Research Article

 

Activity of Phosphatase Enzymes, Concentration of Protein and Divalent Ions in Sheep Sera during different Physiological Status

 

Noor Khalid Zaidan, Wissam Mahmud Mohamed*, Ahmed Salman Hamad

College Veterinary Medicine, University of Fallujah, Iraq.

Editor | Asghar Ali Kamboh, Sindh Agriculture University, Tandojam, Pakistan.

Received | August 23, 2015; Revised | September 01, 2015; Accepted | September 02, 2015; Published | September 29, 2015

*Correspondence | Wissam Mahmud Mohamed, University of Fallujah, Iraq; Email: wissamatea@yahoo.com

Citation | Zaidan NK, Mohamed WM, Hamad AS (2015). Activity of phosphatase enzymes, concentration of protein and divalent ions in sheep sera during different physiological status. J. Anim. Health Prod. 3(4): 78-81.

DOI | http://dx.doi.org/10.14737/journal.jahp/2015/3.4.78.81

ISSN | 2308–2801

Copyright © 2015 Zaidan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

Introduction

 

Sera chemistry is essential on diagnosis, treatment and estimation of diseases. In order to explain biochemical data correctly, the results obtained in the lab must be compared with values alike to healthy material, these called reference values. A big number of factors such as breed, species, age, sex, illness, reproductive state (pregnancy) and seasonal difference can effect on the sera chemistry values (Swanson et al., 2004; Nazifi et al., 2003; Yokus et al., 2006). There are many studies on the effects of different stages of the reproduction cycle on biochemical data in local animal species. In sheep studies were also carried out in relation to lactation and pregnancy (Iriadam, 2007; Giuseppe et al., 2009). These physiological states were considered to regulate metabolism in animals (Krajnicakova et al., 1993). Lactation is a very demanding period for the animal when they have increased nutritional needs (Antunovic et al., 2011).

 

Enzymes are a list of proteins of great biological importance. The presence, or partly the activity, of these catalysts (at times also called ferments) facilitates biochemical processes in the organism, the total of which is referred to as metabolism (Berg et al., 2006; Rochling, 2011; Šoch et al., 2008). Phosphatases enzyme have been classified as alkaline phosphatase (Alp) and acid phosphatase (Acp) depending on optimum pH required for their catalytic activity (Homayon et al., 2009). Alkaline phosphatase (Alp) belongs to hydrolytic category that results the hydrolysis of monoesters of phosphoric acid (Mohr et al., 2007). Alp is made up of many isoenzymes – the, carcinoplacental, placental, intestinal, renal and bone isoezymes (Sato et al., 2005). Alp is present in all tissues of the body, especially in the cell membrane and kidney tubules; and it occurs at high levels in epithelium, liver, intestine, placenta and bone (Beckett et al., 2000; Valocky et al., 2007). Acp is a hydrolytic enzyme, catalyse the hydrolysis of different phosphomonoesters in acidic pH and cause liberation of an inorganic phosphate (Miteva et al., 2010). Phosphatases enzymes are involved in many biological processes such as signal transduction pathways and energy metabolism (Shan, 2002). Divalent ions are essential for vitamin synthesis, enzyme activity, hormone production, collagen formation, tissue synthesis, energy production, oxygen transport and some physiological processes linked to growth, health and reproduction (Gürdoğan et al., 2006). The zinc ion is an important constituent of several enzymes mainly carbonic anhydrase and alkaline phosphatase, in the organic matrix (Gürbüz et al., 2002). Magnesium ion play vital role in the body and is mostly deposited in the skeleton. Magnesium is one of the 4 bulk metals in the body. It is a co-factor for about 300 cellular-enzymes such as alkaline phosphatase (Funda et al., 2004). The present study was accomplished to contrast the levels of metabolites in sera of pregnant and lactating sheep. We measured the levels of alkaline phosphatase, acid phosphatase, total protein, Zn+2 and Mg+2, in addition to calculation of specific activity of Alp and Acp.

 

Material and Methods

 

Study Subjects

This study was applied in 46 healthy female sheep aged 2–4 years with average body weight of 42.1±3.4 kg. Animals were divided into three groups: First group (G1; n = 15) were pregnancy, the second group (G2; n = 16) were lactating and third group (G3; n= 15) were non pregnant and non-lactating (control group).

 

Sample Collection

A 10 ml of blood was collected via jugular venipuncture of each animal, disposable syringes were used and sterile needles 18 gauge x 11/2 inches. The blood samples were placed in glass tubes, sera were separated by centrifugation (10 min) at 3000 rpm. Sera concentrations of total protein and acid phosphatase (Acp) were measured by using a commercial kits (Biolabo SA, France). The serum concentrations of alkaline phosphatase (Alp) was determined using the commercial available kits (BioSystems S.A. Spain) according to a previous report (Sema et al., 2009).

 

Determination of the Divalent Ions

One ml of sera was digested with 3ml of a mixture (6HNO3: 1HClO4) in a glass test tube. The mixture was analysed by atomic absorption spectrophotometer (GBC 933 plus), with air-acetylene flame and hollow cathode lamp (Medhat et al., 2013). Serum Mg and Zn concentrations were determined by using commercial kits with spectrophotometer (Oladipo and Temiye, 2005).

 

Statistical Analysis

Data were statistically analysed by SPSS (Statistical Package for the Social Sciences). All parameters were presented as means ± S.D (Sema et al., 2009).

 

Results

 

Biochemical values obtained from pregnant sheep (G1), lactating sheep (G2) and control group (G3) for different states are summarized in Table 1. Higher (P< 0.05) concentrations of total protein were determined in the serum of sheep in pregnant compared to lactating and control group. The activity and specific activity of Alp were higher (P< 0.01 or P< 0.05) in lactating than pregnant and control group. On the contrary a higher (P< 0.01 or P< 0.05) activity and specific activity of Acp were determined in the blood of pregnant compared to lactating and control group. Results further revealed that the lowest (P< 0.05) serum Zn+2 and Mg+2 levels in pregnant (G1) compared with lactating (G2) and control group (G3).

 

Discussion

 

There were some differences in biochemical values parallel to physiological change ruminants and sheep after and before parturition (Ali et al., 2014). The essential objective of present study was to investigate probable effects of reproductive states on the sera chemistry and divalent ions concentrations in sheep. Higher concentrations of total protein were determined in pregnant compared to lactating and control group. Same concentrations of serum total proteins in pregnant sheep and those in lactating sheep have been found by (Antunović et al., 2002). Decrease of serum total protein over the lactation could be explained by a fast extraction of immunoglobulin from the blood during the last months of pregnancy (Kaneko et al., 2008).

 

Table 1: Phosphatase enzymes, concentration of protein and divalent ions in sheep sera

Group	Acp (U/l)	Alp (U/l)	Total Protein (mg/l)	Specific Activity of Acp (U/ mg)	Specific Activity of Alp (U/mg)	Zn ion (µg/dl)	Mg ion (mg/dl)
G1	18.12 ±2.63a	512.32±36.50b	70.01± 3.02a	0.25 ± 0.02a	7.30 ± 0.20b	48.50 ± 0.41c	2.47±0.41b
G2	14.32± 5.75b	717.53± 53.11a	63.12± 2.06b	0.22± 0.08b	11.37 ± 0.48a	51.02 ± 0.31b	2.94±0.18a
G3	7.24 ± 4.62c	390.22 ± 57.50c	74.03± 3.01a	0.11 ± 0.07c	5.62 ± 0.93c	59.30 ± 0.22a	2.50±0.21b

 

G1= pregnant sheep; G2= lactating sheep; G3= control group; Means with different superscript letters differ significantly (P≤ 0.05); Acp= acid phosphatase; Alp= alkaline phosphatase

 

Our results showing that Alp activity was higher in lactating than pregnant and control group. Increase of Alp activity during lactation is due to an increase in the production of isoenzyme from bone (Sema et al., 2009). The authors (Yokus and Cakmr, 2006; Yokus et al., 2006) suggested that Alp activity in pregnancy were higher than those in lactation period. The result of this study were consistent with some literature (Birgel et al., 1997; Khan et al., 2002; Sema et al., 2009). Higher concentrations of Acp activity were determined in serum of pregnant compared to lactating and control group. Decreased concentrations of Acp activity in lactating sheep have to be considered as a result of constant energy loss with the milk (Antunovi, 2011). Calculate specific activity of enzyme to know the real activity of enzyme in different stages. Specific activity (S. activity) is the number of enzyme units / ml divided by protein concentration in mg per ml.

 

Results revealed that specific activity of Acp was higher in pregnant sheep compared to lactating sheep and control group, whereas a higher specific activity of Alp was determined in serum of lactating compared to pregnant and control group. These results of specific activity exactly matching with the result of enzyme activity. Moreover, present study showed the lowest serum Zn and Mg levels in pregnant (G1) compared with lactated (G2) and control group (G3). The reason for the decrease in the concentration of Zn and Mg ions levels obtained in pregnant sheep compared with lactating could be belong to the foetus requirement. Due to the high Zn+2 and Mg+2 levels being released through involution of the uterus (Gürdoğan et al., 2006). It has been reported (Funda et al., 2004; Seyrek et al., 2006) that the Zn levels of serum in sheep should be between 80-120 µg/dl. Similar results were obtained from control group (59.3 ± 0.22µg/dl). The levels of serum Zn ion in pregnant and lactating sheep were lower than in control group. Higher concentration of Zn and Mg ions in lactating sheep could be connected with higher activity of Alp that could be further contributed to the enzyme requires zinc (Zn+2) and magnesium (Mg+2) or calcium (Ca+2) divalent ions for activity (Steitz, 1999).

 

In conclusion, results of this study showed that values of Acp, Alp activities and total protein could be used as an indicator of pregnancy in sheep.

 

CONFLICT OF INTEREST

 

The authors declare that there is no conflict of interest.

 

ACKNOWLEDGEMENT

 

The authors are grateful to the Department of Biochemistry, School of Veterinary Medicine, University of Fallujah, Iraq for providing the facilities to carry out this work.

 

AUTHOR’S CONTRIBUTION

 

Wissam Mahmud Mohamed designed the study and carried out laboratory work, Ahmed Salman Hamad facilitated the study by collecting and arranging samples Noor Khalid Zaidan carried out laboratory work and finalised the manuscript.

 

References

 

Ali A, Qureshi MS, Adil M, Sikandar A, Ihtesham–Ul–Haq, Awais MM, Hussain A, Khan A (2014). Impact of blood metabolite profile and milk yield on fertility of dairy cows. J. Anim. Health Prod. 2(4): 55 – 59. http://dx.doi.org/10.14737/journal.jahp/2014/2.4.55.59

Antunović Z, Senčić Đ, Marcela Š, Branko L (2002). Influence of the season and the reproductive status of ewes on blood parameters. Small Rum. Res. 45(1): 39-44. http://dx.doi.org/10.1016/S0921-4488(02)00109-8

Antunovi Z, Novoselec J, Sauerwein H, Speranda M, Vegara M, Pavic V (2011). Blood metabolic profile and some of hormones concentration in ewes during different physiological status. Bulgarian J. Agri. Sci. 17(5): 687-695.

Beckett GJ, Walker SW, Alistaire F (2000). Lecture notes on clinical biochemistry. 6thed, Eds Blackwell Science UK.82-83.

Berg JM, Tymoczko J, Stryer L (2006). Biochemistry. ed. 6th. New York : Freeman . W. H. and Company, 1120 p. ISBN 10:071676766.

Birgel EH, Tefen S, Zerbe H (1997). Investigation on the enzyme profile during the preparatory phase of birth in cattle. Praktische Tierarzt. 78: 120-126.

Funda K, Kamil S, Aybeg B, Serap A (2004). Determination of the levels of zinc, copper, calcium, phosphorus and magnesium of chios ewes in the AydÝn Region. Turk. J. Vet. Anim. Sci. 28: 609-612.

Giuseppe P, Giovanni C, Claudia G, Grasso F, Runzo SC, Zumbo A (2009). Selected biochemical serum parameters in ewes during pregnancy, post parturition, lactation and dry period. Anim. Sci. Papers Rep. 27(4): 321-330.

Gürbüz A, Tekin Ş, İbrahim Ç (2002). Effects of zinc oxide administration on body weight gain and some biochemical parameters in lambs. Turk. J. Vet. Anim. Sci. 26(1): 85-90.

Gürdoğan F, Yildiz A, Balikci E (2006). Investigation of serum cu, zn, fe and se concentrations during pregnancy (60, 100 and 150 days) and after parturition (45 days) in single and twin pregnant sheep. Turk. J. Vet. Anim. Sci. 30: 61-64.

Homayon R, Siavash S, Behzad S (2009). Purification and kinetic study of bone and liver alkaline phosphatase isoenzymes in the dog. Comp. Clin. Pathol. 19: 81-84.

Iriadam M (2007). Variation in certain haematological and biochemical parameters during the peri-partum period in Kilis does. Small Rumin. Res. 73: 54-57. http://dx.doi.org/10.1016/j.smallrumres.2006.11.001

Kaneko JJ, Harvey JW, Bruss M (2008). Clinical biochemistry of domestic animals, 6th ed. Elsevier/Academic Press, Amsterdam.

Khan A, Bashir M, Ahmad KM, Javeda MT, Tayyabc KM, Ahmad M (2002). Forecasting neonatal lamb mortality on the basis of haematological and enzymological profiles of Thalli ewes at the pre-lambing stage. Small Rumin. Res. 43: 149 156. http://dx.doi.org/10.1016/S0921-4488(01)00269-3

Krajnicakova M, Bekeova E, Hendrichovscky V (1993). Concentrations of total lipid, cholesterol and progesterone during oestrus synchronization and pregnancy in sheep. Vet. Med. 38: 349 357.

Medhat AS, Emad AT, Sahar AI, Hisham DG, Hanan AM, Nagwa M G (2013). Lead and Cadmium assay in serum and semen of infertile men attending andrology clinic in Assiut university hospital (Rural versus Urban). J. Environ. Sci. Toxicol. Food Technol. 2(4): 38-45. http://dx.doi.org/10.9790/2402-0243845

Miteva R, Zapryanova D, Fasulkov IV (2010). Investigations on acid phosphatase activity in the seminal plasma of humans and animals. Trakia J. Sci. 8: 20–23.

Mohr M, Sharifi K, Eidi S (2007). Hematology and serum biochemistry of Holstein dairy calves: Age related changes and comparison with blood composition in adults. Res. Vet. Sci. 83(1): 30-39. http://dx.doi.org/10.1016/j.rvsc.2006.10.017

Nazifi S, Saeb M, Rowghani E (2003). The influences of thermal stress on serum biochemical parameters of Iranian fattaile sheep and their correlation with triiodothyronine, thyroxine and cortisol concentrations. Comp. Clin. Path. 12: 135–139. http://dx.doi.org/10.1007/s00580-003-0487-x

Oladipo OO, Temiye E O (2005). Serum magnesium, phosphate and calcium in Nigerian children with sickle cell disease. WAJM .24(2): 120-123. http://dx.doi.org/10.4314/wajm.v24i2.28180

Rochling FA (2001). Evaluation of abnormal liver tests. Int. Clin. Cornerstone. 3(6): 1-12. http://dx.doi.org/10.1016/S1098-3597(01)90074-2

Sato J, Kanata M, Yasuda J, Sato R, Okada K, Seimiya Y, Naito Y (2005). Changes of serum alkaline phosphatase in dry and lactional cows. J. Vet. Med. Sci. 67(8): 813-815. http://dx.doi.org/10.1292/jvms.67.813

Sema YG, Gurgoze A, Kafar Z, Nihat O, Hosan I (2009). Investigation of Some biochemical parameters and mineral substance during pregnancy and postpartum period in Awassi ewes. Kafkas Univ. Vet. Fak. Derg. 15: 957-963.

Seyrek K, Pasa S, Kiral F. Bildik A, Babur C, Kilic S (2004). Levels of zinc, copper and magnesium in sheep with toxoplasmosis, Uludag Univ. J. Fac. Vet. Med. 23: 39-42.

Shan J (2002). Dissertation on: Transcriptional regulation of the human prostatic acid phosphatase gene. Research Centre for Molecular Endocrinology. Pp. 15.

Steitz TA (1999). DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274(25): 17395–17398. http://dx.doi.org/10.1074/jbc.274.25.17395

Swanson KS, Kuzmuk KN, Schook LB, Fahey GC (2004). Diet affects nutrient digestibility, hematology, and serum chemistry of senior and weanling dogs. J. Anim. Sci. 82: 1713–1724.

Šoch M, Písek L, Brouček J, Kroupová P, Šilhavá M, Stastna J (2008). Activity of alkaline phosphatase in cattle blood plasma according to stage of pregnancy. Slovak J. Anim. Sci.41: (1): 39 – 41.

Valocky I, Legath J, Lenhardt L, Lazar G, Novotny F (2007). Activity of alkaline phosphatase, acidic phosphatase and nonspecific esterase in the oviducts of puerperal ewes after exposure to polychlorinated biphenyls. Veterinarni Medicina 52: 186-192.

Yokus B, Cakmr DU, Kanay Z, Kanay Z, Gulten T, Uysal E (2006). Effects of seasonal and physiological variations on the serum chemistry, vitamins and thyroid hormone concentrations in sheep. J. Vet. Med A. 53: 271-276. http://dx.doi.org/10.1111/j.1439-0442.2006.00831.x

Yokus B, Cakmr DU (2006). Seasonal and physiological variations in serum chemistry and mineral concentrations in cattle. Biol. Trace Elem. Res. 109: 255-266.

Zvonko A, Josip N, Marcela S, Mensur V, Valentina P, Boro M, Mislav D (2011). Changes in biochemical and haematological parameters and metabolic hormones in Tsigai ewes blood in the first third of lactation. Archiv Tierzucht. 5: 535-545.