Abstract

Background: Although trace amounts of copper (Cu) are necessary to maintain proper body functions, the excess amount can contribute to the development of hepatic dysfunction.

Objectives: This study aimed to investigate the relationship between copper treatment and changes in the serum concentration of high molecular weight alkaline phosphatase (HMW-ALP).

Materials and Methods: Male Wistar rats were injected intraperitoneally (IP) with copper (Cu) as copper chloride (CuCl₂. 4H₂O) 4, 2 and 1 mg/kg for 10, 30 and 60 days respectively. Animals were killed at indicated time and blood samples were collected, and sera was separated and used for alkaline phosphatase activity determinations and also for isoenzymes gel filtration chromatography and Sephacryl S-300 was used.

Results: Obtained data showed that with increasing administration of copper, the ALP activity was elevated significantly. In comparison with the control group the elevations were between 20%-56% using gel filtration chromatography. It was found that the elevation of serum ALP was mostly due to HMW-ALP.

Conclusions: The elevation of HMW-ALP activity in Cu treated animal suggests the occurrence of biliary disease. This may be used as a biomarker for the diagnosis of copper toxicity.

Keywords: Alkaline Phosphatase; Copper; Liver

1. Background

Copper (Cu), a redox active metal, is an essential nutrient for all species studied to date. During the past decades, there was increasing interests in the concept that marginal deficits of this element can contribute to the development and progression of a number of diseases, including cardiovascular disease and diabetes (1). Human Wilson’s disease, an inherited disease of copper metabolism is characterized by a failure of the liver to excrete copper, leading to its accumulation in liver and resulting chronic degeneration (2).

There are two classes of alkaline phosphates isoenzyms in human serum containing: tissue specific (germ cells, placental and intestine) and tissue non-specific (liver, bone and kidney), which encoded by different genes on chromosomes 2 and 1 respectively (3, 4). In pathological terms, a portion of liver isoenzyme that enters the plasma is called high molecular weight alkaline phosphatase. It is separated from other ALP isoenzymes using electrophoresis technique on cellulose acetate and/or other media.

Its estimated molecular weight is 1000 kDa (5). Increase of this isoenzyme in serum or plasma has been suggested as a sensitive test for homeostatic liver disease, as well as a tumor marker for liver cancer (6). This form of ALP isoenzyme may be influenced by some elements (7). Liver dysfunction in those patients (Wilson’s disease) with Cu overload (8) leads us to explore and compare the probable occurrence of high molecular weight ALP in the sera of rats treated with Cu.

2. Objectives

This project aimed to study the effect of copper treatment on the serum level of high molecular weight ALP.

3. Materials and Methods

All chemicals were of reagent grade and obtained from Sigma Chemical Company (USA). Male Wistar rats (200-220 g) were purchased from Pasture Institute (Tehran, Iran) and kept at standard conditions in our department animal house. For each experiment, 4 rats were chosen as controls and 4 rats for the experimental studies. Copper as (CuCl₂.4H₂O) in 4, 2 and 1 mg/kg was administered intraperitoneally for 10, 30 and 60 days daily, respectively. For the preparation of serum samples, rats were decapitated. The sera was then collected and used for the determination of alkaline phosphatase activity and also gel filtration chromatography.

Gel filtration chromatography technique separated high molecular weight ALP from low molecular weight isoenzyme. Serum samples (1 mL) were applied to pre-equilibrated column containing Sephacryl S300 (Pharmacia). The columns were eluted with Tris-HCl buffer (50 mM, pH 7.4) at 10 mL/h, and 2 mL fractions were collected. The activity of alkaline phosphatase in each fraction was determined according to the method of Bessey et al. (9) using P-nitrophenyl phosphate as substrate and 2-amino-2-methyl-1-propanol (AMP buffer, 0.84 mM, pH 10.3).

To study the electrophoretic mobility of high molecular weight alkaline phosphatase, agarose gel (1%) electrophoresis technique was performed. Gels were stained for alkaline phosphatase activity using a solution of α-naphthyl phosphate (50 mg/dL) and fast red (25 mg/dL) in bicarbonate buffer (0.1 M, pH 10.3) containing Mg as magnesium chloride (6).

Analysis of data was performed using SPSS 11 software and independent sample t-test was used to compare mean differences between results. All results were presented as mean ± SD and P < 0.05 were considered statistically significant difference.

4. Results

First series of experiments were established to investigate the effect of copper on the serum total activity of ALP. To achieve this, rats received 4, 2 and 1 mg/kg copper chloride daily for 10, 30 and 60 days. Administration of 4 mg/kg copper led to the elevation of enzyme activity by 20 %, whereas 52% and 58% elevations in the serum total ALP activity was seen following 2 and 1 mg/kg of copper administration for 30 and /or 60 days respectively (Table 1).

Table 1. Dose and Time Dependent Effects of Cu on the Total Activity of Serum Alkaline Phosphatase in Male Rats a,b,c,d

4.1. Gel Filtration Chromatography

In order to investigate the effect of copper on induced serum total ALP activity, gel filtration chromatography technique was used. The sera from copper treated, and untreated controls were loaded on the top of the column, and the column was eluted as mentioned. Figure 1 shows traces of high molecular weight ALP activity only in the sera of control subjects, whereas, a marked elevation (4-8 folds) of high molecular weight ALP was seen following copper treatment. The elevation is highly significant (P < 0.05) in comparison to untreated controls.

Figure 1. The Elution Profile of Serum High and Low Molecular Weight Alkaline Phosphatase of Control and Copper Treated Group

4.2. Electrophoresis Technique

Last experiment was done to separate high and low molecular weight ALP. Using gel electrophoresis technique on 1% agarose, high and low molecular weight ALP eluted fractions from gel filtration chromatography were separately pooled. The prepared high and low molecular weight isoenzymes were electrophoresed on 1% agarose gel. Figure 2 shows the prepared high molecular weight fractions of gel filtration chromatography migrated behind the low molecular weight isoenzyme, when applied to the gel.

Figure 2. Agarose Gel Electrophoresis (1%) of Low and High Molecular Weight Alkaline Phosphatase Isoenzyme

5. Discussion

The serum of patients with copper overload may contain high molecular ALP. The presence of this form of ALP in the serum could be related to the development of the biliary disease (10). Therefore, this form of enzyme can be used as a suitable biomarker for copper toxicity. Data presented in this study showed that treatment of rat with copper has led to a significant (P < 0.05) elevation of total serum ALP activity (Table 1). And this elevation was mostly due to the increased in the level of high molecular weight alkaline phosphatase.

It is now well documented that the existence of this form of ALP in the sera of biliary obstructive and metastatic liver cancer could be a good detector for the diagnosis of the disease (11). Patients suffer form Wilson disease face with copper overload in the liver with subsequent hepatic lenticular degeneration (12). However, elevation of high molecular weight ALP following copper treatment could be considered as a biomarker for the diagnosis of copper toxicity.

It may be also emphasized that early treatment of copper in patients with cupper toxicity may give them more chances for the survival time. Previous reports showed that patients with malignant liver disease had elevated high molecular weight ALP (13). Accumulation of Cu may cause disturbances in the liver of copper treated animals, and it might be due to either stimulation or release of this isoenzyme, particularly by bile duct cells and/or enhancement of biosynthesis of this isoenzyme following copper treatment. More studies are in progress in our laboratory to elucidate the exact molecular mechanism by which copper undertakes its action on the induction of high molecular weight ALP.

Acknowledgments

We thank all laboratory staff of Department of Biochemistry.

Footnotes

References