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Comparison of the Hypertransferasemic Effects of Erythropoietin and U-74389G on Aspartate Aminotransferase Levels

This study compared the effects of the antioxidant drug U-74389G and the cytokine erythropoietin (Epo) on serum aspartate aminotransferase (AST) levels in a rat model of ischemia-reperfusion injury. Two previous studies found that Epo significantly increased AST levels by 19.73% while U-74389G non-significantly increased AST by 16.23%. This study combined the data from the previous studies and found that U-74389G increased AST levels 0.8224656 times less than Epo, indicating it has a smaller hypertransferasemic (AST-increasing) effect. The antioxidant properties of U-74389G were determined to cause less of an increase in

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Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 16
INTRODUCTION
T
he lazaroid U-74389G (L) may be not famous for
its hypertransferasemic[1]
capacity (P = 0.0583).
U-74389G as a novel antioxidant factor implicates
exactly only 260 published studies. The ischemia-
reperfusion (IR) type of experiments was noted in 18.84%
of these studies. A tissue protective feature of U-74389G
was obvious in these IR studies. The U-74389G chemically
known as 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-
piperazinyl]-pregna-1,4,9(11)-triene-3,20-dione maleate
salt is an antioxidant complex, which prevents the lipid
peroxidation either iron-dependent or arachidonic acid-
induced one. Animals’ kidney, liver, brain microvascular
Comparison of the Hypertransferasemic Effects
of Erythropoietin and U-74389G on Aspartate
Aminotransferase Levels
Constantinos Tsompos1
, Constantinos Panoulis2
, Konstantinos Toutouzas3
,
Aggeliki Triantafyllou4
, George C. Zografos5
, Kalliopi Tsarea6
, Maria Karamperi6
,
Apostolos Papalois6
1
Department of Gynecology, General Hospital of Thessaloniki “St. Dimitrios”, Thessaloniki, Hellas, 2
Department
of Obstetrics and Gynecology, Aretaieion Hospital, Athens University, Athens, Attiki, Hellas, 3
Department of
Surgery, Ippokrateion General Hospital, Athens University, Athens, Attiki, Hellas, 4
Department of Biologic
Chemistry, Athens University, Athens, Attiki, Hellas, 5
Department of Surgery, Ippokrateion General Hospital,
Athens University, Athens, Attiki, Hellas, 6
Experimental Research Centre ELPEN Pharmaceuticals, S.A. Inc., Co.,
Pikermi, Attiki, Hellas
ABSTRACT
Aim: This study calculated the effects on aspartate aminotransferase (AST) levels, after treatment with either of two drugs:
The erythropoietin (Epo) and the antioxidant lazaroid (L) drug U-74389G. The calculation was based on the results of two
preliminary studies, each one of which estimated the certain influence, after the respective drug usage in an induced ischemia-
reperfusion (IR) animal experiment. Materials and Methods: The two main experimental endpoints at which the serum
AST levels were evaluated were the 60th
 reperfusion min (for the Groups A, C, and E) and the 120th
 reperfusion min (for the
Groups B, D, and F). Especially, Groups A and B were processed without drugs, Groups C and D after Epo administration,
whereas Groups E and F after the L administration. Results: The first preliminary study of Epo presented a significant
hypertransferasemic effect by 19.73 ± 7.71% (P = 0.0119). The second preliminary study of U-74389G presented a non-
significant hypertransferasemic effect by 16.23 ± 8.59% (P = 0.0583). These two studies were coevaluated since they came
from the same experimental setting. The outcome of the coevaluation was that L is 0.8224656-fold (0.8211631–0.8237701) less
hypertransferasemic than Epo (P = 0.0000). Conclusions: The antioxidant capacities of U-74389G ascribe 0.8224656-fold less
hypertransferasemic effects than Epo (P = 0.0000).
Key words: Aspartate aminotransferase levels, erythropoietin, ischemia, reperfusion, U-74389G
ORIGINAL ARTICLE
Address for correspondence:
Constantinos Tsompos, Department of Gynecology, General Hospital of Thessaloniki “St. Dimitrios,” 2 Elenis
Zografou Street, Thessaloniki 54634, Hellas. Tel.: 00302313322171/00306946674264. Fax: 00302106811215.
E-mail: Tsomposconstantinos@gmail.com
https://doi.org/10.33309/2639-8354.010205 www.asclepiusopen.com
© 2018 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license.
Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G
 Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018
endothelial cell monolayers, and heart models were protected
by U-74389G after IR injury. U-74389G also attenuates
the leukocytes, downregulates the pro-inflammatory gene,
treats the endotoxin shock, produces cytokine, enhances
the mononuclear immunity, protects the endothelium, and
presents anti-shock property.
Erythropoietin (Epo) even if is not famous for its
hypertransferasemic action (P = 0.4430), it can be used as
a reference drug for comparison with U-74389G. Although
Epo is met in over 30,606 published biomedical studies, only
a 3.57% of them negotiate the known type of IR experiments.
Nevertheless, Epo as a cytokine, it is worth of being studied
about its effects on aspartate aminotransferase levels (AST)
levels too.
This experimental work tried to compare the effects
of the above drugs on a rat-induced IR protocol. They
were tested by calculating the serum AST level (sASTl)
augmentations.
MATERIALS AND METHODS
Animal preparation
The Vet licenses under 3693/12-11- 2010 and 14/10-1-2012
numbers, the granting company, and the experiment location
are mentioned in preliminary references.[1,2]
The human animal
care of albino female Wistar rats, 7 days’ pre-experimental
ad libitum diet, non-stop intraexperimental anesthesiologic
techniques, acidometry, electrocardiogram, oxygen supply, and
post-experimental euthanasia are also described in preliminary
references. Rats were 16–18 weeks old. They were randomly
assigned to six (6) groups consisted of n = 10. The stage of
45 min hypoxia was common for all six groups. Afterward,
reperfusion of 60 min was followed in Group A; reperfusion
of 120 min in Group B; immediate Epo intravenous (IV)
administration and reperfusion of 60 min in Group C;
immediate Epo IV administration and reperfusion of 120 min
in Group D; immediate U-74389G IV administration and
reperfusion of 60 min in Group E; and immediate U-74389G
IV administration and reperfusion of 120 min in Group F.
The dose height assessment for both drugs is described at
preliminary studies as 10 mg/kg body mass.
Ischemia was caused by laparotomic clamping the inferior
aorta over renal arteries with forceps for 45 min. The
clamp removal was restoring the inferior aorta patency
and reperfusion. After exclusion of the blood flow, the
protocol of IR was applied, as described above for each
experimental group. The drugs were administered at the
time of reperfusion, through inferior vena cava catheter. The
ASTl was determined at 60th
 min of reperfusion (for A, C,
and E) and 120th
 min of reperfusion (for Groups B, D and F).
Along, a weak relation was rised between ASTl values with
animals’mass (P = 0.2154), so it was no use of calculating the
predicted ASTl values for the animals’ mass.
Statistical analysis
Table 1 presents the (%) hypertransferasemic influence of
Epo regarding reoxygenation time. Furthermore, Table 2
presents the (%) hypertransferasemic influence of U-74389G
regarding reperfusion time. Chi-square tests were applied
using the ratios which produced the (%) results per endpoint.
The outcomes of Chi-square tests are depicted in Table 3.
The statistical analysis was performed by Stata 6.0 software
(Stata 6.0, StataCorp LP, Texas, USA).
Table 1: The % hypertransferasemic influence of erythropoietin in connection with reperfusion time
Hypertransferasemia value (%) ±SD (%) Reperfusion time P value
+29.53 ±31.07 1 h 0.0096
+26.71 ±36.79 1.5 h 0.0235
+23.89 ±39.90 2 h 0.1709
+27.58 ±47.72 Reperfusion 0.0271
+19.73 ±7.71 Interaction 0.0119
SD: Standard deviation
Table 2: The % hypertransferasemic influence of U‑74389G in connection with reperfusion time
Hypertransferasemia (%) ±SD (%) Reperfusion time P value
+33.94 ±44.36 1 h 0.0328
+24.97 ±50.25 1.5 h 0.0593
+16% ±51.91 2 h 0.4077
+21.05 ±64.76 Reperfusion 0.1524
+16.23 ±8.59 Interaction 0.0583
SD: Standard deviation
Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G
Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 18
RESULTS
The successive application of Chi-square tests revealed
that U-74389G augmented the ASTl by 1.149264-
fold (1.006956–1.311683) more than Epo at 1 h
(P = 0.0391), by 0.9347365-fold (0.9334662–0.9360085)
less than Epo at 1.5 h (P = 0.0000), by 0.6695775-fold
(0.5800545–0.7729184) less than Epo at 2 h (P = 0.0000), less
by 0.7631082 (0.7620376–0.7641804) (P = 0.0000) without
drugs, and by 0.8224656-fold (0.8211631–0.8237701)
less than Epo whether all variables have been
considered (P = 0.0000).
DISCUSSION
The unique available study investigating the
hypertransferasemic effect of U-74389G on ASTl was the
preliminary one.[1]
Apart from the most famous activities this
drug has as the neuroprotection and membrane-stabilization
properties, it also accumulates in the cell membrane, protects
the vascular endothelium from peroxidative damage, but
hardly penetrates the blood-brain barrier. It elicits a beneficial
effect in ototoxicity and Duchenne muscular dystrophy. It
increases γgt, superoxide dismutase, and glutathione levels
in oxygen-exposed cells. It treats septic states and acts as
immunosuppressant in flap survival. It prevents the learning
impairments; it delays the early synaptic transmission decay
during hypoxia improving energetic state of neurons. It
shows antiproliferative properties on brain cancer cells and
is considered as a new promising anti-inflammatory drug for
the treatment of reperfusion syndrome in IR injuries.
The same authors confirmed[2]
the short-term
hypertransferasemic effect of Epo preparations in non-iron-
deficient individuals. Li et al. demonstrated[3]
that EPO may
play a protective role against LPS-induced multiple organ
failure by reducing the inflammatory response and tissue
degeneration, possibly through the phosphatidylinositol
3-kinase/AKT and NF-κB signaling pathways restoring the
LPS-induced ASTl in rats. Fu et al. examined[4]
the protective
effect of rHuEPO which is mediated through the activation
of the phosphatidylinositol-3 kinase/AKT/endothelial nitric
oxide synthase (eNOS) signaling pathway, at least in part, by
increasing p-AKT and p-eNOS, and leads to the maintenance
of an elevated level of NO in I/R injury of the liver. Yildar
et al. investigated[5]
the protective effect of 2-aminoethyl
diphenylborinate which significantly reduced the sAST in the
rat kidney I/R injury group. Bayramoglu et al. examined[6]
the use of lycopene, while improvements of the AST values
were partial and dose-dependent (P  0.05) in IR injury of rat
liver. Liu et al. showed[7]
that quercetin significantly reduced
apoptosis rate, improved cardiac function, and decreased the
levels of AST, by inhibiting apoptosis in vivo and PI3K/AKT
pathway involved in the anti-apoptotic effect in SD rats in
myocardial IR injuries in vivo. Bayramoglu et al. noticed[8]
that gallic acid significantly decreased the AST activities in
tissue homogenates than no treatment group (P  0.05) in
oxidative stress generated by hepatic I/R-induced control
rats. Matsuno et al. found[9]
the ASTl in the control versus
hydrogen group in 30, 60, and 120 min after reperfusion
being more by 8.9%, 9.59%, and 3.54%, respectively, in
a porcine liver reperfusion injury. Saïdi et al. compared[10]
the control group with animals treated with tilapia fish oil
which experienced a significant decrease (P  0.05) in ASTl
in reperfusion periods in male Wistar rats subjected to warm
liver IRI. Li et al. significantly ameliorated[11]
the I/R injury
of the liver after Mino treatment, as shown by decreased
Suzuki scores and liver function AST in male Sprague-
Dawley rats liver. Ozkan et al. concluded[12]
that hypothermic
reperfusion and O3
preconditioning might be beneficial in
skeletal muscle IR injury since the ASTl were decreased
than those in the IR group in the rats. Hu et al. found[13]
the
AST activities declined (P  0.05) in tanshinone IIA (TSA)
L-TSA, M-TSA, and H-TSA rat myocardial ischemia groups.
Cahova et al. indicated[14]
that metformin treatment prevented
an acute stress-induced necroinflammatory reaction, reduced
AST serum activity, diminished lipoperoxidation, and
reduced mitochondrial performance but concomitantly
protected the liver from I/R-induced injury in Wistar rats.
Yildiz et al. showed[15]
a decrease in ASTl in the micronized
purified flavonoid fraction-treated rats than hepatic I/R
group rats (P  0.001 for all). Zhang et al. indicated[16]
that
hydrogen inhalation at 2% concentration for 1 h before
liver syngeneic orthotopic transplantation protected from
ischemia/reperfusion injury by activation of the NF-κB
signaling pathway, reducing the sAST activities in rats. Lee
et al. found[17]
hepatocytes AlbCre+/constitutively active
nuclear factor (erythroid-derived 2)-like 2 (caNrf2)+ having
significantly reduced serum transaminases than wild-type
littermate controls in warm hepatic murine IRI. Tang et al.
regarded[18]
Dioscorea nipponica Makino, D. panthaica Prain
et Burkill (DP), and D. zingiberensis C.H. Wright (DZ) as
having the same traditional therapeutic actions, such as TS
Table 3: The U‑74389G/erythropoietin efficacy ratios
on serum aspartate aminotransferase levels after
Chi‑square test application
Odds ratio (95% Conf. interval) P values Endpoint
1.149264 1.006956
1.311683
0.0391 1 h
0.9347365 0.9334662
0.9360085
0.0000 1.5 h
0.6695775 0.5800545
0.7729184
0.0000 2 h
0.7631082 0.7620376
0.7641804
0.0000 Reperfusion
0.8224656 0.8211631
0.8237701
0.0000 Interaction
Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G
19 Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018
Table 4:
A U‑74389G/erythropoietin
efficacy
ratio
meta‑analysis
on
20
hematologic
variables (16
variables
with
balancing
efficacies
and
4
variables
with
opposite
efficacies)
[39,40]
Variable
Endpoint
1
h
P
value
1.5
h
P
value
2
h
P
value
Reperfusion
time
P
value
Interaction
P
value
Variable
WBC
0.957451
0.3782
1.396122
0.0000
1.918237
0.0000
1.71622
0.0000
1.601887
0.0000
RBC
count
0.961059
0.0000
1.733395
0.0000
6.519657
0.0000
1.039524
0.0000
1.309673
0.0000
Hematocrit
38.424
0.0000
9.076658
0.0000
6.222898
0.0000
1.001356
0.2184
12.66419
0.0000
Hemoglobin
1.268689
0.0000
1.839035
0.0000
13.1658
0.0000
1.252422
0.0000
1.94889
0.0000
MCH
151.125
0.0000
4.246814
0.0000
2.709729
0.0000
1.177347
0.0000
4.362893
0.0000
MCV
150.8518
0.0000
4.236722
0.0000
2.704247
0.0000
1.180156
0.0000
4.352528
0.0000
RbcDW
3.306773
0.0000
3.023389
0.0000
2.655885
0.0000
0.2259914
0.0000
2.370353
0.0000
Platelet
count
2.42839
0.0000
6.00238
0.0000
6.1333429
0.0000
3.939027
0.0000
37.62979
0.0000
MPV
145.8532
0.0000
4.053619
0.0000
2.603947
0.0000
1.2334644
0.0000
4.164431
0.0000
Platelet
DW
0.6940233
0.0000
1.319118
0.0000
2.206972
0.0000
2.2484006
0.0000
2.458888
0.0000
Glucose
156.4991
0.0000
4.53659
0.0000
2.81397
0.0000
0.9073196
0.0000
4.660603
0.0000
Urea
158.4209
0.0000
4.50889
0.0000
2.850291
0.0000
0.9017775
0.0000
4.632148
0.0000
Creatinine
168.9034
0.0000
4.872332
0.0000
3.039572
0.0000
1.0262016
0.0000
5.005523
0.0000
Total
proteins
155.9562
0.0000
4.421079
0.0000
2.803573
0.0000
0.8842162
0.0000
4.541934
0.0000
Albumins
0.2457507
0.0073
0.5303472
0.0000
0.6243052
0.0465
1.237477
0.0000
0.5000416
0.0000
ALP
134.0033
0.0000
3.602703
0.0000
2.349961
0.0000
0.7205412
0.0000
3.701187
0.0000
Mean
15.968668
0.0239
3.060916
0.0000
3.093882
0.0028
1.10692926
0.0135
3.6055662
0.0000
Mean
corpuscular
hemoglobin
concentrations
−0.2774225
0.0000
−0.5504722
0.0000
−8522433
0.0000
+3.044774
0.0000
−0.7793243
0.0000
Platelet
crit.
−0.2312044
0.0000
−0.6719365
0.0000
−1.330756
0.0886
+5.620077
0.0000
−0.9771515
0.0000
ALT
+0.5955473
0.0000
−1.157335
0.0000
+7.967324
0.0000
+0.4734427
0.0000
−0.6208232
0.0000
γGT
1
1.0000
+0.5367033
0.0000
−0.9428571
0.8982
+2.146813
0.0000
−0.2683513
0.0000
Mean
−0.4757810
0.0250
−0.9450332
0.0000
−0.6052695
0.2467
+2.0421598
0.0000
−0.5968125
0.0000
Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G
Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 20
groups exhibiting significantly reduced activities ofAST than
the model group (ISO injection only). Yucel et al. noticed
that infliximab significantly reduced[19]
the ASTl in IR liver.
Deng et al. significantly reduced the levels of AST[20]
in the
MT group at each time point than that of the liver function
IR group (P  0.05) in male Sprague-Dawley rats. Lee et al.
acknowledged that[21]
treatment with eupatilin significantly
decreased sAST as well as liver histologic changes in acute
IR-induced hepatic damage. Ozsoy et al. found remarkably
higher sASTl[22]
in IR group than the sham group and the
laboratory tests returned to normal levels in IR+melatonin
(MEL) group after MEL treatment of the hepatic tissue.
Bektas et al. found sAST[23]
significantly different in tadalafil
and pentoxifylline groups than liver IR group. Younis
et al. investigated[24]
the Silymarin preconditioning which
decreased AST and improved hepatic architecture in HIR
injury of insulin-resistant rats. Abdelsameea et al. pretreated
with liraglutide which decreased[25]
AST activities with
attenuation of necrosis and inflammation while enhanced
Bcl-2 expression in liver IR male rats. Liu et al. shown[26]
that
the formula of Guanxin Dhutong capsule which has four main
active ingredients, protocatechuic acid, cryptotanshinone,
borneol, and eugenol, diminished the infarct size and reduced
ASTl in vivo in a rat model of the coronary artery. Hao et al.
noticed that propofol significantly reduced[27]
the activities of
sAST in QSG-7701 cells in vitro of hepatic I/R injury in rats.
Wang et al. revealed[28]
that 2’-O-galloylhyperin effectively
ameliorated CCl4
-induced hepatic damage by reducing AST
activitiesinananimalmodel.Saidietal.inventedthatPistacia
lentiscus oil decreased[29]
both the visible severe intestinal
damage and the sASTl in intestinal IR. Cai et al. pretreated
with salidroside (20 mg/kg/day for 7 days, intraperitoneally)
which significantly decreased[30]
sAST levels after 6 h and
24 h of reperfusion and protected the segmental (70%) warm
hepatic liver against I/R-induced injury. Wang et al. found[31]
that flavonol glycosides which are in high concentrations in
Inula racemosa altered cellular morphology, and cytokines
and AST were returned to near normal level in hepatic
I/R injury. Ardasheva et al. elevated the intra-abdominal
pressure (IAP) above 20 mmHg which led[32]
to progression
of abdominal compartment syndrome that is associated with
organ dysfunction or failure and elevated activities of AST
found in some of the experimental groups than control ones
of animals not subjected to increased IAP and time frame
tested for liver. Zazueta et al. found[33]
that citicoline CDP-
choline reduced ASTl in blood samples from reperfused rats
in I/R rat livers. Miyauchi et al. evaluated the levels of sAST
as well as the scores of liver necrosis[34]
significantly lower
in the antioxidative nutrient-rich enteral diet (Ao diet) group
than the control diet group in liver IR mice. Zhang et al.
showed that[35]
the role of hydrogen-rich saline treatment
significantly decreased serum levels of AST activity and
TUNEL-positive cells in miniature pig model of laparoscopic
HIRI on hepatectomy. Chen et al. observed[36]
significantly
decreased (all P  0.05) the levels of AST in the rosiglitazone
group than the HIRI group in a rat model of hepatic ischemia-
reperfusion injury. Gholampour et al. treated[37]
with remote
ischemic perconditioning and quercetin which reduced
plasma AST activity in the liver after renal I/R. Brandão et al.
observed[38]
separately the protective effects of allopurinol
and the benefits of ischemic post-conditioning by measuring
ASTl with significantly reduced I/R systemic injuries in rats.
According to above, Table 3 shows that U-74389G
has 0.8224656-fold (0.8211631–0.8237701) less
hypertransferasemic effect than Epo (P = 0.0000) whether
all variables have been considered (P = 0.0000), a trend
attenuated along time, in Epo non-deficient rats. A meta-
analysis of these ratios from the same experiment, for 20 other
seric variables, provides comparable results [Table 4].[39,40]
CONCLUSION
The antioxidant agent U-74389G was proved
having 0.8224656-fold (0.8211631–0.8237701) less
hypertransferasemic effect than Epo whether all variables
have been considered (P = 0.0000); a trend attenuated
along the short-term time frame of the experiment in rats.
A biochemical investigation remains about how U-74389G
mediates in these actions.
ACKNOWLEDGMENT
This study was funded by Scholarship by the Experimental
Research Center ELPEN Pharmaceuticals (E.R.C.E), Athens,
Greece. The research facilities for this project were provided
by the aforementioned institution.
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Comparison of the Hypertransferasemic Effects of Erythropoietin and U-74389G on Aspartate Aminotransferase Levels

  • 1. Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 16 INTRODUCTION T he lazaroid U-74389G (L) may be not famous for its hypertransferasemic[1] capacity (P = 0.0583). U-74389G as a novel antioxidant factor implicates exactly only 260 published studies. The ischemia- reperfusion (IR) type of experiments was noted in 18.84% of these studies. A tissue protective feature of U-74389G was obvious in these IR studies. The U-74389G chemically known as 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1- piperazinyl]-pregna-1,4,9(11)-triene-3,20-dione maleate salt is an antioxidant complex, which prevents the lipid peroxidation either iron-dependent or arachidonic acid- induced one. Animals’ kidney, liver, brain microvascular Comparison of the Hypertransferasemic Effects of Erythropoietin and U-74389G on Aspartate Aminotransferase Levels Constantinos Tsompos1 , Constantinos Panoulis2 , Konstantinos Toutouzas3 , Aggeliki Triantafyllou4 , George C. Zografos5 , Kalliopi Tsarea6 , Maria Karamperi6 , Apostolos Papalois6 1 Department of Gynecology, General Hospital of Thessaloniki “St. Dimitrios”, Thessaloniki, Hellas, 2 Department of Obstetrics and Gynecology, Aretaieion Hospital, Athens University, Athens, Attiki, Hellas, 3 Department of Surgery, Ippokrateion General Hospital, Athens University, Athens, Attiki, Hellas, 4 Department of Biologic Chemistry, Athens University, Athens, Attiki, Hellas, 5 Department of Surgery, Ippokrateion General Hospital, Athens University, Athens, Attiki, Hellas, 6 Experimental Research Centre ELPEN Pharmaceuticals, S.A. Inc., Co., Pikermi, Attiki, Hellas ABSTRACT Aim: This study calculated the effects on aspartate aminotransferase (AST) levels, after treatment with either of two drugs: The erythropoietin (Epo) and the antioxidant lazaroid (L) drug U-74389G. The calculation was based on the results of two preliminary studies, each one of which estimated the certain influence, after the respective drug usage in an induced ischemia- reperfusion (IR) animal experiment. Materials and Methods: The two main experimental endpoints at which the serum AST levels were evaluated were the 60th  reperfusion min (for the Groups A, C, and E) and the 120th  reperfusion min (for the Groups B, D, and F). Especially, Groups A and B were processed without drugs, Groups C and D after Epo administration, whereas Groups E and F after the L administration. Results: The first preliminary study of Epo presented a significant hypertransferasemic effect by 19.73 ± 7.71% (P = 0.0119). The second preliminary study of U-74389G presented a non- significant hypertransferasemic effect by 16.23 ± 8.59% (P = 0.0583). These two studies were coevaluated since they came from the same experimental setting. The outcome of the coevaluation was that L is 0.8224656-fold (0.8211631–0.8237701) less hypertransferasemic than Epo (P = 0.0000). Conclusions: The antioxidant capacities of U-74389G ascribe 0.8224656-fold less hypertransferasemic effects than Epo (P = 0.0000). Key words: Aspartate aminotransferase levels, erythropoietin, ischemia, reperfusion, U-74389G ORIGINAL ARTICLE Address for correspondence: Constantinos Tsompos, Department of Gynecology, General Hospital of Thessaloniki “St. Dimitrios,” 2 Elenis Zografou Street, Thessaloniki 54634, Hellas. Tel.: 00302313322171/00306946674264. Fax: 00302106811215. E-mail: Tsomposconstantinos@gmail.com https://doi.org/10.33309/2639-8354.010205 www.asclepiusopen.com © 2018 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license.
  • 2. Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 endothelial cell monolayers, and heart models were protected by U-74389G after IR injury. U-74389G also attenuates the leukocytes, downregulates the pro-inflammatory gene, treats the endotoxin shock, produces cytokine, enhances the mononuclear immunity, protects the endothelium, and presents anti-shock property. Erythropoietin (Epo) even if is not famous for its hypertransferasemic action (P = 0.4430), it can be used as a reference drug for comparison with U-74389G. Although Epo is met in over 30,606 published biomedical studies, only a 3.57% of them negotiate the known type of IR experiments. Nevertheless, Epo as a cytokine, it is worth of being studied about its effects on aspartate aminotransferase levels (AST) levels too. This experimental work tried to compare the effects of the above drugs on a rat-induced IR protocol. They were tested by calculating the serum AST level (sASTl) augmentations. MATERIALS AND METHODS Animal preparation The Vet licenses under 3693/12-11- 2010 and 14/10-1-2012 numbers, the granting company, and the experiment location are mentioned in preliminary references.[1,2] The human animal care of albino female Wistar rats, 7 days’ pre-experimental ad libitum diet, non-stop intraexperimental anesthesiologic techniques, acidometry, electrocardiogram, oxygen supply, and post-experimental euthanasia are also described in preliminary references. Rats were 16–18 weeks old. They were randomly assigned to six (6) groups consisted of n = 10. The stage of 45 min hypoxia was common for all six groups. Afterward, reperfusion of 60 min was followed in Group A; reperfusion of 120 min in Group B; immediate Epo intravenous (IV) administration and reperfusion of 60 min in Group C; immediate Epo IV administration and reperfusion of 120 min in Group D; immediate U-74389G IV administration and reperfusion of 60 min in Group E; and immediate U-74389G IV administration and reperfusion of 120 min in Group F. The dose height assessment for both drugs is described at preliminary studies as 10 mg/kg body mass. Ischemia was caused by laparotomic clamping the inferior aorta over renal arteries with forceps for 45 min. The clamp removal was restoring the inferior aorta patency and reperfusion. After exclusion of the blood flow, the protocol of IR was applied, as described above for each experimental group. The drugs were administered at the time of reperfusion, through inferior vena cava catheter. The ASTl was determined at 60th  min of reperfusion (for A, C, and E) and 120th  min of reperfusion (for Groups B, D and F). Along, a weak relation was rised between ASTl values with animals’mass (P = 0.2154), so it was no use of calculating the predicted ASTl values for the animals’ mass. Statistical analysis Table 1 presents the (%) hypertransferasemic influence of Epo regarding reoxygenation time. Furthermore, Table 2 presents the (%) hypertransferasemic influence of U-74389G regarding reperfusion time. Chi-square tests were applied using the ratios which produced the (%) results per endpoint. The outcomes of Chi-square tests are depicted in Table 3. The statistical analysis was performed by Stata 6.0 software (Stata 6.0, StataCorp LP, Texas, USA). Table 1: The % hypertransferasemic influence of erythropoietin in connection with reperfusion time Hypertransferasemia value (%) ±SD (%) Reperfusion time P value +29.53 ±31.07 1 h 0.0096 +26.71 ±36.79 1.5 h 0.0235 +23.89 ±39.90 2 h 0.1709 +27.58 ±47.72 Reperfusion 0.0271 +19.73 ±7.71 Interaction 0.0119 SD: Standard deviation Table 2: The % hypertransferasemic influence of U‑74389G in connection with reperfusion time Hypertransferasemia (%) ±SD (%) Reperfusion time P value +33.94 ±44.36 1 h 0.0328 +24.97 ±50.25 1.5 h 0.0593 +16% ±51.91 2 h 0.4077 +21.05 ±64.76 Reperfusion 0.1524 +16.23 ±8.59 Interaction 0.0583 SD: Standard deviation
  • 3. Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 18 RESULTS The successive application of Chi-square tests revealed that U-74389G augmented the ASTl by 1.149264- fold (1.006956–1.311683) more than Epo at 1 h (P = 0.0391), by 0.9347365-fold (0.9334662–0.9360085) less than Epo at 1.5 h (P = 0.0000), by 0.6695775-fold (0.5800545–0.7729184) less than Epo at 2 h (P = 0.0000), less by 0.7631082 (0.7620376–0.7641804) (P = 0.0000) without drugs, and by 0.8224656-fold (0.8211631–0.8237701) less than Epo whether all variables have been considered (P = 0.0000). DISCUSSION The unique available study investigating the hypertransferasemic effect of U-74389G on ASTl was the preliminary one.[1] Apart from the most famous activities this drug has as the neuroprotection and membrane-stabilization properties, it also accumulates in the cell membrane, protects the vascular endothelium from peroxidative damage, but hardly penetrates the blood-brain barrier. It elicits a beneficial effect in ototoxicity and Duchenne muscular dystrophy. It increases γgt, superoxide dismutase, and glutathione levels in oxygen-exposed cells. It treats septic states and acts as immunosuppressant in flap survival. It prevents the learning impairments; it delays the early synaptic transmission decay during hypoxia improving energetic state of neurons. It shows antiproliferative properties on brain cancer cells and is considered as a new promising anti-inflammatory drug for the treatment of reperfusion syndrome in IR injuries. The same authors confirmed[2] the short-term hypertransferasemic effect of Epo preparations in non-iron- deficient individuals. Li et al. demonstrated[3] that EPO may play a protective role against LPS-induced multiple organ failure by reducing the inflammatory response and tissue degeneration, possibly through the phosphatidylinositol 3-kinase/AKT and NF-κB signaling pathways restoring the LPS-induced ASTl in rats. Fu et al. examined[4] the protective effect of rHuEPO which is mediated through the activation of the phosphatidylinositol-3 kinase/AKT/endothelial nitric oxide synthase (eNOS) signaling pathway, at least in part, by increasing p-AKT and p-eNOS, and leads to the maintenance of an elevated level of NO in I/R injury of the liver. Yildar et al. investigated[5] the protective effect of 2-aminoethyl diphenylborinate which significantly reduced the sAST in the rat kidney I/R injury group. Bayramoglu et al. examined[6] the use of lycopene, while improvements of the AST values were partial and dose-dependent (P 0.05) in IR injury of rat liver. Liu et al. showed[7] that quercetin significantly reduced apoptosis rate, improved cardiac function, and decreased the levels of AST, by inhibiting apoptosis in vivo and PI3K/AKT pathway involved in the anti-apoptotic effect in SD rats in myocardial IR injuries in vivo. Bayramoglu et al. noticed[8] that gallic acid significantly decreased the AST activities in tissue homogenates than no treatment group (P 0.05) in oxidative stress generated by hepatic I/R-induced control rats. Matsuno et al. found[9] the ASTl in the control versus hydrogen group in 30, 60, and 120 min after reperfusion being more by 8.9%, 9.59%, and 3.54%, respectively, in a porcine liver reperfusion injury. Saïdi et al. compared[10] the control group with animals treated with tilapia fish oil which experienced a significant decrease (P 0.05) in ASTl in reperfusion periods in male Wistar rats subjected to warm liver IRI. Li et al. significantly ameliorated[11] the I/R injury of the liver after Mino treatment, as shown by decreased Suzuki scores and liver function AST in male Sprague- Dawley rats liver. Ozkan et al. concluded[12] that hypothermic reperfusion and O3 preconditioning might be beneficial in skeletal muscle IR injury since the ASTl were decreased than those in the IR group in the rats. Hu et al. found[13] the AST activities declined (P 0.05) in tanshinone IIA (TSA) L-TSA, M-TSA, and H-TSA rat myocardial ischemia groups. Cahova et al. indicated[14] that metformin treatment prevented an acute stress-induced necroinflammatory reaction, reduced AST serum activity, diminished lipoperoxidation, and reduced mitochondrial performance but concomitantly protected the liver from I/R-induced injury in Wistar rats. Yildiz et al. showed[15] a decrease in ASTl in the micronized purified flavonoid fraction-treated rats than hepatic I/R group rats (P 0.001 for all). Zhang et al. indicated[16] that hydrogen inhalation at 2% concentration for 1 h before liver syngeneic orthotopic transplantation protected from ischemia/reperfusion injury by activation of the NF-κB signaling pathway, reducing the sAST activities in rats. Lee et al. found[17] hepatocytes AlbCre+/constitutively active nuclear factor (erythroid-derived 2)-like 2 (caNrf2)+ having significantly reduced serum transaminases than wild-type littermate controls in warm hepatic murine IRI. Tang et al. regarded[18] Dioscorea nipponica Makino, D. panthaica Prain et Burkill (DP), and D. zingiberensis C.H. Wright (DZ) as having the same traditional therapeutic actions, such as TS Table 3: The U‑74389G/erythropoietin efficacy ratios on serum aspartate aminotransferase levels after Chi‑square test application Odds ratio (95% Conf. interval) P values Endpoint 1.149264 1.006956 1.311683 0.0391 1 h 0.9347365 0.9334662 0.9360085 0.0000 1.5 h 0.6695775 0.5800545 0.7729184 0.0000 2 h 0.7631082 0.7620376 0.7641804 0.0000 Reperfusion 0.8224656 0.8211631 0.8237701 0.0000 Interaction
  • 4. Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G 19 Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 Table 4: A U‑74389G/erythropoietin efficacy ratio meta‑analysis on 20 hematologic variables (16 variables with balancing efficacies and 4 variables with opposite efficacies) [39,40] Variable Endpoint 1 h P value 1.5 h P value 2 h P value Reperfusion time P value Interaction P value Variable WBC 0.957451 0.3782 1.396122 0.0000 1.918237 0.0000 1.71622 0.0000 1.601887 0.0000 RBC count 0.961059 0.0000 1.733395 0.0000 6.519657 0.0000 1.039524 0.0000 1.309673 0.0000 Hematocrit 38.424 0.0000 9.076658 0.0000 6.222898 0.0000 1.001356 0.2184 12.66419 0.0000 Hemoglobin 1.268689 0.0000 1.839035 0.0000 13.1658 0.0000 1.252422 0.0000 1.94889 0.0000 MCH 151.125 0.0000 4.246814 0.0000 2.709729 0.0000 1.177347 0.0000 4.362893 0.0000 MCV 150.8518 0.0000 4.236722 0.0000 2.704247 0.0000 1.180156 0.0000 4.352528 0.0000 RbcDW 3.306773 0.0000 3.023389 0.0000 2.655885 0.0000 0.2259914 0.0000 2.370353 0.0000 Platelet count 2.42839 0.0000 6.00238 0.0000 6.1333429 0.0000 3.939027 0.0000 37.62979 0.0000 MPV 145.8532 0.0000 4.053619 0.0000 2.603947 0.0000 1.2334644 0.0000 4.164431 0.0000 Platelet DW 0.6940233 0.0000 1.319118 0.0000 2.206972 0.0000 2.2484006 0.0000 2.458888 0.0000 Glucose 156.4991 0.0000 4.53659 0.0000 2.81397 0.0000 0.9073196 0.0000 4.660603 0.0000 Urea 158.4209 0.0000 4.50889 0.0000 2.850291 0.0000 0.9017775 0.0000 4.632148 0.0000 Creatinine 168.9034 0.0000 4.872332 0.0000 3.039572 0.0000 1.0262016 0.0000 5.005523 0.0000 Total proteins 155.9562 0.0000 4.421079 0.0000 2.803573 0.0000 0.8842162 0.0000 4.541934 0.0000 Albumins 0.2457507 0.0073 0.5303472 0.0000 0.6243052 0.0465 1.237477 0.0000 0.5000416 0.0000 ALP 134.0033 0.0000 3.602703 0.0000 2.349961 0.0000 0.7205412 0.0000 3.701187 0.0000 Mean 15.968668 0.0239 3.060916 0.0000 3.093882 0.0028 1.10692926 0.0135 3.6055662 0.0000 Mean corpuscular hemoglobin concentrations −0.2774225 0.0000 −0.5504722 0.0000 −8522433 0.0000 +3.044774 0.0000 −0.7793243 0.0000 Platelet crit. −0.2312044 0.0000 −0.6719365 0.0000 −1.330756 0.0886 +5.620077 0.0000 −0.9771515 0.0000 ALT +0.5955473 0.0000 −1.157335 0.0000 +7.967324 0.0000 +0.4734427 0.0000 −0.6208232 0.0000 γGT 1 1.0000 +0.5367033 0.0000 −0.9428571 0.8982 +2.146813 0.0000 −0.2683513 0.0000 Mean −0.4757810 0.0250 −0.9450332 0.0000 −0.6052695 0.2467 +2.0421598 0.0000 −0.5968125 0.0000
  • 5. Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 20 groups exhibiting significantly reduced activities ofAST than the model group (ISO injection only). Yucel et al. noticed that infliximab significantly reduced[19] the ASTl in IR liver. Deng et al. significantly reduced the levels of AST[20] in the MT group at each time point than that of the liver function IR group (P 0.05) in male Sprague-Dawley rats. Lee et al. acknowledged that[21] treatment with eupatilin significantly decreased sAST as well as liver histologic changes in acute IR-induced hepatic damage. Ozsoy et al. found remarkably higher sASTl[22] in IR group than the sham group and the laboratory tests returned to normal levels in IR+melatonin (MEL) group after MEL treatment of the hepatic tissue. Bektas et al. found sAST[23] significantly different in tadalafil and pentoxifylline groups than liver IR group. Younis et al. investigated[24] the Silymarin preconditioning which decreased AST and improved hepatic architecture in HIR injury of insulin-resistant rats. Abdelsameea et al. pretreated with liraglutide which decreased[25] AST activities with attenuation of necrosis and inflammation while enhanced Bcl-2 expression in liver IR male rats. Liu et al. shown[26] that the formula of Guanxin Dhutong capsule which has four main active ingredients, protocatechuic acid, cryptotanshinone, borneol, and eugenol, diminished the infarct size and reduced ASTl in vivo in a rat model of the coronary artery. Hao et al. noticed that propofol significantly reduced[27] the activities of sAST in QSG-7701 cells in vitro of hepatic I/R injury in rats. Wang et al. revealed[28] that 2’-O-galloylhyperin effectively ameliorated CCl4 -induced hepatic damage by reducing AST activitiesinananimalmodel.Saidietal.inventedthatPistacia lentiscus oil decreased[29] both the visible severe intestinal damage and the sASTl in intestinal IR. Cai et al. pretreated with salidroside (20 mg/kg/day for 7 days, intraperitoneally) which significantly decreased[30] sAST levels after 6 h and 24 h of reperfusion and protected the segmental (70%) warm hepatic liver against I/R-induced injury. Wang et al. found[31] that flavonol glycosides which are in high concentrations in Inula racemosa altered cellular morphology, and cytokines and AST were returned to near normal level in hepatic I/R injury. Ardasheva et al. elevated the intra-abdominal pressure (IAP) above 20 mmHg which led[32] to progression of abdominal compartment syndrome that is associated with organ dysfunction or failure and elevated activities of AST found in some of the experimental groups than control ones of animals not subjected to increased IAP and time frame tested for liver. Zazueta et al. found[33] that citicoline CDP- choline reduced ASTl in blood samples from reperfused rats in I/R rat livers. Miyauchi et al. evaluated the levels of sAST as well as the scores of liver necrosis[34] significantly lower in the antioxidative nutrient-rich enteral diet (Ao diet) group than the control diet group in liver IR mice. Zhang et al. showed that[35] the role of hydrogen-rich saline treatment significantly decreased serum levels of AST activity and TUNEL-positive cells in miniature pig model of laparoscopic HIRI on hepatectomy. Chen et al. observed[36] significantly decreased (all P 0.05) the levels of AST in the rosiglitazone group than the HIRI group in a rat model of hepatic ischemia- reperfusion injury. Gholampour et al. treated[37] with remote ischemic perconditioning and quercetin which reduced plasma AST activity in the liver after renal I/R. Brandão et al. observed[38] separately the protective effects of allopurinol and the benefits of ischemic post-conditioning by measuring ASTl with significantly reduced I/R systemic injuries in rats. According to above, Table 3 shows that U-74389G has 0.8224656-fold (0.8211631–0.8237701) less hypertransferasemic effect than Epo (P = 0.0000) whether all variables have been considered (P = 0.0000), a trend attenuated along time, in Epo non-deficient rats. A meta- analysis of these ratios from the same experiment, for 20 other seric variables, provides comparable results [Table 4].[39,40] CONCLUSION The antioxidant agent U-74389G was proved having 0.8224656-fold (0.8211631–0.8237701) less hypertransferasemic effect than Epo whether all variables have been considered (P = 0.0000); a trend attenuated along the short-term time frame of the experiment in rats. A biochemical investigation remains about how U-74389G mediates in these actions. ACKNOWLEDGMENT This study was funded by Scholarship by the Experimental Research Center ELPEN Pharmaceuticals (E.R.C.E), Athens, Greece. The research facilities for this project were provided by the aforementioned institution. REFERENCES 1. Τsompos C, Panoulis C, Τοutouzas K, Triantafyllou A, Ζografos G, Papalois A. The effect of the antioxidant drug “U-74389g” on aspartate aminotransferase levels during ischemia reperfusion injury in rats. J Biotechnol Sci Res 2017;4:198-203. 2. 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  • 7. Τsompos, et al.: Hypertransferasemic comparison of Erythropoietin and U-74389G Clinical Research in Hematology  •  Vol 1  •  Issue 2  •  2018 22 hydrogen-rich saline on apoptosis induced by hepatic ischemia reperfusion upon laparoscopic hepatectomy in miniature pigs. Res Vet Sci 2018;119:285-91. 36. Chen J, Liu H, Zhang X. Protective effects of rosiglitazone on hepatic ischemia reperfusion injury in rats. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2018;43:732-7. 37. Gholampour F, Sadidi Z. Hepatorenal protection during renal ischemia by quercetin and remote ischemic perconditioning. J Surg Res 2018;231:224-33. 38. Brandão RI, Gomes RZ, Lopes L, Linhares FS, Vellosa JC, Paludo KS, et al. Remote post-conditioning and allopurinol reduce ischemia-reperfusion injury in an infra-renal ischemia model. Ther Adv Cardiovasc Dis 2018;12:341-9. 39. Τsompos C, Panoulis C, Τοutouzas K, Triantafyllou A, Ζografos CG, Tsarea K, et al. Comparison of the hyperphosphatasemic effects of erythropoietin and U-74389G on alkaline phosphatase levels. Int J Res Stud Sci Eng Technol 2018;5:7-13. 40. Τsompos C, Panoulis C, Τοutouzas K, Triantafyllou A, Ζografos CG, Tsarea K, et al. The diverging effects of erythropoietin and U-74389gon γ-glutamyl transferase levels. Arch Nephrol 2018;1:31-5. How to cite this article: Τsompos C, Panoulis C, Τοutouzas K, Triantafyllou A, Zografos GC, Tsarea K, Karamperi M, Papalois A. Comparison of the Hypertransferasemic Effects of Erythropoietin and U-74389G on Aspartate Aminotransferase Levels. Clin Res Hematol 2018;1(2):16-22.