Rapid real time pcr for Cyp2c19 and itgb3 gene detection to optimize the use of clopidogrel and aspirin for pci stent graft patients

JOURNAL OF MEDICAL RESEARCH

RAPID REAL-TIME PCR FOR CYP2C19 AND ITGB3 GENE
DETECTION TO OPTIMIZE THE USE OF CLOPIDOGREL
AND ASPIRIN FOR PCI STENT GRAFT PATIENTS
Nguyen Thi Trang, Luong Thi Lan Anh , Vu To Giang , Do Duc Huy,
Nguyen Thi Minh Ngoc,
Department of Biomedical and Genetics, Hanoi Medical University, Hanoi, Vietnam
The response to clopidogrel and aspirin in patients is known to be highly variable as bioavailability
is dependent upon the conversion of the prodrug into the pharmacologically active clopidogrel and
aspirin. Blood samples were collected from consenting patients after they were on the maintenance
dose of anti-platelet therapy. The real-time PCR method was developed for the identification of the
specific mutations in the CYP2C19 gene (CYP2C19 *2 and CYP2C19*3) and ITGB3 gene (PlA1/
A2). The real-time PCR method using SYBR green was validated against the Sanger’s sequencing
method described previously and used to determine the frequency and type of mutations of postPCI
patients. The results indicated that patients carrying any CYP2C19 loss-of-function alleles had a
higher event rate (52.5%) of the study group: 10% homozygous and 35% heterozygous (CYP2C19
*2); 7.5% heterozygous carriers (CYP2C19*3). Carriers of the Leu33Pro polymorphism of ITGB3
gene accounted for approximately 10% of the study population. In conclusion, a genotyping variant of
CYP2C19 and ITGB3 provides an excellent opportunity for optimizing the anti-platelet regimen post-PCI.

Keywords: coronary artery disease, CYP2C19, clopidogrel, ITGB3, aspirin resistance

I. INTRODUCTION
Coronary artery disease (CAD) is the
most common type of heart disease. It is the
leading cause of death in the world in both
men and women [1]. In Vietnam, coronary
artery disease has been increasing rapidly
in recent years. Percutaneous coronary
intervention (PCI) is one of the most

common medical procedures performed for
treatment of CAD.
Corresponding author: Nguyen Thi Trang, Department
of Biomedical and Genetics, Hanoi Medical University
Email:
Received: 03 June 2017
Accepted: 16 November 2017

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Dual antiplatelet therapy with aspirin
and a P2Y12 receptor antagonist including
clopidogrel is the standard of care in
patients undergoing PCI and in patients
with acute coronary syndromes (ACS)
because this regimen has markedly
decreased the rate of cardiovascular
events. However, the substantial variability
in pharmacodynamics response, as well
as the moderate antiplatelet efficacy of
clopidogrel and aspirin, has raised major
concerns. Many researches have focused
on the impact of genetic polymorphisms
encoding transport systems or enzymes
involved in the absorption and metabolism
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of these drugs. An ESC Guidelines class

II recommendation has been given for the
management of acute coronary syndromes
patients (2011) to perform genotyping in
high-risk PCI patients if a change in the antiplatelet therapy will ensue based on the test
results [2].
Loss-of-function
polymorphisms
in
CYP2C19 are the strongest individual
variables
affecting
pharmacokinetics
and antiplatelet response to clopidogrel.
CYP2C19 G681A (*2) and CYP2C19
G636A (*3) alleles are associated with CYP
function reduction, impaired clopidogrel
responsiveness and increased subsequent
post-PCI ischemic outcomes [3 - 5].
The GPllb/llla receptor is a key regulator
of platelet aggregation. Consequently,
polymorphisms
within
the
GPllb/llla
receptor have been of great interest with
regard to aspirin resistance, the most

commonly investigated being the PlA1/A2
SNP (Leu33Pro) [6]. Some studies have
suggested an association between the

presence of the PlA2 allele and increased
platelet activity, as determined by platelet
aggregation and/or fibrinogen binding [7 10].
The Realtime - PCR method is an
accurate way of defining polymorphism,
with the advantage of relatively simple,
fast-paced techniques that have opened up
new possibilities for applying this technique
as a routine test before indications of
clopidogrel and aspirin therapy for PCI
patients. The aim of this study was to
investigate the frequencies of CYP2C19
G681A (*2; rs4244285), CYP2C19 G636A
(*3; rs4986893) and PlA1/ PlA2 (T1565C,
rs5918) polymorphisms of CYP2C19 and
ITGB3 gene in study subjects by Realtime
- PCR technique.

II. MATERIALS AND METHODS
1. Sample collection:
40 patients undergoing PCI procedure with coronary artery disease were offered the
genetic test by the treating cardiologist to identify CYP2C19 and ITGB3 genotype. All patients
were treated with an aspirin (325 mg) and clopidogrel (600 mg) loading dose before the
procedure.
2. Method:
About 3 ml of venous blood were collected in a tube containing EDTA. Peripheral blood
leucocytes were separated by centrifugation. DNA was extracted from peripheral blood
with DNA Expression Kit (Lytech, Russia). Realtime - PCR technique was used to identify
polymorphism of CYP2C19 gene (CYP2C19*2 , CYP2C19*3) and ITGB3 gene (PlA1 / PlA2).
The primers used for PCR of each polymorphism are given in Table 1:

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Table 1. FP - Forward primer, ASP - Allele specific primer, RP- Reverse primer.
Gene
CYP2C19*2
(G681A)

CYP2C19*3
(G636A)
Leu33Pro
(T1565C)

Primer
FP

5’ – CCCACTATCATTGATTATTTCTCG – 3’

ASP

5’– CCCACTATCATTGATTATTTCTCA-3’

RP

5’ – ACGCAAGCAGTCACATAACTA – 3’

FP

5’ – GGATTGTAAGCACCCCCAGG – 3’

ASP

5 – GGATTGTAAGCACCCCCAGA – 3’

RP

5’ – TCACCCCATGGCTGTCTAGG – 3’

FP

5’ – GCTCCAATGTACGGGGTAAAC – 3’

ASP

5’ – GCTCCAATGTACGGGGTAAAT – 3’

RP

5’ – GGGGACTGACTTGAGTGACCT – 3’

Realtime - PCR technique was performed on CFX96 (BioRad, USA). The cycling
programmer involved preliminary denaturation at 93°C for 1 min, followed by 35 cycles of
denaturation at 93°C for 10 seconds, annealing at 64°C for 10 seconds, and elongation at
72°C for 20 seconds followed by a final elongation step at 72°C for 5 min.
Using the DNA-express kit for DNA extraction and Realtime Techniques - Genomic
PCR and mutation analysis are time-saving methods: saving an average of 45 minutes for

DNA extraction and 90 minutes for gene polymorphism. This technique is therefore highly
applicable in clinical practice to quickly and accurately identify patients' genotypes prior to the
introduction of the regimen as well as the dose of clopidogrel and aspirin.
Statistical analysis
The continuous data were presented as mean and standard deviation (SD). The statistical
analyses were conducted using SPSS (SPSS Statistics for Windows, Version 17.0. Chicago:
SPSS Inc.)
3. Ethics
All patients were duly informed of the benefit of such a test and were asked to sign an
informed consent form before blood collection. Ethical clearance was obtained from the Hanoi
Medical University Institutional Ethics Committee.

III. RESULTS
1. Characteristics of the study population
There were 40 patients enrolled in the study ranging in age from 39 to 79 years, the
majority were men (85%). Patients with high risk factors for cardiovascular disease were
relatively many, including hypertension (72.5%) and hypercholesterolemia (32.5%) (Table 2).

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Table 2. Characteristics of the study group
Characteristics

n

%

Male

34

85

Female

6

15

Hypertension

29

72.5

Diabetes mellitus

9

22.5

Hypercholesterolemia

13

32.5

Stents ≥ 2 times

7

17.5

Mean

SD

62.6

9.5

Gender

 
Age(Year)

2. CYP2C19 and ITGB3 polymorphism detection rates using Realtime – PCR
technique.
Table 3 indicated that the rates of CYP2C19*2 and CYP2C19*3 polymorphisms in the
study group were relatively large: 52.5% of patients carrying at least one CYP2C19 allelic
variant reduced the function of clopidogrel metabolism. In addition, 10% of patients had
ITGB3 polymorphism associated with aspirin resistance.
Table 3. CYP2C19 and ITGB3 polymorphism detection rates using Realtime - PCR
Gene

 

CYP2C19
CYP2C19 *2, *3

Characteristic
 

ITGB3
PlA1/ PlA2

Gender

n

%

n

%

Male

16

47.1

3

8.8

Female

4

66.7

1

16.7

Total

21

52.5

4

10

3. Genotype frequencies of study subjects.
Table 4 shows that the number of patients with CYP2C19*2 allele accounted for 45%,
mainly heterozygous (35%), homozygotes (10%). 7.5% of patients with CYP2C19*3 allele, all
of them are heterozygous. 10% of patients carry PlA2 alleles - all of them are heterozygous.

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Table 4. Genotype frequencies of study subjects.
Genotype frequency
Wild
type

Gene

Allele frequency 

Hetero zygous

Homozygous 

Wild type allele 

Mutant allele 

n

%

n

%

n

%

n

Freq.

n

Freq.

CYP2C19*2
(G681A)

22

55.0

14

35.0

4

10

681G

0.725

681A

0.275

CYP2C19*3
(G636A)

37

92.5

3

7.5

0

0

636G

0.9625

636A

0.0375

PlA1/PlA2
(T1565C)

36

90.0

4

10.0

0

0

1565T

0.95

1565C

0.05

IV. DISCUSSION
The combination of aspirin and
clopidogrel is the mainstay anti - platelet
strategy for preventing ischemic events after
PCI. However, many studies have shown
resistance to aspirin and clopidogrel, which
reduces the effectiveness of treatment.
According to Boris T. et al. (2009), the rate
of no responsiveness to aspirin varied
from 5% to 60%, with clopidogrel 6% -25%
and both drugs 10.4% [11]. In Vietnam,
according Do Quang Huan (2013), in 174
patients with coronary artery disease
enrolled in the study, the prevalence of

nonresponse to aspirin and clopidogrel
were 21.3% and 26.4% [12]. According
to Ibrahim O. (2013), aspirin resistance/
non-responders in their study at acute
coronary syndrome patients accounted for
4.69% while non-responders to clopidogrel
accounted for 21.9% [13]. Thus, studies on
factors related to clopidogrel and aspirin
resistance, particularly genetic factors, are
essential in the prevention and treatment of
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coronary heart disease.
Several polymorphisms of the CYP2C19
gene have been identified and they produce
an inactive enzyme. Two inactive genetic
variants (CYP2C19*2 and CYP2C19*3)
account for more than 95% of cases of poor
metabolism of the relevant medications [14].
The genotypes were distributed as good
or normal metabolizers (CYP2C19*1/*1;
also called extensive metabolizers in
literature), intermediate (*1/*2; *1/*3,
*2/*17, and *3/*17), poor (*2/*2 and *3/*3),
rapid (*1/*17), and ultra-rapid metabolizers
(*17/*17).
In our study, more than half of the study
population carriers of at least one CYP2C19
loss-of-function allele (CYP2C19 * 2 or
CYP2C19 * 3). The results of our study were

similar to those found in Liu Mao’s study
(2013): about 55% of Asians have one or
more loss-of-function allele of CYP2C19
[15]. Among them, the frequencies of
heterozygous CYP2C19 G681A (*2) in our
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study was 35% (14 patients), the frequencies
of heterozygous CYP2C19 G636A (*3)
was 7.5% (3 patients). Poor metabolizers,
with both alleles being mutated and hence
unable to metabolize clopidogrel, formed
10% of the population (4 patients), all of
them are homozygous CYP2C19 *2. Thus,
the intermediate metabolizers and the poor
metabolizers of clopidogrel are relatively
large. As recommended by the American
Association for Clinical Pharmacology and
Clinical Pharmacy (ASCPT) in 2013 for the
use of platelet antiplatelet drugs [16]: it is
necessary to consider alternative treatment
sor treatment strategies in patients identified
as CYP2C19 intermediate metabolizers and
poor metabolizers. Therefore, identifying
the genotype of an individual before taking
clopidogrel is necessary to achieve its
optimal anti-platelet activity.
In addition, our study identified that

4 patients (10% of study population)
were PLA1/A2 heterozygotes. No PLA2
homozygotes were found in the study
group. No patients carry the PlA2 / PlA2
homozygous genotype. According to a
study by Sperr W. R. et al. (1998), in central
Europeans, the PlA1/A2 allele is present in
20-30 % of people and the PlA2/A2 allele
is present in 1-3 % of people [8]. It has
been shown that platelets containing PlA1/
A2 or PlA2/A2 alleles are more reactive
than homozygous PlA1/A1 platelets with
enhanced thrombin formation and a lower
threshold for activation, granule release,
and fibrinogen binding, and therefore will
have a variable response to the antiplatelet
effects of aspirin [9; 10].
Results from our study indicate high
6

prevalence of mutations that would alter
the normal function of the CYP2C19 gene
in metabolizer clopidogrel. Our study also
found that the small rate of individuals
with the PlA2 allele of SNP rs5918 of the
ITGB3 gene reduces the response to
aspirin. Variability or resistance to aspirin or
clopidogrel has been demonstrated using
various in vivo biomarkers and ex vivo
platelet function tests. Accumulating data

suggested that patients with resistance are
at high risk for ischemic events, including
stent thrombosis. Thus, cardiologists have
focused much attention on the adequacy of
antiplatelet regimens.
Study limitations: First and foremost,
among the limitations of the present study
is its small sample size and absence it a
control group, although we tried to overcome
this shortcoming by matching the cases. We
calculated our sample size based on the
frequency of the CYP2C19*2 alleles in the
Vietnam population. However, our achieved
power was markedly reduced due to the
low frequency of the non-functional allele
in our study population. Moreover, while
we focused on CYP2C19*2, CYP2C19*3
and PlA2 as the most important alleles
responsible for the resistance to clopidogrel
and aspirin therapy, we did not study
the prevalence of other non-functional
CYP2C19 alleles. Further cohort studies
with larger samples and on different
ethnicities in the Vietnam population are
required to determine the effects of the
CYP2C19 and ITGB3 polymorphism on
the prognosis of CAD patients who have
undergone PCI and are receiving dual antiplatelet therapy.
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V. CONCLUSION
In our study, carriers of at least one
CYP2C19 loss-of-function allele (CYP2C19
* 2 or CYP2C19 * 3) approximately 52,5% of
the study population and 10% of the study
population were PLA1/A2 heterozygotes of
ITGB3 gene. Therefore, the prevalence of
antiplatelet therapy nonresponse is higher
in patients undergoing PCI. The Realtime PCR technique is a fast, accurate and costeffective way to identify polymorphisms of
CYP2C19 gene and ITGB3 gene, that are
suitable for Vietnamese conditions, and is
clinically relevant. Using this method, the
common mutations that cause changes in
the bioavailability of clopidogrel and aspirin
may be reliably identified and the medication
and dosage adjusted accordingly. Thus, this
technique could be used as a routine test
before treatment of dual – platelet for PCI
patients.

Acknowledgments
The authors would like to take this
opportunity to extend our sincere thanks to
Center of Genetics Couseling for providing
financial support for the study. We also are
grateful for National Cardiovascular Institute
for supporting us in identifying CYP2C19

and ITGB3 mutations.

REFERENCES
1. Shanthi Mendis, Pekka Puska,
Bo Norrving, (2011). Global Atlas on
cardiovascular disease prevention and
control. World Stroke Organization, 3 - 9.
2. C.W. Hamm, J.P. Bassand, S.
Agewall et al. (2011). ESC guidelines

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for themanagement of acute coronary
syndromes in patients presenting without
persistent ST-segment elevation: The task
force for the management of ACS (acute
coronary syndromes) in patients presenting
without persistent ST-segment elevation of
the ESC (European Society of Cardiology),
Eur. Heart J. 32 (23): 2999 - 3054.
3. Brant J.T., Close S.L, Iturria S.J.
et al. (2007). Common polymorphysm
of CYP2C19 and CYP2C9 affect the
phamacokinetic and pharmacodynamic
respone to clopidogrel but not prasugrel. J
Thromb Heamost. 5: 2429 - 2436.
4. Mega J. L., Close S. L., Wiviott
S. D., et al. (2009). Cytochrome p-450
polymorphisms and response to clopidogrel.
N Engl J Med, 360(4), 354 - 362.

5. Collet J. P., Hulot J. S., Pena
A., et al. (2009). Cytochrome P450 2C19
polymorphism in young patients treated
with clopidogrel after myocardial infarction:
a cohort study. Lancet, 373(9660), 309 317.
6. Newman P. J., Derbes R. S.,
Aster R. H. (1989). The human platelet
alloantigens, Pl(A1) and Pl(A2), are
associated with a leucine(33)/proline(33)
amino acid polymorphism in membrane
glycoprotein IIIa, and are distinguishable by
DNA typing. J. Clin. Invest. 83: 1778 - 1781.
7. Sirotkina OV, Khaspekova SG,
Zabotina AM, Shimanova YV, Mazurov
AV. (2007). Effects of platelet glycoprotein
IIb-IIIa number and glycoprotein IIIa
Leu33Pro polymorphism on platelet
aggregation and sensitivity to glycoprotein
IIb-IIIa antagonists. Platelets. 18: 506 – 14.

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JOURNAL OF MEDICAL RESEARCH
8. Sperr W. R., Huber K., Roden M.
et al (1998). Inherited platelet glycoprotein
polymorphisms and a risk for coronary
heart disease in young central Europeans.
Thromb Res. 90: 117 - 123.
9. Undas A., Brummel K., Musial J.

et al (2001). Pl(A2) polymorphism of beta3 integrins is associated with enhanced
thrombin
generation
and
impaired
antithrombotic action of aspirin at the site of
microvascular injury. Circulation. 104: 2666
- 2672.
10. Michelson A. D., Furman M.I.,
Goldschmidt-Clermont P. et al (2000).
Platelet GP. IIIa- PlA polymorphisms
display different sensitivities to agonists.
Circulation. 101: 1013 - 1018.
11. Boris T. I., Mareike Sausemuth
H. I., Evangelos G. et al (2009). Dual
Antiplatelet Drug Resistance Is a Risk
Factor for Cardiovascular Events after
Percutaneous
Coronary
Intervention.
Clinical Chemistry. 55: 1171 - 1176
12. Đỗ Quang Huân, Hồ Tấn Thịnh
(2013). Tỷ lệ không đáp ứng với điều trị
thuốc chống kết tập tiểu cầu trên bệnh nhân
được can thiệp động mạch vành qua da.

8

Tạp chí Y học thực hành (878), số 8/2013,
9 - 13.

13.Ibrahim O, Oteh M, A Syukur
A et al (2013). Evaluation of Aspirin and
Clopidogrel resistance in patients with Acute
Coronary Syndrome by using Adenosine
Diposphate Test and Aspirin Test. Pak J
Med Sci, 29(1): 97 – 102.
14. Yuanyuan Dong, Huasheng Xiao,
Qi Wang et al (2015). Analysis of genetic
variations in CYP2C9, CYP2C19, CYP2D6
and CYP3A5 genes using oligonucleotide
microarray. Int J Clin Exp Med 8(10): 18917
– 18926.
15. Liu Mao et al (2013). Cytochrome
CYP2C19 polymorphism and risk of
adverse clinical events in clopidogreltreated patients: A meta-analysis based on
23035 subjects. Archives of Cardiovascular
Disease. 106: 517 - 527.
16. Scott S. A., Sangkuhl K., Stein C.
M. et al (2013). Clinical Pharmacogenetics
Implementation Consortium guidelines
for CYP2C19 genotype and clopidogrel
therapy: 2013 update. Clin Pharmacol Ther.
94(3), 317 - 323.

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STUDY ON THE HYPOGLYCEMIC ACTION OF CF2 IN

INDUCED TYPE 2-LIKE DIABETIC MICE MODEL
Ho My Dung¹, Vu Thi Ngoc Thanh¹, Pham Thi Van Anh¹,
Nguyen Thi Thanh Ha¹, Nguyen Thi Minh Hang²
¹Hanoi Medical University
²Institute of Marine Biochemistry - Vietnam Academy of Science and Technology
To investigate the hypoglycemic action of CF2 in a type 2-like diabetic mice model induced

by high

fat diet (HFD) combined with streptozotocin (STZ) injection. Method: Model: mice were fed with HFD
for 8 weeks, then injected with a dose of STZ (100 mg/kg body weight, intraperitoneal injection).
Animal used: Swiss male mice. Drugs: type 2-like diabetic mice with HFD and STZ treated with either
gliclazide 80 mg/kg body weight, CF2 2 m/kg or CF2 4 mg/kg body weight daily by oral route of administration during 14 days. Result: CF2 has blood glucose-lowering effect in type 2-like diabetic mice
for 14 days treatment at doses of 2 mg/kg and 4mg/kg daily (p < 0.05). The blood glucose- lowering
effect of CF2 at dose of 2 mg/kg daily is similar to gliclazide 80 mg/kg daily. CF2 at dose of 4mg/kg
is more effective than that of 2 mg/kg and gliclazide 80 mg/kg in reducing blood glucose level. Conclusion: CF2 has effect on lowering blood glucose level at doses of 2 mg/kg and 4 mg/kg daily for
14 days in type 2-like diabetic mice, induced by HFD and STZ 100 mg/kg intraperitoneal injection.

Keywords: CF2, Callisia fragrans, ecdysteroid, type 2-like diabetic mice, STZ, HFD,
blood glucose

I. INTRODUCTION
The prevalence of diabetes mellitus is
increasing at an alarming rate globally. According to a World Health Organization report an estimated 422 million adults globally
were living with diabetes in 2014, compared
to 108 million in 1980 [1]. The number of
people with diabetes aged 20 - 79 years was
Corresponding author: Ho My Dung, Hanoi Medical
University.
Email:

Received: 15 June 2017
Accepted: 16 November 2017

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predicted to rise to 642 million by 2040 [2].
The health consequences of diabetes can
overwhelm the health care systems due to
the severity of the long term complications
of diabetes [1]. Several oral hypoglycemic
agent are available to lower blood glucose
levels in diabetics. However, their administration may cause side effects in patients [3;
4]. Therefore, there is on urgent need to find
new prevention strategies and treatments
for diabetes. Recently, many plant-based
natural products that contain certain phytochemicals are being investigated anti-di9


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abetic potential for their [5]. Using an herbal
remedy as an alternative therapy for diabetes treatment would reduce individuals dependence on synthetic oral hypoglycemic
agents [6]. The plant kingdom offers a wide
field of possible effective oral hypoglycemic
agents.
Basket Plant (whose scientific name
is Callisia fragrans) has been used as a traditional therapy for pain, fever, digestive
disorders, heart diseases, diabetes, cancers and many other airments [7; 8]. CF2
powder is extracted from the leaves, stems
and shoots of Basket Plant. CF2 powder
contains the active component ecdysteroid

which can be used in treating diabetes;
preventing inflammation and osteomalacia;
protecting the nervous system; and improving the immune system [9; 10]. However,
adequate characterization of CF2 effect is
yet to be done and no study has been performed using a type 2 diabetes model. The
objective of this study was to evaluate the
hypoglycemic action of CF2 in a model of
induced type 2-like diabetic mice. Type-2
like diabetes was individual by high fat diet
(HFD) combined with streptozotocin (STZ)
injection.

II. MATERIALS AND METHODS
1. Materials
Experimental Medicine

CF2 powder extracted from leaves,
stems and shoots of Basket Plant was supplied by Institute of Marine Biochemistry,
Viet Nam.
Experimental Animals
Swiss male white mice, from 6 - 8 weeks

10

of age, and weighing between 23 - 27
grams, were used for this study. The mice
were obtained from National Institute of Hygiene and Epidemiology, Viet Nam. The experimental animals were caged individually
and acclimatised to laboratory conditions
for 2 weeks prior to the experiment. The
study was carried out at the Pharmaceutical

Department of Hanoi Medical University.
Machines and Chemicals
- Streptozotocin 1 g (Sigma-Aldrich, Singapore), Buffer solution Citrate pH 4.5
- Diamicron (gliclazide) tablets 30 mg
(Servier ,France).
- Blood glucose monitoring system On
Call EZII (ACON Biotech, USA)
- Animal blood counter Vet Exigo (Bonle
Medical AB, Sweden)
- Chemistry analyzer Erba and Test
strips: blood triglyceride, HDL-C, cholesterol (Transasia, India).
2. Method
The study was divided into two stages
[5]:
* The first stage:
Before ending the study, all mice baseline fasting glucose levels checked from peripheral blood samples.
+ Group 1: Control condition (n = 10)
mice were randomized to one of two group:
Normal fat diet regime (NFD) for 8 weeks.
+ Group 2: Diabetic condition (n = 70):
High fat diet regime (HFD) in 8 weeks following Fabiola and Srinivasan method with
43% saturated fat combined siro fructose
55% [6].
After 8 weeks, the fasting glucose level
in all mice was checked. Mice in group 2
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were injected intraperitoneally with STZ at

a dose of 100 mg/kg. Mice in group 1 were
injected intraperitoneally with citrate pH 4.5.
After 72 hours (t0), the blood of all mice
of group 2 was checked. The mice were
considered diabetic if their blood glucose
concentration was more than 10.0 mmol/L
at that time. Diabetic mice were then randomized into four groups from, group 2 to
group 5.
* The second stage: The study was carried out in a continuous 14-day period. Mice
were divided into five groups of ten animals:
- Group 1: NFD regime + drinking distilled water;
- Group 2: HFD regime + STZ injection
100mg/kg + drinking distilled water;
- Group 3: HFD regime + STZ injection
100 mg/kg + drinking gliclazide 80 mg/kg/
day;
- Group 4: HFD regime + STZ injection
100 mg/kg + drinking CF2 2 mg/kg/day

(low-dose group);
- Group 5: HFD regime + STZ injection
100 mg/kg + drinking CF2 4 mg/kg/day
(high-dose group).
After 7 days (t1) and 14 days (tc) of treatment, the fasting glucose level of all mice
were tested. After 14 days of treatment, all
animals blood lipid index (total cholesterol (TC), triglyceride (TG), HDL-Cholesterol,
LDL-Cholesterol) were checked. Animals
were subjected to a full gross necrospy.
From 30% of each group, mices livers and
pancreas were removed for histopathology

examination.
Statistical analysis
Data was analyzed using Microsoft Excel software version 2010. The levels of significance between groups were determined
using student's t-test and Avant-après test.
Data is shown as mean ± standard deviation. All data was considered significant at
p < 0.05.

III. RESULTS

Figure 1. Wight of mice over time
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The results in Figure 1 showed that the weight of mice in the HFD eating regime was significantly higher than weight of mice in the NFD eating regime at the sixth week (p < 0.001).
Hower, 72 hours after STZ injection in the NFD eating group, the weight of the mice in the
HFD group was slightly reduced.
Table 1. The change in blood glucose level of mice in type 2-like diabetes model
Blood glucose level (mmol/l)
( X ± SD) (n = 10)
Time

p2-1

Group 1: Normal
control

Group 2: Diabetic
control

(t- test Student)

Before studying

5.55 ± 0.74

5.79 ± 0.88

> 0.05

After 8 weeks

5.45 ± 0.74

5.66 ± 1.04

> 0.05

After STZ injection 72
hours

5.36 ± 0.81

16.55 ± 5.48 *

< 0.001

*: p < 0.05: In comparison to before studying
Show in Table 1, the blood glucose level of mice in HFD regime group showed no differences compared to NFD regime group at the time before studying and after 8 weeks (p >
0.05). However, after STZ injection 72 hours, the blood glucose level of mice in group 2 was
significantly increased compared to level in group 1 (p < 0.001).
Table 2. Effect on blood glucose level of mice after 2 weeks of treatment
Blood glucose level (mmol/l)
Group

( X ± SD) (n = 10)
t0 (before
treatment)

t1 (after 7 days
of treatment)

tc (after 14 days
of treatment)

Group 1: NFD + distilled water

5.22 ± 0.73

5.34 ± 0.68

5.35 ± 0.76

Group 2: HFD + STZ + distilled
water

16.31 ± 3.32*

19.41 ± 3.77*

18.68 ± 1.26*

↑ 21.3

↑ 20.43

15.49 ± 4.08**

12.74 ± 3.32***

↓ 4.07

↓ 19.45

% in change in comparison to t0
Group 3: HFD + STZ + gliclazide
80 mg/kg/day
% in change in comparison to t0

12

16.45 ± 4.40*

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Group 4: HFD + STZ + CF2 2 mg/
kg/day

16.64 ± 4.53*

15.09 ± 4.92**

13.76 ± 3.11***

↓ 6.97

↓ 13.1

11.82 ± 3.76***

10.73 ± 3.84***

↓ 27.02

↓ 31.00

% in change in comparison to t0
Group 5: HFD + STZ + CF2 4 mg/
kg/day

16.89 ± 5.87*

% in change in comparison to t0
*: p < 0.001: In comparison to group 2
**: p < 0.05: In comparison to group 2

CF2 powder at two oral doses (2 mg/kg/day and 4 mg/kg/day) used continuously after 14
days had effect to reduce blood glucose level in comparison to diabetic control group 2 (p <
0.05 and p < 0.001). Glucose reducing effect of CF2 at dose of 2 mg/kg/day was similar to
reducing effect of gliclazide dose 80 mg/kg/day seen in group 3 (p > 0.05). CF2 at dose of 4
mg/kg/day was more effective than CF2 at dose of 2 mg/kg/day and gliclazide 80 mg/kg/day
(p < 0.05).
4.5

Concentration (mmol/l)

4
3.5
3

Group 1

2.5

Group 2

2

Group 3
Group 4

1.5

Group 5

1
0.5
0

TC

TG

HDL-C

LDL-C

Figure 2. Effect on blood lipid content of mice after 2 weeks of treatment
The result of figure 2 shows lipid disorder conditions TC, TG, HDL-C, LDL-C were in
creased in group 2 (diabetic control) in comparison to group 1 (normal control). Blood lipid disorders at gliclazide group and CF2 groups were improved, with reduction in TC, TG,
LDL-C and increase of HDL-C content in comparison to diabetic control group. However,
none of these were significant differences (p > 0.05).
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Histopathological examination of liver and pancreas shows that all mice in group 2 (diabetic control) were in degenerate condition when compared to group 1 (normal control).
Histopathological examination of liver and pancreas of mice treated with gliclazide and two
doses of CF2 were improved significantly in comparison to samples from group 2. The results
are shown in Figure 3 and Figure 4.

Figure 3. Histopathological image of mice liver after 2 weeks of treatment

Figure 4. Histopathological image of mice pancreas after 2 weeks of treatment
14

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IV. DISCUSSION
After 8 weeks of consuming a high fat
diet regime, the blood glucose level in mice
was increased but there was no significant
difference when compared to normal control mice. After mice with HFD eating regime
were injected with STZ at a dose of 100 mg/
kg, the blood glucose level nearly 3 times
higher than before the injection. This model was stably maintained during 2 weeks
of treatment and similar to the model of
ShaoYu [7]. These results prove a rich energy eating regime combined with a low-dose
STZ injection has an effect on hyperglycemia and successfully creates model of type
2 like diabetes.
After 7 days and 14 days of treatment, gliclazide 80 mg/kg/day, CF2 at a low dose (2
mg/kg/day), and a high dose (4 mg/kg/day)
effectively in reduced blood glucose levels
in type 2-like diabetic mice when compared
to diabetic control group. The blood glucose-lowering effect of CF2 at dose of 2 mg/
kg daily was similar to gliclazide 80 mg/kg
daily. CF2 at dose of 4mg/kg daily was more
effective than the dose of 2 mg/kg and gliclazide 80 mg/kg in reducing blood glucose
level. CF2 powder is there a contains ecdysteroid which was proved to reduce blood
glucose level in experimental animals. In Kizelsztein’s study (2009), 20-hydroxyecdyson at dose of 10 mg/kg/day reduces blood

glucose level in type 2-like diabetic mice
after 13 weeks through increasing circulating adiponectin levels [8]. Another study by
Sumdaram showed that the administration
of 20-OH-ecdysone results in a significant
restoration towards normal levels of plasma
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glucose, insulin, HbA1c, and key carbohydrate an enzymes [9].
Besides reducing blood glucose, CF2
effected lipid disorder conditions. CF2 at
two doses of 2 mg/kg/day and 4 mg/kg/
day reduced total cholesterol, triglyceride
and LDL-C and increased HDL-C, however,
differences were not significant. This was
similar to gliclazides 80 mg/kg/day effect
on regulating lipid disorders. This is consistent with previous research showing 20-hydroxyecdyson in Quinoa extract decreased
total cholesterol and triglyceride in diet-induced obesity mice [10].
The result of histopathological examination showed that there was positive change
in liver and pancreas structure of mice after 2-week of treatment with either dose of
CF2. The experimental medicine improved
lipid disorders which is possibly low, it also
reduced the degenerate condition of the liver. Another possible explanation for CF2 is
regenerative effect on reason for recreating
pancreas structure is the antioxidant activity
of Basket Plant [11].

V. CONCLUSION
CF2 lower blood glucose level at doses
of 2 mg/kg and 4 mg/kg daily over 14 days
in type 2-like diabetic mice when compared

to a control group.
The high dose was more effective than
both the low dose and the glyceride 80mg/
kg dose.
Besides reducing blood glucose effect,
CF2 improved lipid disorder conditions. It
lowered total cholesterol, triglyceride and
LDL-C and increased HDL-C.

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Acknowledgements
I would like to express my deepest gratitude to the Department of Pharmacology,
Hanoi Medical University for supporting us
in this study.

REFERENCES
1. World Health Organization (2016).
Global report on diabetes 2016. WHO.
2. K. Ogurtsova, J. D. da Rocha Fernandes, Y. Huang et al. (2017). IDF Diabetes Atlas: Global estimates for the prevalence of diabetes for 2015 and 2040.
Diabetes Res Clin Pract, 128, 40 - 50.
3. Katzung B.G. (2012), "Chapter
41: Pancreatic Hormones & Antidiabetic
Drugs". Basic and clinical Pharmacology,
The Mc GrawHill company, 743 - 765.
4. Standards of Medical Care in Diabetes (2017). Summary of Revisions. Diabetes Care, 40(Suppl 1), S4 - S5.
5. Malviya N Jain S and Malviya S.

(2010). Antidiabetic potential of medicinal
plants. Acta Poloniae Pharmaceutica, Drug
Research, 67 (2), 113 - 118.
6. Suchý V., Zemlicka M., Svajdlenka
E. et al (2008). Medicinal plants and diabetes mellitus. Ceska Slov Farm, 57(2), 78 84.
7. Joash Ban Lee Tan, Wei Jin Yap,
Shen Yeng Tan et al (2014). Antioxidant
Content, Antioxidant Activity, and Antibacterial Activity of Five Plants from the Commelinaceae Family. Antioxidants, 3(4), 758
- 769.
8. Zilfikarov I.N. Olennikov D.N., Toropova A.A. et al (2008). Chemical composition of Callisia fragrans Wood. Juice and
its antioxidant activity (in vitro). Chemistry
of plant raw material, 4, 95 - 100.
9. L. Dinan (2009). The Karlson Lecture.
Phytoecdysteroids: what use are they?.

16

Arch Insect Biochem Physiol, 72(3), 126 41.
10. Lafont R and Dinan L. (2009). Innovative and future applications for ecdysteroids. Ecdysone: structures and functions.
Springer Science, New York, 551 – 578.
11. K. Srinivasan, B. Viswanad, L. Asrat et al (2005). Combination of high-fat diet-fed and low-dose streptozotocin-treated
rat: a model for type 2 diabetes and pharmacological screening. Pharmacol Res,
52(4), 313 - 320.
12. Escalona C.N., Rivera R.F., Garduno S.L. et al (2011). Antiobesity and hypoglycaemic effects of aqueous extract of
Ibervillea sonorae in mice fed a high fat diet
with fructose. Journal of Biomedicine and
Biotechnology, 12, 3 - 10.
13. Shao Yu WU, Guang fa WANG,
Zhong LIU et al (2009). Effect of geniposide, a hypoglycemic glucoside, on hepatic regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin.
Acta Pharmacol Sin, 30(12), 202 - 208.

14. Kizelsztein P., Govorko D.,
Komarnytsky S. et al. (2009). 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model.
Am J Physiol Endocrinol Metab, 296, 433
– 439.
15. R. Sundaram, R. Naresh, P. Shanthi et al. (2012). Efficacy of 20-OH-ecdysone on hepatic key enzymes of carbohydrate metabolism in streptozotocin induced
diabetic rats. Phytomedicine, 19(8-9), 725
- 9.
16. A. S. Foucault, V. Mathe, R. Lafont et al. (2012). Quinoa extract enriched
in 20-hydroxyecdysone protects mice from
diet-induced obesity and modulates adipokines expression. Obesity (Silver Spring),
20(2), 270 - 277

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THE IMPACT OF SEMINAL ZINC AND FRUCTOSE
CONCENTRATION ON HUMAN SPERM CHARACTERISTIC
Vu Thi Huyen, Nguyen Thi Trang, Luong Thi Lan Anh,
Vu To Giang, Bui Bich Mai, Nguyen Xuan Tung
Department of Biomedical and Genetics, Hanoi Medical University, Hanoi, Vietnam
This study assessed the association between fructose and zinc concentration and various seminal
characteristics. Fructose and zinc in semen reflect the secretory function of seminal vesicles. These
tests may help in assessing the diagnosis and the management of male infertility. Seminal plasma
was gathered from 180 males who averaged 31.1 ± 3,6 in age. A specific complexant was used
to form a stable coloured complex with fructose or zinc. The colour intensity of the complex in a
determining wavelength is proportional to the amount of fructose or zinc present in the sample.
The study found that seminal fructose concentration was significantly lower in the oligozoospermic
group and the azoospermic group in comparison to the with normozoospermic group. There

were also many significant differences in zinc’s concentration in semen when two of three groups
were compared with one another. In conclusion, the role of seminal fructose concentration lie
not only in the assessing seminal vesicle dysfunction, but also, in conjunction with other seminal
properties could give a useful indication of male reproductive function, whilst seminal zinc
concentration might not be most appropriate for the assessment of male reproductive dysfunction.

Keywords: infertility, seminal fructose, seminal zinc, azoospermia.

I. INTRODUCTION
As many reasons cause male infertility, it
is essential to identify appropriate methods
to diagnose the underlying cause many
tests have been applied previously, such
as semen analysis, genetic tests and
hormone methods. Recently, some of these
biochemical markers zinc and fructose, are
like increasingly recognized as important for
Corresponding author: Nguyen Thi Trang, Department
of Biomedical and Genetics,Hanoi Medical University.
Email:
Received: 03 June 2017
Accepted: 16 November 2017

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diagnosing the cause of male infertility.
Fructose is essential for spermatozoa
metabolism and motility. Fructose is
an energy source of spermatozoa. It is
produced by the seminal vesicles with

some contribution from the ampulla of the
ductus deferens [1; 2]. Absence of fructose
in semen is indicative of ejaculatory duct
obstruction or seminal vesicle dysfunction
[3; 4].
Apart from fructose, zinc is another factor
that is essential for the male reproductive
system. Deficiency of zinc in the reproductive
system causes hypogonadism and gonadal
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hypofunction [5; 6]. Many studies have
shown that zinc plays an important role in
sperm mobility an the normal development
of the testicles and prostate [2; 7; 8].
However, in Vietnam, knowledge about
the relationship between seminal zinc and
fructose concentration in human sperm is
scare. Therefore, the purpose of this study
was to determine the association between
fructose and zinc concentration and various
seminal characteristics in men.

II. SUBJECTS AND METHODS
1. Subjects
The study design was descriptive.
Fructose and zinc concentration was
measured in the seminal plasma of 180

patients, who visited the Fertility Department
of Hanoi Medical University Hospital from
March, 2016 to March, 2017 after semen
analysis tests showing abnormal seminal
characteristics (sperm concentration, total
count, motility, progressive motility). All the
samples were analyzed according to the
World Health Organization criteria (1992).
On the basis of the assessed parameters,
sperm concentration and sperm motility
were considered as the most important
parameters.
2. Method
Measuring the concentration of
fructose and zinc
After semen analysis, samples were
centrifuged at 1500 x g for 10 min and
zinc and fructose concentrations assayed
from the supernatant (i.e. seminal
plasma). Zinc concentration was assessed
using spectrophotometry (5- Br- PAPS
18

method) – direct colorimetric test without
deproteinization of the sample. At pH
8.6, in a buffered media, zinc react with
specific complexant 5-Br-PAPS form a
stable color compound Fructose content
in seminal plasma was determined by the
resorcinol method where fructose reacts

with resorcinol in concentrated hydrochloric
acid (HCl) solution to form a red compound.
Measure the coloric complex of Zinc and
Fructose at a wavelength of 560 nm against
blanks (ROE, 1976).
Statistical analysis
Statistical analysis was performed
using SPSS version 16.0. The means were
compared using student t test. The statistical
tests were considered to be significant at
the p ≤ 0.05 level.
3. Ethics
Ethical approval to conduct the study
was sought from the Hanoi Medical
University. Permission to use data from
the Hanoi Medical University Hospital was
sought from the hospital authority. All the
information from the database was kept
under strict confidentiality. No names were
recorded.

III. RESULTS
Fructose concentration and seminal
parameters
Table 1 shows that seminal fructose in
oligozoospermia was significantly higher
than normozoospermia (p < 0.05). Besides,
the mean sperm concentration (133.808
± 48.215 billion/ mL), and the mean
vitality (86.483 ± 3.218 %) and the mean

progressive motility (11.250 ± 10.157 %) in
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males with normozoospermia were significantly higher than that in males with oligozoospermia
(5.633 ± 4.992 billion/ mL and 58.183 ± 18.14 % and 11.250 ± 10.157 % respectively) (p <
0.01).
Table 1. Seminal fructose and some characteristics of the semen
(Independent sample T – test)
Normozoospermia
Mean± SD

Oligozoospermia
Mean± SD

p-value of
fructose test

Fructose

1.601 ± 0.604

1.881 ± 0.640

< 0.05

Sperm concentration
(billion/ml)

133.808 ± 48.215

5.633 ± 4.992

< 0.01

Vitality (%)

86.483 ± 3.218

58.183 ± 18.114

< 0.01

Progressive motility
(%)

54.667 ± 9.278

11.250 ± 10.157

< 0.01

Some sperm characteristics and seminal fructose concentration are shown in the graph
below. The results suggest is a significant correlation at the 0.05 level (2-tailed) between
seminal fructose concentration and sperm progressive motility (z = -0.183; p < 0.05)
(Spearman test) (Figure 1, Figure 2 and Figure 3).

Figure 1. Correlation between seminal fructose concentration (g/l)
and sperm concentration (billion/ml)

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Figure 2. Correlation between seminal fructose concentration (g/l)
and sperm vitality (%)

Figure 3. Correlation between seminal fructose concentration (g/l)
and sperm progressive motility (%)
Zinc concentration and seminal parameters
Table 2 shows the following:
• The progressive mobility of the low zinc concentration group was 16.87 ± 10.67%,
lower than that of the normal zinc concentration group (49.93 ± 15.35%). This difference is
statistically significant (z= -11.481, p < 0.01)
20

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•
There was no statistically significant difference in mean non- progressive motility
of males with low zinc concentration compared to males with normal zinc concentration (p=
0.19)

Table 2. Seminal zinc concentration and motility of the sperm

(Mann – Whitney test)
Low zinc
concentration
(n = 84)

Normal zinc
concentration (n = 96)

z

p-value

Progressive
motility (%)

16.87 ± 10.67

49.93 ± 15.35

- 11.481

< 0.01

Non- progressive
motility (%)

3.64 ± 2.07

4.07 ± 4.63

- 1.301

> 0.05

Immotile (%)

73.00 ± 21.42

44.07 ± 15.43

10.433

< 0.01

The low zinc concentration group had an immotile percentage of 73.00 ± 21.42% was
higher than the normal zinc concentration group (44.07 ± 15.43%) (z = 10.433). This difference
was statistically significant with p < 0.001.
Seminal zinc concentration showed a significant positive correlation (r = 0.596) with sperm
progressive motility (p < 0.01) Negative correlations with sperm immotile (r = - 0.527) which
were observed reached statistical significance (p < 0.01).

Figure 4. Correlation between seminal zinc concentration (g/l)
and sperm progressive motility (%) (r = 0.596; p < 0.01) ( Spearman test)

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Figure 5. Correlation between seminal zinc concentration (g/l)
and sperm immotile (%) (r = - 0.527; p < 0.01) ( Spearman test)

IV. DISCUSSION
Fructose is a main carbohydrate source
in seminal plasma and necessary for
sperm motility [9; 10]. The measurement
of seminal fructose has been used in
many laboratories. Therefore, the World
Health Organization manual recommends
measurement of seminal fructose as a
marker of seminal vesicular function [11].
Methods for determination of seminal
fructose include gas chromatography,
indole coloration, and resorcinol coloration.
In particular, the resorcinol method has
been used widely in clinical andrology
laboratories for its simplicity operational,
and high specificity.
Fructose is the primary source of
energy for all sperm activities. The higher
the sperm concentration, vitality, and,
motility the lower fructose will be [2; 4]. Lu

22

(2007) reported that when sperm motility
increased, fructose decreased, and in vitro,
sperm continued using fructose [4]. Normal

seminal fructose concentration confirms
normal levels of testosterone and function
of vesicles and vas deferens [12]. Biswas et
al. (1978) also reported that when seminal
fructose concentration decreased, sperm
concentration and mobility increased [13].
Furthermore, Lewis Jones et al.,1996 found
that fructose concentrations were inversely
ratio to sperm motility with R = - 0,062 (p
< 0.05) [7]. However, Andrade Rocha
(2001) found contrary evidence that that
seminal fructose concentration was related
to sperm concentration, survival, motility
and morphology, but the association was
not statistically significant [14]. In Amidu
(2012), seminal fructose concentration
negatively correlated with sperm motility

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(R = - 0.04) but was also not statistically
significant [15]. Fructose concentration was
inversely celated to sperm concentration (R
= - 0.21) anh this correlation was significant
at 0.05 [16]. Determination of seminal
fructose concentration has been used in
the examination of obstructive azoospermia
and inflammation of male accessory glands

[11; 12; 15]. Inflammation may lead to
atrophy of the seminal vesicles and low
seminal fructose concentration. When
ejaculatory ducts are blocked, fructose
concentration in seminal plasma usually
decreases and may become undetectable
[12; 17]. Additionally, determination of
seminal plasma fructose concentration is
useful for auxiliary diagnosis of obstructive
and nonobstructive azoospermia. Seminal
fructose concentration in non-obstructive
azoospermia is usually higher than or
equal to that in males of normal fertility
[9]. However, the fructose concentration
in seminal plasma of patients with
obstructive azoospermia is usually absent
or significantly lower than that in men of
normal fertility [12; 15]. Absence of seminal
fructose has also been found in patients with
congenital vas deferens-seminal vesicle
developmental defect (Kise et al., 2000;
Kumar et al., 2005). Therefore, our results
are consistent with those reported studies
in other international.
One of the biochemical processes
related to genital fluid mixing is the
regulation of the fraction of free seminal
zinc, which can interact with spermatozoa.
Zinc is first secreted in prostatic fluid in 2
forms available for sperm cells (free zinc and

zinc-citrate complex). During ejaculation,
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however, a partial redistribution of the ion
from citrate to very high affinity vesicular
ligands reduces the unbound zinc fraction
[18 - 20].
The measurement of zinc in human
seminal plasma is important in the
evaluation of male infertility. In the present
study, the level of zinc in seminal plasma
was found to be mor frequently immotile
in the zinc concentration group was higher
(73.00 ± 21.42%) than in the group with
normal zinc concentration (44.07 ± 15.43%)
(z = 10.433). A positive correlation between
zinc levels and sperm concentration and
motility was also observed in our study. This
isin accordance with previous studies of
Doshi et al., Hussain et al., Badade et al.,
Atig et al., and Abed [21 - 25]. Eliasson and
Lindholme et al., in contrast could not find
any correlation between zinc concentration
and sperm density, motility, or morphology
[26].
Fuse et al., found a positive correlation
between zinc and sperm concentration
and motility, but no correlation with sperm
morphology was observed [27]. Mankad
et al., found a positive correlation between

zinc and sperm count, but no significant
correlation between zinc and sperm motility
[28].
Thus, it seems that zinc is important for
semen quality. The low zinc levels in infertile
men in our study might be attributed to
disorders in the prostate excretory function
or possibly to asymptomatic prostate
infection.
Omu (1998), Hadwan (2013), and others
found that sperm motility increased after
treatment with zinc supplementation [29
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- 33]. However, Omar F. Abdul-Rasheed
(2009) found no correlation between zinc
concentrations in semen and sperm motility
[34].

V. CONCLUSION
The seminal fructose concentration of
the normozopermia group is significantly
lower than oligozoospermia group. Fructose
seminal concentration correclated with
sperm motility.
The progressive motility in the low zinc
concentration group is significantly lower
than that of the normal zinc concentration

group. The number of immotile sperm in the
low zinc concentration group is significantly
higher than that of the normal zinc
concentration group. Zinc concentration has
a positive correlation with sperm progressive
motility and a negative correlation with
immotile both are statistically significant.

Acknowledgements
The authors would like to take this
opportunity to extend their sincere thanks to
the Ministry of Health for providing financial
support for the study. They also are grateful
for the technical support form the Hanoi
Medical University Hospital for assaying of
seminal fructose and zinc.

REFERENCES
1. Schoenfeld C, Amelar RD, Dubin
L, Numeroff M (1979). Prolactin,fructose,
and zinc levels found in human seminal
plasma. Fertil Steril 32(2) , 206 - 208.
2.Biswas S., Ferguson K.M.,
Stedronska J. et al (1978). Fructose and
hormone levels in semen: their correlations
24

with sperm counts and motility. Fertility and
Sterility, 2, 200 - 204.
3. Aumuller G, Riva A (1992).

Morphology and functions of the human
seminal vesicle. Andrologia 24(4): 183 196.
4. Lu C.J (2007). Standardization and
quality control for determination of fructose
in seminal plasma. Journal of Andrology, 28
(2), 207 - 213.
5.Sandstead HH, Prasad AS,
Schulert AR, Farid Z, Miale A, Jr., Bassilly
S, et al. (1967). Human zinc deficiency,
endocrine manifestations and response to
treatment. Am J Clin Nutr 20(5):422 - 442.
6. Omu A.E, Dashti H, Al Othman S
(1998). Treatment of asthenozoospermic
with zinc sulphate: andrological ,
immunological and obstetric outcome. Eur J
Obstet Gynecol Reprod Biol, 79, 179 - 184.
7. Lewis Jones D.I., Aird I.A., Biljan
M.M. (1996). Effects of sperm activity
on zinc and fructose concentrations in
seminal plasma. Oxford Journals, Human
Reproduction, 11 (11), 2465 – 2467.
8. Basil Oied Mohammed Saleh,
Nawal Khiry Hussain, Ali Yakub Majid
et al (2008). Status of Zinc and Copper
Concentrations in Seminal Plasma of Male
Infertility and Their Correlation with Various
Sperm Parameters. The Iraqi postgraduate
medical journal, 7, 76 - 80.
9. Buckett WM, Lewis-Jones DI
(2002). Fructose concentrations in seminal

plasma from men with nonobstructive
azoospermia. Arch Androl; 48 , 23 – 27.
10. Santiani A., Huanca W., Sapana
R., Huanca T., Sepulveda N., Sanchez

JMR 111 E2 (2) - 2018


JOURNAL OF MEDICAL RESEARCH
R.(2005). Effects on the quality of frozenthawed alpaca (Lama pacos) semen using
two different cryoprotectants and extenders.
Asian J Androl, 7 , 303 – 309.
11. World Health Organization (1999).
Laboratory Manual for the Examination of
Human Semen and Sperm-Cervical Mucus
Interaction. 4th ed. Cambridge, United
Kingdom: Cambridge University Press; 1 –
10.
12. WHO (1987). Laboratory manual
for the examination of human semen
and semen - cervical mucus interaction,
Cambridge University Press.
13.
Biswas S., Ferguson K.M.,
Stedronska J. et al (1978). Fructose and
hormone levels in semen: their correlations
with sperm counts and motility. Fertil Steril,
2, 200 – 204.
14. Andrade Rocha F.T. (2001). Sperm
parameters in men with suspected infertility.

Sperm characteristics, strict criteria sperm
morphology analysis and hypoosmotic
swelling test. J. Reprod Med., 46, 577 –
582.
15.Amidu N., Owiredu W.K.B.A,
Bekoe M.A.T. (2012). The impact of seminal
zinc and fructose concentration on human
sperm characteristic. Journal of Medical
and Biomedical Sciences, 1 (1), 14 – 20.
16. Yassa D.A., Idriss W.K., Atassi
M.E. et al (2001). The diagnostic value of
seminal α – glucosidase enzyme index for
sperm motility and fertilizing capacity. Saudi
Medical Journal, 22 (11), 987 – 991.
17. Coppens L (1997). Diagnosis and
treatment of obstructive seminal vesicle
pathology. Acta Urol Belg, 65 (2): 11 - 19.
18.Björndahl L, Kvist U (1990).
JMR 111 E2 (2) - 2018

Influence of seminal vesicular fluid on the
zinc content of human sperm chromatin. Int
J Androl, 13, 232 - 237.
19. Arver S, Eliasson R (1982). Zinc
and zinc ligands in human seminal plasma
II contribution by ligands of different origin
to the zinc binding properties of seminal
plasma. Acta Physiol Scand, 115, 217 - 224.
20. Kvist U, Kjellberg S, Björndahl
L, Soufir JC, Arver S.(1990), Seminal

fluid from men with agenesis of the wolffian
ducts: zincbinding properties and effects on
sperm chromatin stability. Int J Androl,13,
245 - 252.
21. Doshi H, Heana O, Hemali T, Minal
M, Sunil K (2008). Zinc levels in seminal
plasma and its relationship with seminal
characteristics. Journal of Obstetrics and
Gynecology of India, 58, 152 - 155.
22. Hussain NK, Rzoqi SS, Numan
AW, Ali DT (2011). A comparative study of
fructose, zinc and copper levels in seminal
plasma in fertile and infertile men. Iraqi
Journal of Medical Sciences. 9 (1):48.
23. Badade ZG, More KM, Narshetty
JG, Badade VZ, Yadav BK (2011). Human
seminal oxidative stress: correlation with
antioxidants and sperm quality parameters.
Annals of Biological Research, 2 (5), 351 –
59.
24. Atig F, Raffa M, Habib BA,
Kerkeni A, Saad A, Ajina M(2012). Impact
of seminal trace element and glutathione
levels on semen quality of Tunisian infertile
men. BMC Urol, 12, 6.
25. Abed AA (2013). Essence of some
trace elements in seminal fluid and their role
in infertility. Int J Chem and Life Sciences,
02(6):1179–84.
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