Avicenna J Med Biochem. 2014 September; 2(1): e18255.
DOI: 10.17795/ajmb-18255
Research Article
Published online 2014 September 25.
High Molecular Weight Alkaline Phosphatase Changes Following Animal
Copper Treatment
1,*
2
Jamshid Karimi ; Ali Asghar Moshtaghie ; Bahram Haghighi
2
1Department of Biochemistry and Nutrition, Hamadan University of Medical Sciences, Hamadan, IR Iran
2Department of Biochemistry, Isfahan University of Medical Sciences, Isfahan, IR Iran
*Corresponding author: Jamshid Karimi, Department of Biochemistry and Nutrition, Hamadan University of Medical Sciences, Hamadan, IR Iran. Tel: +98-8138380462, Fax: +988138380208, E-mail: [email protected]
Received: February 20, 2014; Revised: April 14, 2014; Accepted: April 21, 2014
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
3.1. Reagents
All chemicals were of reagent grade and obtained from
Sigma Chemical Company (USA). Male Wistar rats (200220 g) were purchased from Pasture Institute (Tehran,
Iran) and kept at standard conditions in our department
Implication for health policy/practice/research/medical education:
The elevation of high molecular weight alkaline phosphatase activity during copper (Cu) treatment of animals may suggest the occurrence of biliary
disease. This may be used as a biomarker for the diagnosis of copper toxicity.
Copyright © 2014, Hamadan University of Medical Sciences; Published by Hamadan University of Medical Sciences. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.
Karimi J et al.
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.
tigate 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).
4.1. Gel Filtration Chromatography
3.2. Column 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.
Gel filtration chromatography technique separated
high molecular weight ALP from low molecular weight
isoenzyme. Serum samples (1 mL) were applied to preequilibrated 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-amino2-methyl-1-propanol (AMP buffer, 0.84 mM, pH 10.3).
3.3. Electrophoresis
Figure 1. The Elution Profile of Serum High and Low Molecular Weight
Alkaline Phosphatase of Control and Copper Treated Group
25
23
21
19
17
15
13
11
9
7
1
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.
H
5
3.4. Statistical Analysis
L
40
38
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
3
ACTIVITY(IUL)
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).
FRACTION NO
CONTROL
CU2+(2mg/Kg)
CU2+(1mg/Kg)
CU2+(4mg/Kg)
Sera from both Cu-treated and untreated controls were loaded on the top
of column containing Sephacryl S-300. The column was eluted as mentioned in methods. High (H) and low (L) molecular weight ALP were separated, and enzyme activity was measured.
4. Results
First series of experiments were established to inves-
Table 1. Dose and Time Dependent Effects of Cu on the Total Activity of Serum Alkaline Phosphatase in Male Rats a,b,c,d
Group
A
B
C
CuCl2.4H2O, mg/kg
Exposure Duration, d
4
ALP, IU/L
Control (n = 4)
Treated (n = 4)
10
95.6 ± 3.1
120.9 ± 44c
2
30
93.2 ± 5.2
142.3 ± 2.1c
1
60
90.1 ± 3.2
144.4 ± 3.2c
a Abbreviation: ALP, alkaline phosphatase.
b Data presented as mean ± SD.
c P < 0.05 compared to corresponding control.
d Rats were treated with copper for different period of times. Animals were decapitated at indicated times. Sera were collected and alkaline phosphatase
total activity was measured.
2
Avicenna J Med Biochem. 2014;2(1):e18255
Karimi J et al.
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
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.
Acknowledgements
We thank all laboratory staff of Department of Biochemistry.
Authors’ Contributions
Performing in vitro and vivo experiments, data interpretation, preparing the manuscript: Jamshid Karimi;
Planning and supervising all experiments: Ali Asghar
Moshtaghie; and Planning of experiments: Bahram
Haghighi.
Funding/Support
This research was financially supported by Isfahan University of Medical Sciences.
High (lane 1) and low (lane 2) molecular weight ALP eluted fractions from
gel filtration chromatography were separately pooled. H and L show the
location of high and low molecular weight enzymes on the gel.
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