CERULOPLASMIN ACTIVITY AND ITS RELATION TO COPPER LEVEL IN BLOOD SERUM OF FRIESIAN DAIRY COWS.

CERULOPLASMIN ACTIVITY AND ITS RELATION TO COPPER LEVEL IN BLOOD SERUM OF FRIESIAN DAIRY COWS.

GHADA A.E. MOHAMED; EMAN M.A. EL-NASER and HANAN K. ELSAYED^*

^*Dept. of Animal Med., Fac.of Vet. Med., Assiut Univ. Assiut. Egypt; Animal health Research Institute, Assiut, Egypt.

Email: dr_kada2012@yahoo.com

INTRODUCTION

Copper (Cu) is one of the essential and important trace elements for the normal health and growth of animals (Rucker et al., 2008). Clinical copper deficiency occur as a primary deficiency due to lack of copper intake or as secondary deficiency due to inhibition of normal copper homeostasis by the antagonistic minerals as molybdenum, sulphur and iron (Suttle, 1991).

Copper has an important role in the immune system helping to fight off infections and diseases. Inadequate copper status may be related to an increased incidence of infections at calving. Serum copper concentration is a reliable indicator of copper deficiency (Weiss, 2010).

Ceruloplasmin (Cp) is a metalloenzyme with oxidase activity that is associated with iron and copper homeostasis (Szczubiał et al., 2008). Each molecule of ceruloplasmin contains six to eight atoms of copper which influence its biological activity. Cereloplasmin has been used diagnostically to confirm copper deficiency (Laven and Livesey, 2007).

Ceruloplasmin (Cp) functions include transportation of copper in the blood to various tissues, oxidizing minerals most notably iron and manganese, and scavenging oxygen radicals to protect cells. Copper is involved in the antioxidant system through its presence in several significant proteins. Copper is present most commonly in the proteins ceruloplasmin and superoxide dismutase (SOD). Ceruloplasmin activity is diminished or absent without sufficient copper (Gropper et al., 2005).

Ceruloplasmin is released into the blood from the liver and constitutues about 60% of the circulating copper in the blood after meals (Smith and Cipriano, 1987) as a modulator of the inflammatory process; ceruloplasmin serves as anacute-phase protein. Acute phase proteins rise in the blood with infection and other inflammatory events (Ceron et al., 2005 and Gropper et al., 2005). Ceruloplasmin (Cp) evaluation gives an indication of the health status of cattle and could be used as a marker of animal health and welfare (Skinner et al., 2001 and Martinez-Subiela    et al., 2002). The percentage of copper associated with ceruloplasmin was 55% in serum (Kincaid et al., 1986).

Laven, et al. (2007) used serum Cp/serum Cu (sCu) for evaluation of copper status in cattle as marginal and low. The lower end of the normal range for serum copper concentration is reported to be 10 µmol/L, but serum copper concentrations were considerably lower than this when cases of copper deficiency were discovered (Laven et al., 2007). While serum copper concentration is an index of copper deficiency, it does not reflect dietary intake except when intake is below a certain level. Serum copper concentration increases under a number of conditions due to increased concentrations of ceruloplasmin (Mackenzie et al., 2001).

The aim of the present study was to evaluate the relationship between ceruloplasmin activity and copper concentration at three different stages of lactation in serum of dairy Friesian cows. Furthermore, ceruloplasmin to copper ratios was also investigated.

MATERIALS and METHODS

Animals

A total number of 60 Friesian dairy cows were included in this study. All cows were classified randomly according to their lactation stages into three different groups as early (4-6 weeks post-partum), mid (17-22 weeks) and late (25-30 weeks) post-partum. The age of cows ranged from 4 to 7 years. Cows were clinically healthy, and any cows that had diseases such as lameness or mastitis were excluded from the study depending on the clinical examination made before the collection of blood samples (Skinner et al., 1991 and Horadagoda et al., 1999). The copper status of all cows was categorized according to their Cp/Cu ratios into ‘adequate’, ‘marginal’ or ‘low’ as >19, 15–19 or <15 mg/μmol, respectively, according to Telfer et al. (2004).

Blood Sampling

For separation of serum samples, blood samples were collected from each cow from the jugular vein into plane tubes without anticoagulants. The blood samples were left to clot, then centrifuged to provide non hemolysed serum and frozen at −20°C until analysis.

Biochemical Assays

The activity of ceruloplasmin was measured by Assay Max Ceruloplasmin ELISAKit (Catalog No. EC4001-1 Assaypro) A polyclonal antibody specific for ceruloplasmin had been pre-coated into a specific microplate with removable strips. Ceruloplasmin in standards and samples is competed with a biotinylated ceruloplasmin sandwiched by the immobilized antibody and streptavidin- peroxidase conjugate. All unbound material was then washed away and a peroxidase enzyme substrate was added. The color development was stopped and the intensity of the color was measured. Serum was tested for copper using spectrophotometer (UV/ VIS spectrophotometer Optizem 3220 uv MECASYS Co., LTD, Korea.

Statistical Analysis

Values are presented as means ± standard deviation (SD) of the mean. For statistical evaluations, all data were processed via the statistical package for social science (SPSS 2000). At first, analysis of variance was carried out to study the effects of lactation stage and sample type on concentrations of copper and activities of ceruloplasmin. The statistical significance of differences between group means was estimated by the least significant difference test at P < 0.05.

RESULTS

Table 1showes concentrations of copper and activities of ceruloplasmin in serum and ceruloplasmin to copper ratios (Cp/ Cu ratios) in different lactation stages included in the study. Serum copper (Cu) concentrations were higher in the group of early lactation stage than the other groups in mid-lactation stage and late- lactation stage; the difference was significant (P < 0.05). Serum ceruloplasmin (Cp) activity was higher in the group of early lactation than the mid-lactation and late lactation stage groups (P < 0.05); Lactation stage has significant effect on ceruloplasmin to copper ratios (P <0.05).

Table 1: Effect of Lactation Stage on Mean Serum Copper Concentrations, Mean Serum Ceruloplasmin Activity, and mean Cp/Cu ratio.

* Different means in the same row differ significantly, P < 0.05, SD Standard deviation,

w.p.p. week(s) postpartum, CI confidence interval, sCp/sCu serum ceruloplasmin to serum copper

Table 2 summarises the pooled data of the three groups' of lactation stages used in this study (early, mid and late) Cu concentrations and Cp activities in serum. Serum ceruloplasmin activity and sCu were significantly correlated (r = 0.62, P < 0.01).

Table 2: Values of Mean ± SD, Number (n), range and 95% CI of Pooled Data of Copper and Ceruloplasmin in serum.

Table 3 showes the copper status of dairy cows based on sCp/sCu, the use of sCp/sCu identified more animals as marginal and low (P < 0.001).

The copper status of all cows was categorized according to their Cp/Cu ratios into ‘adequate’, ‘marginal’ or ‘low’ as >19, 15–19 or <15 mg/μmol, respectively, according to Telfer, et al. (2004).

Table 3: Copper status of dairy cows based on serum Cp/ serum Cu ratio.

DISCUSSION

The significant increase of ceruloplasmin activity in the group of early lactation suggested that parturition is associated with a physiologic acute response as a result of oxidative stress around calving, as has been described before (Koets et al., 1998; Castillo et al., 2005 and Hussein and Staufenbiel, 2012). Increased concentration of copper in the group of early lactation may be secondary to increased ceruloplasmin activity as most of copper is associated with this protein. Dairy cows undergoing stressful periods have physiologically increased blood levels of copper (Ward and Spears, 1999) and ceruloplasmin (Hussein et al., 2011).

Ceruloplasmin status should therefore be interpreted with caution in the days following parturition because it could be difficult to distinguish between the physiologic acute response of calving and a pathologic inflammatory process.

In comparison with those in plasma in the other researches recorded by (Paynter, 1982 and Kincaid    et al., 1986), decreased copper concentrations and ceruloplasmin activities in serum might be due to trapping of some ceruloplasmin and its copper in the fibrin clot formation.

The low level of sCp/sCu could be attributed to sequestration of ceruloplasmin and the copper associated with this protein into the clot during clot formation by attachment of the enzyme to the blood cellular fraction (Laven and Livesey, 2006).

Using sCp/sCu in calculating Cp/Cu ratio significantly altered individual sample status (P < 0.001). Using sCp/sCu identified more animals as low and marginal.

It can be concluded that, stage of lactation had a noticeable effect on the concentration of serum Cu, and the lowest concentration was found within few weeks before dry off. The highest concentration was in the early stage of lactation and the levels in the mid-lactation varied (Laven et al., 2007). Concerning the serum ceruloplasmin activity, the obtained results revealed that there was a highly significant (p≤0.01) decrease in ceruloplasmin activity in –late lactation-group. This finding offers diagnostic possibilities for copper deficiency.

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