Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
726
PURIFICATION OF LACTATE DEHYDROGENASE-C4 FROM SEMINAL
PLASMA OF MALE INFERTILITY
Shihab A. Al-Bajari* , Nashwan I. Al-Lehebe**, Sarmad A. Hazzaa***
*Medical Analysis ,Mosul Technical Institute,Northern Technical University,Mosul, Iraq
.**Department of chemical.College of Education ,University of Mosul ,Mosul, Iraq
***Community Health ,Medical Baghdad Institute ,Middle Technical University ,Baghdad, Iraq
Key words:Seminogram,Lactate dehydrogenase, male infertility.
Corresponding Author; Shehab.unv.79@gmail.com
ABSTRACT
Male factor infertility contributes partially and solely to the problem of childlessness in
around 50% of the cases. Unfortunately, 30 -- 50% of the etiologies of male infertility are unknown
and therefore, no specific therapy can be instituted. Sperm count and sperm motility are prime
parameters that determine the functional ability of spermatozoa.This study included determination
of lactate dehydrogenase-C(LDH) activity in male infertility (n=23)and its relationship with the
different parameters of the seminogram. Also deals with partial purification lactate
dehydrogenasefrom semen plasma of male infertility patients by dialysis, and ion exchange DEAEcellulose
techniques. There was statistically significant correlation was found between LDH activity
and Sperm Count , morphology % and Motility % (r=0.486), (r=0.263)and (r=0.444) in fertile group
respectively, also that the correlation in infertile group was statistically significant between LDH
activity and Sperm Count , morphology % and Motility % (r=0.431), (r=0.671)and (r=0.336)
respectively.
Oneproteinous peak of lactate dehydrogenaseactivity is obtained with specific activity of 0.861u\mg
protein and purification fold 6.10 compared to crude enzyme. The kinetic characteristics of partially
purified lactate dehydrogenaseare studied. The maximum activity is obtained at 250μg of enzyme,
Tris-Hcl buffersolution at pH 7.9, temperature 37ᵒC, incubation time 5 min., with 10mMK+, Vmax
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
727
and Km values of partially purified lactate dehydrogenase1.176 unit/ml and 27.02 mM with the
substrate sodium pyruvateare found respectively.
INTRODUCTION
Male infertility represents a very challenging area of clinical medicine. Many different types
of medications have been tried and very few have had satisfactory results. There is a huge need to
advance and develop andrologic diagnostic techniques, focusing on the metabolomics and
proteomics of the sperm, seminal plasma, and testicular tissue. Clarification of the causes of
idiopathic male infertility and the discovery of novel molecular targets will help guide future
innovative development of new pharmacologic agents(1). Infertility is defined as the lack of ability
to conceive within one year of unprotected intercourse with the same partner. It is estimated that
nearly 8–12% of couples are infertile, and approximately 30–40% of infertility cases are caused by
male factors(2). Several risk factors are involved in the pathogenesis of infertility, some of which
include alterations in spermatogenesis due to testicular cancer, aplasia of the germinal cells,
varicocele, defects in the transport of sperm, or environmental factors(3.5).As well as congenital
anomalies, infectious diseases, bilateral spermaducts, pregnancy-related infections, alterations in the
characteristics of semen such as a decrease in sperm motility and sperm count, the presence of
antisperm antibodies (ASAs), and nutritional deficiency of trace elements such as selenium and zinc
(Zn). (3).The lactic dehydrogenase C4 (LDH-C4), also named LD H -X, is an isoenzyme of lactic
dehydrogenase (EC 1.1.1.27) specific in mature testicles and sperm , whose beginning is closely
associated with the starting of active spermatogenesis, being specific for certain types of germinal
cells of the seminal epithelium (4). Reports have been published in which LDH activity in the
seminal fluid is shown to be the highest for all corporal fluids, LD H -C 4 making the major
contribution to the total LDH activity in fertile individuals. The present study was carried out to
demonstrate the practical usefulness of the quantitative analysis ofLDH-C4 activity in seminal
plasma as a tracer of germinal activity and to prove with a simple and slight noninvasive test that it
is possible to get an idea of in-depth physiological events that differentiate the spermatogenesis in
fertile and infertile men(5)
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
728
MATERIAL AND METHODS
The study was conducted in Al-Mosul governorate, 23 infertile menpatients with different
infertility potential. Those selected infertile men group include 23 patientswith age range (33-41)
years with primary infertility and sperm count less than 4 million/milliliter (grade A according to
World Health Organization (1999).The control group include 25 apparently healthy fertile
volunteers with normal seminal parameters according to WHO (1999)(6).All semen samples were
obtained by masturbation after 3 to 4 days of abstinence. The samples were collected at the
laboratory and examined after liquefaction at 37°C. The initial analysis included assessment of the
volume, viscosity, pH, number, motility, viability (expressed as stained spermatozoa percentage)
and morphology of the spermatozoa. Seminal plasma was obtained by centrifugation for 15 minutes
at 400 X g and then for 5 minutes at 6000 Xg.(5)
Measurement of Seminal Plasma lactate dehydrogenase–Cactivity:
LDH activity was measured by the method of Brown et al 1975.The assay mixture, directly made in
a 3 mL cuvette, contains 1.8 ml of Tris-buffer; 1.0 ml of 1.8 mM sodium pyruvate; and 0.1 ml of 5
mM NADH. The reaction is initiated by the addition of 0.1 ml of diluted enzyme from the different
purification steps and the decrease in A340 as a function of time is monitored(7).
Purification of LDH:
Purification of LDH from seminal plasmaof Infertile Men.
Step I: Dialysis
Ten milliliter of serum was dialyzed against 10mM Tris-Hcl buffer (pH 8.6).The solution was
stirred overnight with a magnetic stirrer at 4oC. The buffer was changed every 6 hrs. during dialysis
(8).
Step II: Ion Exchange Chromatography
Ten milliliter of dialyzed enzyme solution was applied on DEAE cellulose anion exchanger column
(2.5x40) cm, followed by Tris-Hcl buffer (pH 8.6). Elution of the protein was carried out at a flow
rate (50) ml/hour with a definite time (6) min, using Tris-Hcl buffer as eluent. The fractions were
collected and the protein in each fraction was detected by following the absorbance at wavelength
280 nm. peak was combined separately from the plot of an absorbance versus elution volumes and
LDH was determined in each fraction (9)
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
729
Lyophilization: The fractions which contain LDH activity were collected and concentrated by
lyophilizer at -20'C.
Properties of the purified LDH:
Enzyme concentration: The partially purified LDH was added to the reaction mixture with
different concentrations to study the best concentration of enzyme. The partially concentratedelution
volume used were 50,100,150,200,250,300and 350 μl.
Buffers type: The partially purified enzyme reaction mixture were incubated with the following
buffers: Tris-HCl, Na-Na-phosphate, Na-K-phosphate، Citric acid, Sodium acetate.
pH range: The purified enzyme reaction mixture was incubated at different pH values 6.3,6.9,7.3,
7.9,8.5and 9.1using more effective buffer above on enzyme reaction.
Temperature: The partially purified LDH activity was measured at 10,20، 30،37,50,60,70and
80C`.
Incubation time: The partially purified enzyme is incubated at different times
(3,4,5,6,7,8,9,10,11,12 and 13 min) to study the best time for reaction
Metal ions: All these metal ions Na+l, K+1, Mg+2 and Ca+2 were applied on the reaction mixture
with a concentration of 10mM.
Substrate concentration: The substrate, Sodium pyruvatewas applied on the reaction mixture at
different concentrations (10,,20,30,40 and 50mM).
Statistical analysis: All values are reported as mean ± SEM. Statistical significance was assessed
using Student's t-test. P value less than 0.05 was accepted as the significance level. The correlation
coefficient was analyzed statistically using spss-program (p<0.001).
RESULTS AND DISCUSSION
Levels of some biomarkers in fertileand infertile men . The concentration of seminal LDH was
significantly decreased in infertile males 1792U/mg against 820U/mg in fertile males.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
730
Table 1: Seminal fluid analysis and LDH parameters[Mean ± SD] of fertile and infertile groups.
Semen parameter
Fertile group
N=25
Infertile group
N=23
Age (years) 36±4.3 35±5.4
Volume (ml) 3.77±0.78 3.42±0.6
Sperm Count (Million/ml) 60± 8.9 3.3±0.7*
morphology % 43.6±3.5 17.9 ±3.7*
Motility % 60.6±6.29 31.9±7.03*
LDH (U/mg) 1792±68.55 820±183.0*
Data are reported as mean ± SD. *P<0.05 compared to control (Student t-test).* significant.
There is controversy about the role of LDH activity in sperm viability. Different studies
have reported that: absent and diminished LDH activity in seminal plasma was associated with
male infertility and there was a strong positive correlation between LDH-C4 activity and sperm
density (10).Human seminal plasma contains several enzymatic systems that play an important role
in the normal function of sperm. Many studies suggested that decreased levels of this enzyme in
seminal plasma might be a potential cause of infertility but there were always contradictory between
reports (11). In our research we tried to explore the seminal activity of LDH in seminal plasma of
our collected samples , we noted that the activity of seminal LDH was lower in abnormal groups
than normal groups. Noguera 1993 showed also that the seminal LDH activity from healthy subjects
wasthree times greater than that from infertile males. Efficiently, the increased activity of LDH in
seminal plasma of normospermic men suggested that higher activity of this enzyme catalyzes the
ROS which might protect sperm against per-oxidative damage (5)and also plays role in sperm
maturation from the early events up to the onset of fertilization (10). Reduction of LDH in seminal
plasma may lead to reduce fertilizing capacity and defective sperm quality (12).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
731
Significantly lower levels of lactate dehydrogenase have been observed in infertile males.Also a
significant positive correlation was found between LDH activity and Sperm Count , morphology %
and Motility % (r=0.486), (r=0.263)and (r=0.444) in Fertile group respectively , also that the
correlation in Infertile group was a significant positive between LDH activity and Sperm Count ,
morphology % and Motility % (r=0.431), (r=0.671)and (r=0.336) respectively.
Purification LDH: A representative purification profile of the LDH active fractions is summarized
in Table 3. male infertilityLDH was purified to about 6.10 fold compared to crude enzyme with
173% activity recovery. From the elution profile of proteins, which is shown in Fig.1, LDH active
fractions were represented in the fractions between 24 to 35.
Table 3: LDH-C4 purification steps from seminal plasma of Infertile Men.
Purification
steps
Volume
(ml)
Totalprotein
(mg)
Total activity
(U)*
Specific activity
(U/mg protein)
Yield %
Purification
fold
Crude 10 93 13.2 0.141 100 1
Dialysis 9 81.9 13.9 0.169 105 1.19
Ion exchange 82 26.61 22.93 0.861 173 6.10
U: A unit is defined as that amount of enzyme which reduce I micrornole of LDH per min.
Fig. 1: Elution profile of LDH purification enzyme activity seminal plasma of Infertile Menby
DEAE- cellulose chromatography column (40x2.5 cm). LDH activity at 270nmΔ , ProteinO
Protein
activity
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20 25 30 35 40 45 50
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3
Absorbance(Δ A) at 340
nm
Fraction No.
Absorbance at 280 nm
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
732
Effect of enzyme conc. on reaction velocity: Fig. (2) showed that LDH activity was linearly
proportional to the amount of protein up to250μg .
(Fig.2) Effect of enzyme concentration on the activity of the partiallyLDH-C4
Effect of buffers and pH on the reaction velocity: LDH-C4 activity assayed with different
buffers. It was found that LDH activity exhibited maximum activity Tris-Hcl buffer Fig. (3)
Fig.3: Effect of pH values on the activity of the partially purified LDH-C4
When assayed the LDH-C4 activity at different pH (6.3 to 9.1) of Tris-Hcl buffer, it exhibited
optimum activity at pH:7.9Fig. (4)This value was different from those of human pH 7.5 (13)and
liver pH 7.8 (14).The optirnum pH for different enzymes varies depending on the nature of catalytic
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
733
groups. The stability of the tertiary and I or quaternary structures of the enzyme may also be pH
dependent and may affect the velocity of the enzyme reaction, especially at extreme alkaline or
acidic pH values (11).
Fig. 4: Effect of buffer type on the activity of the partially purified LDH
Effect of temperature on the reaction velocity: The activity of LDH-C4 was determined at
different temperature (10-70)ᵒC. It is clear from the results presented in Fig. (5) that LDH exhibited
a maximum temperature at 37ᵒC .
Fig. 5: Temperature effect on the activity of the partially purified LDH-C4
The optimum temperature for the enzyme activity from the heart of Karamanos 2014 was around
40'c (15). The enzyme reaction has optimum temperatures and then rapidly decrease with further
temperature increase. The loss of activity at the higher temperatures is due to thermal
conformational (denaturation) changes of the enzyme. Most enzymes are inactivated at
temperatures above 55-60.(16).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
734
Effect of enzyme incubation time on the reaction velocity: The activity of LDH-C4 was
determined every 1 min. The maximum activity was at 5 min (Fig.6)
Fig.6 Effect of incubation time on the activity of the partially purifiedLDH-C4
Effect of ions on the activity of LDH :LDH activity was determined with different ions (1-Na+1 ,2-
K+1,3-Mg+2, 4-Ca+2) conc. at l0mM. As shown in Fig (7)
Fig.7: Effect of ions (10mM) on the activity of the partially purifiedLDH-C4
Effect of substrate concentration on enzymatic activity: The effect of different substrate
concentration was tested by incubating different substrate concentration (10 to 50mM) with the
same amount of the enzyme. The enzyme activity was plotted against substrate concentration. The
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
735
results in Fig.(8) demonstrated that 30 mM of substrate was the best of the optimal LDH
activity.Our study illustrated the values of Vmax and Km were 1.176 U/ml and 27.02mM
respectively .
Fig. 8: Linewaver-Burk plot of LDH-C4
The Km value of pig heart LDH was 6mM by using pyruvate، as substrate (15),The velocity
increases with the increase in substrate concentration up to a certain point and then becomes
constant and reaches a maximum velocity. The high value of Km indicates that there is a low
enzyme affinity toward substrate. The Km value is affected by substrate, pH and temperature (17)
CONCLUSION
In conclusion, the LDH activities in sperm and semen were significantly correlated with seminal
qualityinfertile samples . A seminal LDH stimulation may be a useful tool for determining sperm
fertilization potential. However, the results of this study could provide a database for further
research into the effects of LDH on sperm. fertility is a complex process involving many factors.
The real roles played by LDH in sperm quality merit further investigation.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
736
REFERENCES
1- Hamada J.A., Montgomery M. and Agarwal A.(2012) .Male infertility: a critical review of
pharmacologic management .Expert Opin. Pharmacotherp72(10):747-54.
2- Cardona-Maya W., Velilla P. A., Montoya C. J., Cadavid Á. &Rugeles M. T.(2011). In
vitro human immunodeficiency virus and sperm cell interaction mediated by the mannose
receptor. J ReprodImmunol 92, 1–7.
3- Mahdi B. M.(2011). Frequency of antisperm antibodies in infertile women. J .ReprodInfertil 12,
261–5.
4- Mahat (.2016) . Risk Factors and Causes of Male Infertility-A Review, Biochem Anal Biochem
2016, 5:2.
5- Tsutsumi A, Yamamoto Y, Okamura K, Oki Y, Hamada K, Ishizu H.(1984) LDH (lactate
dehydrogenase) isoenzyme in human semen and seminal stains. Hanzaigaku Zassi;50: 41-8.
6- Noguera Velasco (1993). Lactic dehydrogenase-C4 activity in seminal plasma and male in fertility.
Fertility and Sfertility; 60:(2),333-335.
7- WHO (1999). Laboratory manual for the examination of human semen and sperm-cervical
mucus interaction. 4th ed. Cambridge University Press, Cambridge. pp12-34.
8- Brown A., Constance P. and Robin L.(1975)"Purification,Characterization,and Regulation of a
Nicotinnamide Adenine Dinucleotide-Dependent Lactate Dehydrogenase from
Actinomycesviscosus" J of Bacteriology1126-1135.
9- Awasthi, Y. C.; Beutler, E. and Srivastava, S. K. (1975) .Purification and properties of
human erythrocyte glu- tathione peroxidase. J. Biol. Chem., 250,5144 - 5149.
10- Hsieh YY, Chang CC and Lin CS(2006). Seminal malondialdehyde concentration but not
glutathione peroxidase activity is negatively correlated with seminal concentration and motility. Int
J Biol Sci. 2 (Suppl 1): 23-29.
11- Sawane M.(2005). Seminal LDH-C4 isozyme: relationship with routine seminal parameters.
Journal of Datta Meghe Institute of Medical Sciences University1(1):15-19.
12- Robyt, F.J. and White, J.B. (1987). Biochemical techniques, theory and practice .
Brookes/Cloe Publishing Company, Monterey, California, pp. 115-118, 294-295.
13- Faanyetal.(2011)" Lactate Dehydrogenase C and Energy Metabolism in Mouse Sperm"
BiolReprod 85(3): 556–564.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
737
14- Royal J., Robert B. and George N.(1998) .Optimum Reaction Conditions for Human Lactate
Dehydrogenase lsoenzymes as They Affect Total Lactate Dehydrogenase Activity.GAY ET AL.
Clinical Chemistry.Vol. 14. No. 8.
15- JennferA. .(2017) .Lactate dehydrogenase kinetics and inhibition using a microplate reader
Biochemistry and Molecular Biology Education, 10(2):1-6.
16- Daniel R.M. .(2010) The molecular basis of the effect of temperature on enzyme
activity.Biochem. J. 425, 353–360.
17- Karamanos Y.(2014) Purification and characterisation of lactate dehydrogenase: an
undergraduate biochemistry laboratory experiment.Advances in Biochemistry; 2(1): 14-23
18- Ahmed, T.Y. and Alhilali, L.A. (2010). Biochemistry . Part I, IbnALatheer Publishing, Mosul
university, Iraq, p278. (In Arabic)
