Dicle Tıp Dergisi / Dicle Med J (2019) 46 (2) : 315 - 320
Özgün Araştırma / Original Article
Thiol – Disulphide Homeostasis in Polycythemia Vera
Aysun Şentürk Yıkılmaz1, Şule Mine Bakanay2, Sema Akinci3, Senem Maral4,
Fuad Mustafayev5, Murat Alisik6, Özcan Erel7, İmdat Dilek8
1 Department of Hematology, Yıldırım Beyazıt University Medical Faculty, Ankara, Turkey ORCID: 0000-0001-5281-5955
2 Department of Hematology, Yıldırım Beyazıt University Medical Faculty, Ankara, Turkey ORCID: 0000-0003-3941-0368
3 Department of Hematology , Ataturk Training and Research Hospital, Ankara, Turkey ORCID: 0000-0003-4237-3342
4 Department of Hematology , Ataturk Training and Research Hospital, Ankara, Turkey ORCID: 0000-0003-4766-1861
5 Department of Internal Medicine, Ataturk Training and Research Hospital,Erzurum, Turkey ORCID: 0000-0003-0254-1402
6 Department of Medical Biochemistry, Yildirim Beyazıt University Medical Faculty,Ankara, Turkey ORCID: 0000-0003-0434-3206
7 Department of Medical Biochemistry, Yildirim Beyazıt University Medical Faculty,Ankara, Turkey ORCID: 0000-0002-2996-3236
8 Department of Hematology, Yıldırım Beyazıt University Medical Faculty, Ankara, Turkey ORCID: 0000-0001-8316-446
Received: 13.12.2018; Revised: 06.03.2019; Accepted: 02.04.2019
Abstract
Background: Thiol-disulphide homeostasis has vital role in cell signalling mechanisms, regulation of transcription factors and
enzymatic activities, signal transduction and regulation of proliferation rate, apoptosis and detoxification and antioxidant
protective mechanism.
Objective: This study aims to demonstrate in Polycythemia Vera (PV) patients the thioldisulphide homeostasis which is known to
play a role in cell proliferation, apoptosis and various steps of cell cycle.
Design: Descriptive prospective cross-sectional study.
Settings: Yildirim Beyazıt University Hospital Ankara, Turkey between 2016-2018.
Method: Forty-two PV patients and 43 healthy controls were included in the study. Serum total (–SH + –S–S–) and native (–SH)
thiol levels were measured in all subjects. The amount of dynamic disulphide bonds and, the ratio of (–S–S–) and (–S–S–) × 100/(–
SH), (–S–S–) × 100/(–SH + –S–S–), and –SH ×100/(–SH + –S–S–) were calculated with automatic method. The data obtained from
the patient group were compared with the control group.
Main outcome measures: The amount of dynamic disulphide bonds and, the ratio of (–S–S–) and (–S–S–) × 100/(–SH), (–S–S–) ×
100/(–SH + –S–S–), and –SH ×100/(–SH + –S–S–) were calculated with automatic method in PV patieint and healthy control group.
Results: Both groups were similar in terms of age and gender distribution. Compared with the control group, PV group had
significantly higher native thiol, total thiol and nativ/total thiol levels.
Limitation: The generalizability of the study's findings were limited by the small sample size.
Conclusions: In accordance with the nature of the disease, thiol balance in PV patients was in favor of proliferation. Increased total
thiol (–SH + –S–S), native thiol (–SH) levels and native thiol/total thiol ratio might be associated with uncontrolled proliferation.
The balance of the thiol-disulphide homeostasis shifted to reductive thiol side in the PV. This change can provocate proliferation
status of the disease and/or may be secondary to the disease.
Conflict of Interest: No conflict of interest was declared by the authors.
Keywords: Polycythemia Vera, thiol, disulphide bonds, myeloproliferative neoplasms
DOI: 10.5798/dicletip
Yazışma Adresi / Correspondence: Aysun Şentürk Yıkılmaz, Department of Hematology,Yıldırım Beyazıt University, Bilkent Yolu 3. Km. Çankaya
Ankara 06010, Turkey, e-mail: senturkaysun@gmail.com
Yıkılmaz A.Ş., Bakanay Ş.M., Akinci S., Maral S., Mustafayev F., Alisik M., Erel Ö.,Dilek İ.
Polistemia Vera Olgularında Thiol-Disülfid Homeostasis
Öz
Giriş: Bu çalışmanın amacı, Polistemia Vera (PV) olgularında; hücre siklusunun çeşitli basamaklarında, apoptozis ve
hücre proliferasyonunda önemli rol oynadığı bilinen tiyol-disülfid homeostasisini incelemeyi amaçladık.
Yöntemler: Kırkiki PV olgusu 43 sağlıklı kontrol grubuyla karşılaştırıldı. Serum total (–SH + –S–S–) ve nativ (-SH) tiyol
seviyeleri her iki grupta ölçüldü. Dinamik disülfid bağlarının yüzdesi ve (–S–S–) and (–S–S–) × 100/(–SH), (–S–S–) ×
100/(–SH + –S–S–), ve –SH ×100/(–SH + –S–S–) oranları Erel ve arkadaşlarının yeni metodu ile hesaplandı. PV olguları
ve sağlıklı kontrol grubunun verileri kıyaslandı.
Sonuçlar: Her iki grubun yaş ve cinsiyet dağılımları benzerdi. PV grubunda nativ tiyol, total tiyol ve nativ/total tiyol
seviyeleri istatiksel olarak anlamlı artmıştı.
Tartışma: PV hastalığının doğasıyla uyumlu olarak, PV olgularında dengenin proliferasyon yönüne kaydığı
gözlemlendi. Total tiyol (–SH + –S–S), nativ tiyol (–SH)ve nativ /total tiyol oranındaki artışı PV hastalığındaki
kontrolsüz proliferasyonla açıklayabiliriz. PV olgularında tiyol/disülfid dengesinin indirgeyici tiyol tarafında olduğunu
gösterdik. Tüm bu değişiklikler PV hastalığının patofizyolojisinde olan kontrolsüz proliferasyonla ilişkili olabilir.
Anahtar kelimeler: Polistemia vera, tiyol, disülfid bağ, myeloproliferatif hastalık.
INTRODUCTION
Thiols are organic compounds which contain
sulphydryl group. Albumin and proteins
constitute the major part of plasma thiol while
low molecular weight thiols such as cystein,
cystein glycine, glutathione, homocystein and
gamma glutamyl cystein constitute a lower
proportion1,2. These compounds have high
reduction capacity and are known as good
nucleophiles because they can easily get into
many reactions3. Thiols are open to both
reversible and irreversible modifications and
can provide new end products by forming
reversible bonds with disulphides as a result of
oxidation reactions. The formed disulphide
bonds can be reduced back to thiols. Thus,
thiol-disulphide homeostasis is maintained.
Thiol-disulphide homeostasis has vital role in
cell signalling mechanisms, regulation of
transcription factors and enzymmatic activities,
signal transduction and regulation of
proliferation rate, apoptosis and detoxification
and antioxidant protective mechanisms4.
Increased intracellular thiol has been
associated with proliferation while thiol levels
have been found to be decreased in apoptosis.
Thus thiols are important compounds in cell
cycle regulation and the control of cell
division5. Additionally, it has been shown that
some chemotherapeutic agents which use the
thioredoxin system exert their anti-cancer
effects through thiol homeostasis6.
Thiol-disulphide homeostasis show different
pattern in various diseases. In degenerative
vascular
diseases
such
as
coronary
atherosclerosis7,8,
diabetes
mellitus9,
preeclampsia10 and cerebral ischemia11, thiol
levels are decreased and disulphides are found
to be increased. On the other hand, in
proliferative diseases, thiols are expected to be
increased12-14.
Chronic Myeloproliferative disorders (CMPD)
comprise a group of diseases in which one or
more of pluripotent hematopoietic stem cells in
the bone marrow have proliferation advantage.
Disregulation of proliferation cycle of
hematopoietic stem cells have a role in the
exaggerated production of mature blood cells.
Policytemia Vera (PV) is one of the CMPD and is
characterized by clonal proliferation of
erythroid lineage and presence of JAK 2 V617F
mutation in most cases15.
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Dicle Tıp Dergisi / Dicle Med J (2019) 46 (2) : 315-320
There is in fact proliferation in all three
lineages and continuous proliferation which
may result in post-PV myelofibrosis and
transformation to acute leukemia16-20. Various
abnormalities have been discovered in the
signal
transduction
pathways
in
the
hematopoietic stem cell cycle of PV patients21.
Although it is well known that tyrosine kinase
activation results in JAK2V617F mutation and
increased sensitivity to hematopoietic growth
hormones and cytokines, the exact mechanism
of uncontrolled cell proliferation in PV is still
incompletely understood22. To our knowledge,
thiol-disulphide homeostasis has not been
studied in PV patients before. In this study we
have aimed to investigate the levels of native
thiol, total thiol and disulphide, and the ratios
of disulphide/native thiol, disulphide/total
thiol, and native thiol/total thiol in PV patients
using a novel, automated method that
determines
dynamic
thiol/disulphide
homeostasis.
METHODS
Study population
Forty-two patients diagnosed with PV and 43
healthy volunteers were enrolled in the study.
Forty-two patients with PV were compared
with 43 healthy control cases. The control
group was constituted of cases who applied for
check up and who did not have any systemic
diseases or drug use. Patients with diabetes,
severe renal or liver diseases, active infectious
or inflammatory diseases, previous stroke,
rheumatologic diseases, or malignancy were
excluded from the study.
This study has been designed in accordance
with 2013 Brazil version of Helsinki
Declaration and was approved by the local
Ethics Committee. All participants have
provided written consent.
Biochemical parameters
Venous blood samples were taken from each
patient into tubes containing ethylenediamine
tetraacetic acid (EDTA) after 8 hours of fasting.
Collected
samples
were
immediately
centrifuged at 3483,6 g value for 10 minutes to
separate the serum, then the samples were
stored at -80oC until analysis.
Thereafter, all parameters were analyzed at
the
same
time.
The
thiol/disulphide
homeostasis were determined with the more
recently developed automated method. Firstly,
short disulfide bods were reduced with sodium
borohydride to form free functional thiol
groups. To prevent reduction of DTNB (5.5’dithiobis-[2-nitrobenzoic] acid), reductive
sodium borohydride was removed and
consumed with formaldehyde. All of the thiol
groups, including reduced and native thiol,
were measured after DTNB reduction. The
dinamic disulfide value was defined as half of
the difference between total and native thiols.
After determining native and total thiols,
disulphide level, disulphide/total thiol percent
ratios, native thiol/total thiol percent ratios
and disulphide/native thiol percent ratios were
calculated2. Measurements were made by an
Autocobas 501 autoanalyser (Roche-Hitachi,
Mannheim,
Germany).
The
analyzer
automatically detects lipemic-icteric and
hemolytic serums and does not work without
approval. Hemolysis does not interfere
positively with results.
Statistical Analyzis
Normality distributions of study groups were
evaluated by the Kolmogorov-Smirnov test. The
parametric values were given as mean ± SD,
non-parametric values were given as median
(Inter Quartile Range). Comparisons were done
with Student’s t-test in cases of normal
distribution and with Mann-Whitney U test in
cases of abnormal distribution. The Spearman
and polyserial correlation coefficients were
calculated to evaluate the relationship between
the measurements. The P value <.05 was
regarded as significantly.
Yıkılmaz A.Ş., Bakanay Ş.M., Akinci S., Maral S., Mustafayev F., Alisik M., Erel Ö.,Dilek İ.
Table 1: The PV and Control group complete blood count
values and thiols levels.
Ethics Statement
Ethics committee approval was received for
this study from the Institutional Review Board
of Ankara Atatürk Training and Research
Hospital (No. K-18-727). Written informed
consent was obtained from all patients and
controls.
PV (n=42)
Age
Hemoglobin (g/dl)
Red Blood Cell (RBC) (M/ uL)
Mean Corpusculer Vol. (MCV) (fL)
Platelet (K/uL)
White Blood Cells (K/uL)
Basophils (K/uL)
Lymphocytes (K/uL)
Native Thiol (μmol/L)
Total thiol (μmol/L)
Disülfide (μmol/L)
%SS/SH (μmol/L)
%SS/totalSH (μmol/L)
%SH/totalSH (μmol/L)
RESULTS
The median age of the study cases was 59.7
(14.6) years (range 30-83 years) and the
median age of the control cases was 58.2 (13.4)
years (range 43-84). There was no differences
between the median age of the control cases
and the median age of the study cases. Gender
of cases were 18 males and 24 females. The
distribution of the genders was similar in the
control cases and study cases. There were 11
(18.3%) cases with thrombosis history and 6
(10%) cases with hemorrhage history in cases
of PV. There were 38 (90%) cases with JAK
positivity in the PV group. There were 31
(51.7%) cases with splenomegaly in the PV
group. The mean spleen size of the PV group
was measured as 127 (30.8) mm.
Mean native thiol levels (SH), total thiol levels
(SH+SS) and the native thiol/total thiol ratio
(SH/ SH+SS) were higher in PV group
compared to the control group (P< .001, P =
.001 and P = .02, respectively), which is
described in Table 1 and Figure 1.
The basal complete blood counts and white
blood cell differential were compared in PV and
control groups. The age and gender distribution
was similar between the groups. The mean
levels of hemoglobin, white blood cell count
and platelets were statistically significantly
higher in the PV group as expected (Table 1).
59.7 (14.6)
18.2 (2.3)
6.14 (1.25)
93 (12)
500 (295)
12230 (3337)
70 (130)
2098 (714)
471.3 (55.2)
509.2 (61.2)
19.8 (11.4)
4.27 (2.46)
3.82 (2.00)
92.73 (4.32)
Control
(n=43)
59.5 (9.6)
13.2 (1.7)
5,2 (0.98)
89 (8)
350 (185)
7500 (1800)
10 (2.3)
2010 (658)
428.6 (37.8)
470.0 (38.5)
20.7 (6.4)
4.76 (2.66)
4.41 (1.38)
91.31 (4.43)
P
value
0.920
<.001
.165
.321
<.001
<.001
<.001
.402
<.001
.001
.648
.106
.119
.020
Figure 1: Native thiols for PV group and Control
There was no statistically significant difference
between thiol and disulfide values among those
with and without JAK positivity in the PV group.
Those with and without hemorrhage history or
thrombosis history. Among PV cases, cases with
a history of hemorrhage or thrombosis
compared cases with no history of hemorrhage
or thrombosis and, there was no statistically
significant difference between thiol and
disulfide values. There was no statistically
significant correlation between spleen size and
thiol-disulfide parameters of PV group.
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Dicle Tıp Dergisi / Dicle Med J (2019) 46 (2) : 315-320
DISCUSSION
The results of this study have shown that both
native and total thiol levels as well as the
native/ total thiol ratio (SH/SH+SS) are
elevated in PV patients indicating that thiol
disulphide homeostasis has shifted towards
proliferation. To our knowledge, this is the first
study in the literature investigating the thiol
disulphide homeostasis in PV. One study has
demonstrated increased levels of reduced
glutathione (GSH) which is one of the low
molecular weight thiols, in the red blood cells
of patients with myeloproliferative neoplasia
compared with healthy controls. Additionally,
they noted that the elevated GSH containing
compounds is independent of the age of red
blood cell23. Additionally, in another study
demonstrated previous studies which utilized
Erel and Neşelioğlu method, found that plasma
thiol disulphide homeostasis was increased in
degenerative diseases, such as diabetes,
obesity, pneumonia, and in the case of asthma,
whereas it was reduced in proliferative
diseases such as multiple myeloma, renal cell
carcinoma, lung cancer, and renal cancer24-30. In
this study we could not find any differences
between the PV and control groups in terms of
the amount of plasma disulphide bonds. This
indicates clearly that the balance has shifted
towards proliferation.
In summary; thiol-disulphide homeostasis was
enlarged and the balance shifted to reductive
thiol side in the disease. This change can
provocate proliferation status of the disease
and/or may be secondary to the disease.
Conflicts of interest: The authors have no
conflict of interests to declare.
Financial Disclosure: The authors declared
that this study has received no financial
support.
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