
s a is ically signi ican di e ence be ween MT le els in di e en umou s. We also did no
ind any co ela ion be ween MT le els and esponse o he apy o clinical s ages.
Howe e , we ound a posi i e co ela ion be ween MT le els and age (p = 0.009) and a
nega i e co ela ion wi h absolu e lymphocy e numbe (p = 0.001). The ac ha pa ien s
who had ea ly disease ecu ence had lowe MT le els du ing he ea men (comple e
OPEN ACCESS
In . J. Mol. Sci. 2013, 14 12171
emission 2.67 s. ecu ing 2.34, p = 0.001) seems o be impo an o clinical p ac ice.
Acco dingly we belie e ha he e is bene i in u he s udies o se um MT le els
in umou s.
Keywo ds: se um me allo hioneins; child en; solid umou s; ea men ; di e en ial pulse
ol amme y B dicka eac ion
1. In oduc ion
Me allo hioneins (MTs) a e low molecula weigh cys eine ich p o eins, which ha e na u ally
occu ing Zn2+ in bo h binding si es [1]. They a e ound in he cy oplasm and a ious subcellula
o ganelles, pa icula ly in li e , kidney and in es inal cells [2,3]. Fou majo me allo hionein iso o ms
ha e been iden i ied in mammals (MT 1–4). MT-1 and 2 a e ubiqui ous, and hei main unc ions a e
he egula ion o coppe and zinc me abolism and he de oxi ica ion o hea y me als. They pe o m
se e al unc ions o gas oin es inal ac , a e in ol ed in cell ansc ip ion, and play a ole in immune
unc ion. MT-3 (also called neu onal g ow h-inhibi o y ac o ) is ound in he b ain and small le els
a e p esen in he panc eas and in es ines. MT-3 is syn hesized p ima ily in he as ocy es o co ex,
b ains em and spinal co d [4]. I s main unc ion is a g ow h inhibi o y ac o in b ain [5]. MT-4 is
loca ed in he epi helial cells o skin, ongue and s omach. I pa icipa es in he egula ion o s omach
acid pH, as e and ex u e disc imina ion o he ongue, and helps o p o ec he skin agains damage
om UV ligh [6,7]. Expe imen al s udy has shown educed neu onal p o ec ion agains oxida i e
s ess-induced damages in MT knockou mice [8]. Howe e , mice wi h dele ions o bo h he MT-1 and
MT-2 genes do no exhibi an al e ed pheno ype unde no mal condi ions [9–12]. MT-1 and MT-2,
unlike MT-3 and MT-4, a e highly inducible in mammalian cells by di e en s imuli (hea y me als,
cy okines). A signi ican associa ion be ween me al le els, MT exp ession, and disease was ound in
a ious issues [6,7].
The e a e some expe imen al and clinical p eceden s o sugges ing ha MTs can be expo ed and
aken up by cells h ough a ecep o -media ed mechanism [13]. High le els o MTs we e de ec ed in
sec e ed panc ea ic juice a e piloca pine s imula ion in mice [10]. Bilia y exc e ion o MT-1 ose in
a s ed wi h a high coppe die [12]. MTs we e eleased a e he induc ion o di e en ia ion o
ib oblas ic p eadipocy es in o adipocy es in i o) [11]. MTs we e de ec ed in human seminal plasma
sec e ed p edominan ly om he p os a e [14].
MTs could be e y impo an o pa ien s who a e unde going an i-cance he apy. They ha e
an iapop o ic, an ioxidan , p oli e a i e and p oangiogenic e ec s ha a e impo an in oncogenesis,
umou p og ession and esponse o an icance he apy [1,3,15–18]. MTs a e also in ol ed in
esis ance o cy os a ic d ugs, because hey educe d ug up ake, inc ease d ug e lux and pa icipa e in
DNA epai [17–22]. The e is a g ea deal o in o ma ion abou cellula MT con en in di e en
cance s and abou i s p ognos ic signi icance [1,23]. Howe e , he e is limi ed knowledge abou se um
le els in cance pa ien s and so a , he e a e no conclusi e da a on he signi icance o MT se um le el
in child pa ien s. MT-2A is he mos exp essed MT human iso o m because o he binding abili y o
In . J. Mol. Sci. 2013, 14 12172
enhance s in he MT-2A p omo e egion [24]. Howe e , he in o ma ion on se um le els o indi idual
iso o ms is s ill lacking.
The aim o his s udy is o explo e ela ionships be ween MT se um le els and ype o umou ,
s ages and esponse o he apy in child en su e ing om solid umou s. We also we e looking o
co ela ion be ween MT le els and o he labo a o y indings.
2. Resul s
2.1. MT in Se um
The mean MT le el in child en wi h malignan umou s was 2.67 ± 0.5 μM. The e we e no s a is ically
signi ican di e ences be ween di e en umou s in ou g oup o pa ien s (Figu e 1). The highes mean
MT le els we e ound in ge m cell umou s (MTs 2.94 ± 0.9 μM) and he second highes le els we e
de e mined in b ain umou s (2.82 ± 0.5 μM) bu he di e ences a e no s a is ically signi ican and a e
p obably caused by age dis ibu ion. We p e iously published a e age MT le els in se um o heal hy
adul olun ee s de ec ed also using he B dicka me hod. In he sample (mean age 27 yea s), MT le el
was 0.52 ± 0.2 μM [25]. Milne owicz and Bizoń ound simila esul s in heal hy adul olun ee s using
enzyme-linked immunoso ben assay [26] and Singh and Hanson ound lowe MT le els in 50 no mal
child en (aged 2–11 yea s) by immunoassay (app ox. 2 mg/L) [27]. Somewha highe we e MT le els
de ec ed by di e en ial pulse ol amme y–B dicka eac ion ound in a g oup o child en (age 3–17
yea s) wi h a ious enal diseases wi h no mal c ea inine le els (MTs 1.42 ± 0.09 μM) [28]. Those
le els in heal hy adul con ols a e app oxima ely i e imes lowe han he a e age (2.67 ± 0.5 μM)
MT le els ound in ou g oup o pa ien s. The MT le els in he g oup o child en wi h enal diseases
we e oughly hal ha o alues in child en su e ing om malignan umou s. Bo h hose di e ences
we e s a is ically signi ican (T- es , p < 0.01). The MT le els ound in a g oup o child en wi h
nonmalignan kidney diseases we e app oxima ely hal o le els in ou g oup wi h malignan umou s.
We ound highly signi ican posi i e co ela ion be ween MT le els and age ha was independen o
umou ype. Figu e 1 depic s a gene al linea model wi h dependen a iable MT le el, be ween
subjec s ac o ype o umou and co a ia e age, displaying signi icance o co a ia e e ec age
p = 0.009 (F = 7.05, d = 1), pa ame e es ima e o age B = 0.023 (posi i e).
2.2. MT Rela ionship o Cou se o he Disease
We did no ind any di e ences in MT le els among pa ien s wi h umou s be o e s a ing he apy,
wi h umou and/o me as ases du ing he apy o wi h p og essi e disease (g oup o “pa ien s wi h
ac i e disease”) and pa ien s in comple e emission (g oup wi hou “ac i e disease”). The e we e no
s a is ically signi ican di e ences in MT le els in pa ien s wi h me as a ic diseases in compa ison wi h
le els in pa ien s wi h localized cance . The majo i y pa ien s displaying lowe MTs le els had ea ly
ecu ence o disease. A e age MT le els du ing all ollow up pe iod be o e de ec ion o ecu ence
was 2.34 μM, e sus comple e emission 2.67 μM (p = 0.001). These esul s a e shown in Figu e 2.
We, howe e , ound signi ican dec ease o MT le els in child en du ing ollow up ≥2 mon hs a e he
end o he ea men as compa ed wi h le els a he end o he apy (p = 0.001, Figu e 3).
In . J. Mol. Sci. 2013, 14 12173
Figu e 1. MT le el/mean ± s.d./in di e en childhood malignan umou s. Ha ched column:
all umou s; do ed columns: heal hy adul olun ee s. The e we e no s a is ically signi ican
di e ences be ween di e en umou s, di e ence be ween MT le els in all malignan
umou s and in heal hy olun ee s was signi ican . MMT: So issue sa coma (Malignan
mesenchymal umou s).
Figu e 2. Rela ion o MT le el/mean ± s.d./ o clinical pa ame e s. N.S.: no signi ican .
p = 0.001: s a is ically signi ican on he le el o 0.1%. The e we e no s a is ically
signi ican di e ences be ween MT le els in pa ien s wi h ac i e disease and in emission
o in pa ien s wi h gene alized disease and wi h cance s wi hou me as asis.
Figu e 3. MT le els/mean ± s.d./in g oup o pa ien s <2 mon hs a e inishing
chemo he apy compa ed o pa ien s ≥2 mon hs a e inishing chemo he apy [pai ed T- es
( = 3.53, d = 75) p = 0.001].
In . J. Mol. Sci. 2013, 14 12174
2.3. MT Le els and Chemo he apy
MT le els in pa ien s wi h ac i e disease we e highe in hose ea ed wi h chemo he apy han
wi hou (p = 0.02). The e was no signi ican di e ence in MT le els in pa ien s ea ed by p o ocols
con aining pla inum cy os a ics and ea ed wi hou hose cy os a ics (Figu e 4).
Figu e 4. MT le els in ela ion o chemo he apy. N.S.: no signi ican . p = 0.02: s a is ically
signi ican on he le el o 2%. The only signi ican di e ence was ound be ween le els in
pa ien s wi h ac i e umou (be o e he apy, pa ial emission, p og essi e disease) wi h and
wi hou chemo he apy. The e was no ela ionship o chemo he apy wi h pla inum cy os a ics.
2.4. Co ela ion o MT Le els wi h O he Biochemical Pa ame e s
The e was a nega i e co ela ion be ween MT le els and absolu e lymphocy e coun (p = 0.001)
and posi i e co ela ion be ween MT and c ea inine le els (p = 0.003). Co ela ion be ween
haemoglobin and MT le els was below he le el o s a is ical signi icance (p = 0.085). Pa ien s wi h
CRP abo e no mal le els exhibi ed no di e ence in MT le el compa ed o hose wi h no mal CRP
(p = 0.912). Summa y o he esul s ob ained is shown in Table 1. Co ela ions o MT le els wi h
o he labo a o y indings men ioned in Ma e ials and me hods we e no signi ican .
Table 1. Co ela ions be ween labo a o y pa ame e s.
Co ela ions
MT ANL CRP Hgb
MT Pea son 1 −0.252 ** 0.009 0.132 #
Sig. (2- ailed) - 0.001 0.912 0.085
N 172 171 152 171
ANL Pea son −0.252 ** 1 −0.126 −0.040
Sig.(2- ailed) 0.001 - 0.119 0.599
N 171 174 154 174
CRP Pea son 0.009 −0.126 1 0.039
Sig. (2- ailed) 0.912 0.119 - 0.635
N 152 154 154 154

In . J. Mol. Sci. 2013, 14 12175
Table 1. Con .
Co ela ions
MT ANL CRP Hgb
Hgb Pea son 0.132 −0.040 0.039 1
Sig. (2- ailed) 0.085 0.599 0.635 -
N 171 174 154 174
C ea Pea son 0.273 −0.381 0.170 0.445
Sig. (2- ailed) 0.003 0.000002 0.036 0.000001
N 170 173 153 173
ANL: absolu e lymphocy e numbe ; CRP: C- eac i e p o ein, Hgb: haemoglobin, C ea: c ea inine. ** p < 0.01,
# nea he le el o s a is ical signi icance.
3. Discussion
Nume ous s udies ha e been ca ied ou o unde s and he ela ionship be ween MT in cells and
cance . Mos o hese ha e ocused on adul s [6,18,29–33] and a ewe on child en [34–37]. S udies
in adul s ha e shown ha cellula MT could se e as p ognos ic ma ke s in some umou s [17,38–43].
Rela ionships ha e been desc ibed be ween MT and p53 s a us [1], inc eased umou g ade [44,45] and
me as ases de elopmen [1,46]. Howe e wha has been p o ed in adul s is no so clea in childhood
cance s. The e is one s udy in child en wi h os eosa coma, whe e he au ho s ound di e en ial
exp ession o MT in biopsies [36] and hey sugges ed ha MT migh play an impo an ole in
de elopmen o disease. Ano he s udy on child en su e ing om leukaemia showed ha pa ien s wi h
acu e lymphoblas ic leukaemia whose blas s exp ess MT ended o ha e sho e disease- ee su i al
compa ed wi h he MT nega i e ones [37]. A u he epo on pa ien s wi h neu oblas oma demons a ed
only limi ed alue o MT o p edic ion o he apeu ic esponse [34].
MT le els in se um ha e seldom been esea ched in cance pa ien s. They ha e been used o
moni o ing in oxica ion by me als (lead, cadmium) [47]. P e iously, ou g oup has published h ee
publica ions on MT in se um in adul cance . Inc eased MT se um le els in p os a e cance pa ien s
we e ound o be nega i ely co ela ed wi h hei Gleason sco e [48]. MT le els in pa ien s su e ing
om head and neck spinocellula cance demons a ed co ela ion wi h umou g ade and clinical
s age [49,50]. In ano he s udy hepa ocellula ca cinoma se um MT le els we e dec eased compa ed o
he con ol g oup [51]. To ou knowledge no wo k has ye been published on MT se um le els in child
cance pa ien s.
Ou s udy co e ed a wide ange o child cance s. MTs we e ound no o ac as a p ognos ic ma ke
o ea men esponse in any o hese diseases. Pa ien s wi h me as ases o p og essi e disease did no
exhibi di e en MT le els ei he . Di e ences be ween di e en ypes o umou s we e no signi ican .
The highes mean MT le els we ound we e in ge m cell umou s (MT 2.94 ± 0.9 μmol·dm−3) mos
likely because hese included a highe p opo ion o olde pa ien s. The second highes we e in b ain
umou s (2.82 ± 0.5 μmol·dm−3)—p obably because he b ain, in gene al, con ains highe amoun s o
MT [16,52,53], bu age dis ibu ion may also play a ole. Du ing an icance he apy he e a e many
di e en pa hological eac ions ( issue damage induced by cy os a ics and/o adio he apy, suppo i e
he apy including an ibio ics and an imyco ics, e y h opoe in and/o ilg as im, in ec ions, umou
beha iou , e c.). Each o hese con ibu es o a ious and hence con inuously a ying le els o
In . J. Mol. Sci. 2013, 14 12176
oxida i e s ess. MTs a e known o unc ion as one o he main an ioxidan de ence sys ems [1,54,55].
Changes o MT le els caused by he apy and/o i s complica ions men ioned abo e a e p obably he
main eason why co ela ion be ween umou ac i i y and MT le els is no signi ican . We ha e no
ound signi ican co ela ion be ween umou ac i i y and MT le els.
The inding ha low MT le els du ing ea men we e closely co ela ed wi h ea ly elapses may be
impo an o clinical p ac ice. Peye e e al. ound ha up o 80% o child en wi h ecu ence o
ependymoma ha e down- egula ed MT-3 genes, no caused by MT gene dele ion o p omo e
me hyla ion. MT-3 exp ession was es o ed by his ondeace ylase inhibi o o zinc ea men [35]. One
may sugges ha down- egula ion could be a key cause o low MT le els in pa ien s su e ing om
ecu ence, al hough om wel e ecu en umou s, only h ee we e b ain umou s and i is no known
i o he MT iso o ms exp ession is also down egula ed in ecu en umou s o o he o gans.
The explana ion o slow decline (2 mon hs) o MT le els a e comple ing he apy is long eco e y
eac ions in he o ganisms a e he end o ea men . We choose a wo mon h limi because i is an
in e al in which haema opoiesis and immuni y is usually econs i u ed a e con en ional doses o
chemo he apy, and one may specula e ha o he sys ems a e also no malized du ing his in e al [56].
This sugges s ha chemo he apy in luences MT le els by mul iple mechanisms e.g. he damage o
di e en o gans and sys ems, he inc easing isk o in ec ions, and/o by he p oduc ion o eac i e
oxygen species. I will be necessa y o obse e hese pa ien s o e longe pe iods o de e mine when
MT le els e u n o no mal. We sugges ha changes o MT le els could be conside ed as a p omising
ma ke o la e elapses. In ou s udy we did no ha e pa ien s who su e la e ecu ence mo e han 2
yea s a e he end o ea men . The ac ha MT le els declined du ing he wo mon hs ollowing he
end o ea men suppo s ou iew ha he an icance and suppo i e he apy signi ican ly a ec s he
se um le els o MT.
MT plays an impo an ole in chemo esis ance and no only o me al con aining d ugs. Thei
exp ession may be inc eased no only by me als bu by o he s imuli e.g. glucoco icoids, ca echolamines,
ee adicals, umou nec osis ac o α, in e leukins-1, -2 and -6 [1,17]. P oduc ion o some hose
MT-inducing biologically ac i e molecules is s imula ed by chemo he apy. This may explain inc eased
MT le els in pa ien s wi h “ac i e umou s” ea ed wi h chemo he apy.
The indings o inc eased MT le els in pa ien s wi h ac i e disease (wi h umou s be o e s a ing
he apy, wi h umou and/o me as ases du ing he apy o wi h p og essi e disease) bu no in emission
ea ed by cy os a ics may be explained by he p oduc ion o MT by cance cells s imula ed by
chemo he apy. MTs a e able o bind pla inum-based cy os a ics and he eby educe hei cy o oxic
e ec . Chemo esis ance o pla inum an i- umou compounds is media ed h ough se e al mechanisms.
One o hem is he ans e o pla inum om cispla in and ca bopla in o MT ha esul s in inac i a ion
o hose d ugs. Cul i a ion o neu oblas oma cells esis an o cispla in in medium wi h cispla in o
ca bopla in, has been shown o signi ican ly inc ease in acellula MT le els [1]. Howe e in a
sensi i e cell line only insigni ican inc eases in MT we e de ec ed a e cul i a ion wi h he same
concen a ions o cispla in o ca bopla in [1]. Ano he s udy showed ha a cispla in- esis an o a ian
cance cell line exposed o cispla in mani es ed a nuclea MT exp ession [57]. In hepa oblas oma
pa ien s ea ed wi h ca bopla in, i was e i ied ha non- esponde s had a highe pe cen age o
MT-posi i e umou cells [58]. The signi icance o MT exp ession o esis ance o gas ic cance o
cispla in was e i ied by Suganuma e al. [59]. MT up egula ion was de ec ed in medulloblas oma and
In . J. Mol. Sci. 2013, 14 12177
habdomyosa coma cells wi h induced esis ance o he alkyla ing d ug BCNU [60]. Esophageal
ca cinomas which do no exp ess MT, espond well o chemo adio he apy (5- luo ou acil and
cispla in) while cance s wi h high MT exp ession a e esis an [61]. Women wi h b eas ca cinoma
ea ed wi h chemo he apy (cyclophosphamide, me ho exa e, 5 luo ou acil o doxo ubicin) had
signi ican ly longe su i al i hei umou s had lowe MT exp ession [42]. Hishikawa and co-wo ke s
ound ha MT nega i e pa ien s wi h oesophageal cance ea ed wi h cispla in expe ienced inc eased
su i al compa ed o hose wi h MT posi i e umou s [62].
I has been ound ha MT exp ession ini ially inc eases wi h age, bu hen dec eases in people o e
se en y [63] and he inducibili y o MT inc ease wi h age in in an a model was desc ibed by
Baue ly [64]. Co ela ion o se um MT le els ound in ou s udy may also indi ec ly con i m his
phenomenon. Na ale e al. showed in animal expe imen s ha low MT explains enhanced suscep ibili y o
neu onal loss a e inju y in imma u e b ains [53]. I is common knowledge ha chemo he apy is mo e
oxic in e y young child en compa ed o olde child en. Ou indings o low MT in in an pa ien s
could be one o he possible easons o mo e se ious side e ec s o chemo he apy and adio he apy in
in an s compa ed o olde child en.
The possible explana ion o dec eased MT in pa ien s wi h highe c ea inine le els may be kidney
damage, as hey a e one o he majo p oduce s o MTs. MT-1 and MT-2 a e p oduced pa icula ly in
kidney, li e , panc eas and in es ine [1]. The posi i e co ela ion o MT le els and lymphocy e coun
in cance pa ien s do no ha e a clea explana ion. Due o signi ican co ela ions be ween se um
c ea inine and lymphocy e coun s and haemoglobin le els, i is di icul o assess whe he he
ela ionship be ween MT and c ea inine is p ima y, o whe he he e a e p ima y ela ionships o
haema ological pa ame e s. I is possible ha he induc ion o MT by cy okines such as in e leukin-1,
-2 and -6 o umou nec osis ac o , which a e p oduced by lymphocy es, may play a possible ole.
I has been ound ha zinc supplemen a ion in luences lymphocy e p oduc ion [63,65]. The au ho s
looked a changes in la ge numbe s o genes ha we e in ol ed in zinc homeos asis in pe iphe al
blood leukocy es o child en wi h sep ic shock. MT exp ession in child en who died was also
inc eased. I was sugges ed ha decline in zinc concen a ions among c i ically ill child en was ela ed
o shi s in MT exp ession and low plasma zinc le els we e associa ed wi h he deg ee o o gan
ailu e [64–66]. Child pa ien s who ha e low lymphocy e coun also ha e inc eased isk o sepsis. In
ou s udy we no iced ha pa ien s wi h low lymphocy e coun had inc eased MT le els. Th ee o ou
pa ien s had li e h ea ening sepsis du ing leucopoenia and high se um MT le els—2.1 imes highe
han mean (da a no shown), which is in ag eemen wi h he s udies men ioned abo e. The ac ha
CRP did no co ela e wi h MT le els sugges s ha MTs a e no acu e-phase p o eins, and may be a
labo a o y ma ke ha is independen o acu e-phase.
4. Expe imen al Sec ion
4.1. Pa ien s
This p ospec i e s udy in ol es examina ion o 865 samples om 172 pa ien s wi h malignan
umou s ea ed a he Depa men o Paedia ic Haema ology and Oncology, Uni e si y Hospi al
Mo ol om 2008 o 2011. Samples we e collec ed be o e s a ing chemo he apy, du ing chemo he apy
In . J. Mol. Sci. 2013, 14 12178
and a e . Diagnoses and age dis ibu ion see Table 2. The g oup included 71 gi ls (41%) and 101 boys
(59%). O he clinical pa ame e s we e: me as a ic disease 93 (54.1%), dea h du ing ollow up 32 (18.6%),
ecu ence du ing ollow up 12 (7%). The minimal ollow up was 18 mon hs and median ollow up
was 39 m (Table 2).
Table 2. Clinical cha ac e is ics o pa ien s g oup.
Diagnosis No. o
pa .
Median
age
Minimum
age
Maximum
age Recu ence Me as a ic Chemo he apy/
P cy os aic **
Neu oblas oma 33 1 y 8 m 1 m 15 y 3 24 30/24
B ain umou s 27 9 y 2 m 8 m 15 y 4 m 3 3 * 27/16
Lymphoma 24 15 y 10 m 9 m 18 y 4 m 2 / 24/0
Ewing sa coma 20 12 y 7 m 1 y 5 m 18 y 3 m 0 3 20/0
Ge m cell umou s 14 16 y 4 m 6 m 19 y 6 m 1 10 14/14
Os eosa coma 12 13 y 1 m 6 y 5 m 16 y 11 m 1 3 12/12
So issue sa coma 12 10 y 8 m 1 y 8 m 16 y 6 m 1 1 12/0
Neph oblas oma 7 1 y 9 m 9 m 6 y 10 m 1 3 7/2
O he malignan
umou s 23 16 y 4 m 6 m 19 y 6 m 0 9 16/2
All 172 9 y 11 m 1 m 19 y 6 m 12 56 162/70
* spinal me as ases, ** no o ea ed by any cy os a ic/no o ea ed by P con aining cy os a ic (cispla in and/o ca bopla in).
In ou s udy we in es iga ed he co ela ion be ween MT le els and clinical pa ame e s (age,
diagnosis, clinical s age, ecu ence and esponse o an icance he apy). We also compa ed MT le els
examined be o e, du ing and a e he apy wi h o he labo a o y indings: o al blood coun and u ea,
c ea inine, u ic acid, lac a e dehyd ogenase, ansaminases, bili ubin, e i in, o al p o ein, and C- eac i e
p o ein (CRP) de e mina ion. To al blood coun was examined a he Depa men o Clinical
Haema ology, and o he biochemical es s a Depa men o Clinical Biochemis y and Pa hobiochemis y,
Uni e si y Hospi al Mo ol, acco ding o s anda d p o ocols.
4.2. De e mina ion o Me allo hioneins
Samples we e p epa ed by hea ea men using an au oma ed pipe ing sys em epMo ion 5075
(Eppendo , Hambu g, Ge many) and kep a 4 °C hen ans e ed o he 96 well pla es (Eppendo ,
Hambu g, Ge many) oge he wi h 0.2 M phospha e bu e pH 7. This mix u e was kep a 99 °C o
15 min. The las s ep was cooling down o samples o 4 °C. Hea ing dena u es and emo es he high
molecula weigh p o eins om samples [67]. MT quan i ica ion was de e mined by elec ochemical
de ec ion. Di e en ial pulse ol amme ic B dicka eac ion measu emen s we e pe o med using a
747 VA S and ins umen connec ed o a 693 VA P ocesso and 695 Au osample (Me ohm, He isau,
Swi ze land), using a s anda d cell wi h h ee elec odes, a cooled sample holde and measu emen
cell. Measu emen s we e aken on samples cooled o 4 °C (Julabo F25, Julabo, Seelbach, Ge many). A
hanging me cu y d op elec ode (HMDE) wi h a d op a ea o 0.4 mm2 was he wo king elec ode. An
Ag/AgCl/3M KCl elec ode was he e e ence and pla inum elec ode was auxilia y. Fo da a
p ocessing VA Da abase 2.2 by Me ohm was employed. The analysed samples we e deoxygena ed
p io o measu emen s by pu ging wi h a gon (99.999%) sa u a ed wi h wa e o 120 s. A B dicka
In . J. Mol. Sci. 2013, 14 12185
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me allo hionein de ec ion in human blood se um and cadmium- ea ed umou cell lines a e
do - and elec oblo ing. Elec opho esis 2009, 30, 3726–3735.
75. K iziko a, S.F. I; Adam, V.; Kukacka, J.; P usa, R.; Cha is, G.J.; T nko a, L.; S nadel, J.;
Ho ak, V.; Kizek, R. U ilizing o adso p i e ans e s ipping ecchnique b dicka eac ion o
de e mina ion o me allo hioneins le el in melanoma cells, blood se um and issues. Senso 2008,
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