Advances in Bioscience and Biotechnology, 2012, 3, 574-579 ABB
http://dx.doi.org/10.4236/abb.2012.35074 Published Online September 2012 (http://www.SciRP.org/journal/abb/)
Investigation of minerals, testosterone, and transaminases
in the semen and serum of fertile and infertile men belongs
to different age groups
Zahed Mahmood1*, Muhammad Riaz1, Imtiaz Mahmood Tahir1, Abdul Shakoor1, Zia Ur Rahman2,
Muhammad Usman Qamar Saeed1
1Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan
2Department of Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan
Received 24 June 2012; revised 28 July 2012; accepted 10 August 2012
ABSTRACT
The present study was aimed to assess the potential of
infertility to induce the adverse effects with reference
to testosterone, Triiodothyronine (T3), Thyroxine (T4),
Alanine Aminotransferase (ALT), Aspartate Ami-
notransferase (AST), zinc, copper and iron. All the
samples were divided into four groups according to
age and disorder (Group 1, 10 infertile men of 25 - 40
years; Group 2, 10 fertile men of 25 - 40 years; Group
3, 10 infertile men of 41 - 60 years and Group 4, 10
fertile men of 41 - 60 years). Semen and blood sam-
ples were analyzed by atomic absorption spectropho-
tometry to determine minerals while, Testosterone, T3
and T4 were determined by enzyme immunoassay
kits. ALT and AST were determined using standard
kit assay method. The levels of testosterone and T3
and AST in the fertile semen of 41 - 60 years age
group were increased significantly (P ≤ 0.001) as
compared to that of fertile semen of 25 - 40 years age
group. While, the level of T4 in the fertile semen of 41
- 60 years age group was decreased significantly (P ≤
0.001) as compared to that of fertile semen of 25 - 40
years age group. In case of fertile serum, only the
level of testosterone was significantly decreased (P ≤
0.05) in the 41 - 60 years age group as compared to 25
- 40 years age group. The levels of testosterone and
Cu in the infertile serum of 41 - 60 years age group
were decreased significantly (P ≤ 0.001). While, the
levels of T3, T4, ALT and Fe in the infertile serum of
41 - 60 years age group were increased significantly (P
≤ 0.05) as compared to that of infertile serum of 25 -
40 years age group.
Keywords: Semen; Reproduction; Infertility; Minerals;
Testosterone; Transaminases
1. INTRODUCTION
Infertility primarily refers to the biological inability of a
person to contribute to conception. According to World
Health Organization [1], infertility is a period of two
years without conception. After one year of infertility
many couples seek a medical opinion because modern
artificial reproduction techniques like intra-cytoplasmic
sperm injection (ICSI) can help couples to overcome
infertility [2]. Sub fertility is the failure to conceive after
1 year of regular, unprotected intercourse with the same
partner. Sub fertility is caused by sperm defects or dys-
function [3].
Testosterone is a principal male sex hormone and is
primarily secreted in the testes of males. In men, testos-
terone plays a crucial role in the development of male
reproductive tissues such as the testis and prostate as
well as promoting secondary sexual characteristics such
as increased muscle, bone mass and the growth of body
hair [4]. The thyroid hormones, T3, and T4 are tyrosine
based hormones formed by the thyroid gland mainly re-
sponsible for regulation of metabolism [5]. ALT and AST
are enzymes located in liver cells that leak out into the
general circulation during the injury of liver cells [6].
Among the metal trace elements Zinc is one of the
most interesting nutritional traces in the reproductive
system. In man it is a cofactor of more than 200 met-
allo-enzymes and plays an important role in the normal
testicular development, spermatogenesis, and sperm mo-
tility [7]. Copper is an essential trace element required in
the diet because it is the metal cofactor for a variety of
enzymes (amine oxidase, superoxide dismutase, cyto-
chrome oxidase and tyrosinase). However, excess copper
can cause problems because of its capacity to oxidize
proteins and lipids, bind to nucleic acids and enhancing
the production of free radicals. Therefore, it is important
to maintain the amount of copper in the body within
normal limits. Copper reduces sperm motility by reduc-
*Corresponding author.

Z. Mahmood et al. / Advances in Bioscience and Biotechnology 3 (2012) 574-579 575
ing oxidative processes and glucose consumption [8].
Iron is a vital component of a group of heme proteins
that function in oxygen transport or as enzymes in redox
systems. A small amount of iron is enclosed in several
non heme metalloenzymes. The chief complexes coordi-
nating iron with the cell are heme and heme containing
proteins, hemosiderin and ferritin [9,10]. The aim of this
study was to assess the potential causes of infertility and
old age to induce the adverse effects with reference to
testosterone, T3, T4, ALT, AST, zinc, copper and iron.
2. MATERIALS AND METHODS
2.1. Sample Collection
Semen and blood samples were collected from each in-
dividual patient of each group. Semen samples were col-
lected by masturbation in a clean specimen container
after sexual abstinence for 3 - 5 days followed by lique-
faction at 37˚C. The semen samples were evaluated ac-
cording to World Health Organization recommendations
(ejaculate volume, pH, time to liquefaction, sperm con-
centration, motility and morphology). The remaining se-
men samples were centrifuged at 3000 × g for 10 min to
obtain the seminal plasma. The separated seminal plasma
was stored at –80˚C until further analysis for the detec-
tion of testosterone, Zn, Cu, Fe, T3, T4, ALT and AST
level. Blood samples were centrifuged at 4000 × g for 15
min after complete coagulation and supernatant serum
was collected in Eppendorf tubes and frozen until further
analysis for the detection of testosterone, Zn, Cu, Fe, T3,
T4, ALT and AST level.
2.2. Measurement of Minerals
Zinc, iron and copper in the samples were measured by
atomic absorption spectrophotometer. The samples were
aspirated to the instrument and reading was recorded at
the Monochromator wavelength 213.9, 285.2 and 342.7
nm for zinc, iron and copper respectively.
2.3. Measurement of Testosterone
Testosterone was determined with the help of DRG Tes-
tosterone Enzyme Immunoassay Kit.
2.4. Measurement of T3 & T4
Triiodothyronine (T3) and Thyroxine (T4) were deter-
mined by using J D Biotech T3 and J D Biotech T4 En-
zyme Immunoassay Kit respectively.
2.5. Measurement of ALT
Human Gasellschaft fur Biochemica and diagnostica
mbH (EC 3.1.3.1) kit was used to determine the level of
ALT as described by Saher et al. [11].
2.6. Measurement of AST
The AST was evaluated by adopting the methodology as
described by Reitman and Frankel [12].
2.7. Statistical Analysis
The results were expressed as mean ± SEM for all con-
tinuous variables. Mean ± SEM have been prepared in
Microsoft® Excel Windows Version 2007. The data ob-
tained were analyzed by applying student t-test using
Minitab 15 Windows Version to assess the significant
differences between all selected age groups.
3. RESULTS AND DISCUSSION
The levels of testosterone and T3 and AST in the fertile
semen of 41 - 60 years age group were increased signifi-
cantly (P ≤ 0.001) as compared to that of fertile semen of
25 - 40 years age group. While, the level of T4 in the
fertile semen of 41 - 60 years age group was decreased
significantly (P ≤ 0.001) as compared to that of fertile
semen of 25 - 40 years age group. There was no signifi-
cant difference among the levels of ALT, Zn, Cu and Fe
between the fertile semen of 25 - 40 years age group and
41 - 60 years age group (Figure 1).
In case of fertile serum, only the level of testosterone
was significantly decreased (P ≤ 0.05) in the 41 - 60
years age group as compared to 25 - 40 years age group.
While, the levels of T3, T4, ALT, AST, Zn, Cu and Fe
had no significant difference between the fertile serums
of 25 - 40 years age group and 41 - 60 years age group
(Figure 2).
In the infertile semen of 41 - 60 years age group, the
levels of testosterone, T4, Zn and Cu were decreased
significantly (P ≤ 0.001, P ≤ 0.05) as compared to that of
infertile semen of 25 - 40 years age group. While, the
levels of T3, ALT and AST were increased significantly
(P ≤ 0.001, P ≤ 0.05) in the infertile semen of 41 - 60
years age group as compared to that of infertile semen of
25 - 40 years age group (Figure 3).
The levels of testosterone and Cu in the infertile serum
of 41 - 60 years age group were decreased significantly (P
≤ 0.001) as compared to that of fertile semen of 25 - 40
years age group. While, the levels of T3, T4, ALT and Fe
in the infertile serum of 41 - 60 years age group were in-
creased significantly (P ≤ 0.001, P ≤ 0.05) as compared to
that of infertile serum of 25 - 40 years age group (Figure
4).
4. DISCUSSION
The lower level of testosterone in the fertile serum of 41
- 60 years age group as compared to that of fertile serum
of 25 - 40 years age group indicates that testosterone
level is decreased in older age resulting in the decreased

Z. Mahmood et al. / Advances in Bioscience and Biotechnology 3 (2012) 574-579
576
Figure 1. Biochemical parameters evaluation in fertile semen of age group 2 & 4.
Figure 2. Biochemical parameters evaluation in fertile serum of age group 2 & 4.

Z. Mahmood et al. / Advances in Bioscience and Biotechnology 3 (2012) 574-579 577
Figure 3. Biochemical parameters evaluation in the infertile semen of age group 1 & 3.
Figure 4. Biochemical parameters evaluation in the infertile serum of age group 1 & 3.

Z. Mahmood et al. / Advances in Bioscience and Biotechnology 3 (2012) 574-579
578
sperm production. In consequence, the testosterone con-
centration is increased in the fertile semen of 41 - 60
years age group as compared to that of fertile semen of
25 - 40 years age group to increase the sperm production.
Significantly lower level of testosterone in the infertile
serum of 41 - 60 years age group represents that testos-
terone deficiency is more pronounced in older age. This
may be due to atrophy of glandular tissue of pituitary or
testes resulting in significantly lower level of testoster-
one in the infertile semen of 41 - 60 years age group.
This may also be due to prostatic inflammation and ob-
struction or blockage of the male reproductive tract.
Hence testis not functioning properly and testosterone
production is affected. Zinc deficiency in infertile semen
of 41 - 60 years age group also causes lower testosterone
production as zinc is involved in steroidogenesis, tes-
ticular development and testosterone synthesis [3]. The
similar results were found by Khan et al. [13]. They ob-
served the decrease in testosterone concentration in the
infertile individuals. Omrani et al. [14] and Weedin et al.
[15] also found the decreased testosterone level in the
infertile men.
[3] Ebisch, I.M.W., Thomas, C.M.G., Peters, W.H.M., Braat,
D.D.M. and Steegers-Theunissen, R.P.M. (2007) The im-
portance of folate, zinc and antioxidants in the patho-
genesis and prevention of subfertility. Human Reproduc-
tion Update, 13, 163-174.
Significantly lower level of zinc in the infertile semen
of 41 - 60 years age group in the present study might be
due to excessive ROS production. After ejaculation the
abnormal spermatozoa are sources of oxidants which
bind with the zinc and reduce its concentration in semi-
nal plasma. Hence, zinc is considered to be an important
antioxidant protecting the spermatozoa from oxidative
stress. Zinc deficiency decreases testicular weight and
causes shrinkage of the seminiferous tubules. Zinc defi-
ciency also induces atrophy of the seminiferous tubules
and causes failure of the spermatogenesis. Low seminal
zinc levels have been correlated with decreased fertility
potential [16]. Ali et al. [17] observed the low zinc level
in the infertile subjects and found that low zinc level
affected the semen parameters. Colagar et al. [18] found
that poor Zn nutrition may be an important risk factor for
low quality of sperm and idiopathic male infertility.
Significantly lower level of Copper in the infertile se-
men and serum of 41 - 60 years age group may be due to
liver toxicity as it is absorbed by the liver, bound to al-
bumin and excreted into the bile. It is also involved in
protein synthesis during certain enzymatic reactions act-
ing as a cofactor. Nutritional deficiency may also lower
its level in infertile men. Abdul-Rasheed [19] reported a
significant decrease in seminal plasma copper levels in
azoospermic patients. In the present research work, sig-
nificant increase of iron in the infertile serum might be
due to oxidative stress in infertile men. Aydemir et al.
[20] observed the increased iron level in the infertile men.
Eghbali et al. [10] reported that seminal plasma iron
content is related with the motility and viability of the
spermatozoa after ejaculation. Presence of iron in the
seminal plasma will assist spermatozoa to sustain their
functions as long as it is needed for semen fertility.
5. ACKNOWLEDGEMENTS
The authors express gratitude to the lab staff of Physiology and Phar-
macology Department, Faculty of Veterinary Sciences and Staff of Bio-
medical Lab, University of Agriculture Faisalabad, Pakistan. On pro-
viding lab facilities authors are also grateful to the Mian Muhammad
trust Hospital Faisalabad and Biocare Labs, Aziz Fatima Hospital Fais-
alabad, Pakistan.
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