Comparative characterization and biochemical    analysis  of alkaline phosphatase from Echinococcus granulosus cysts and  Human serum 

Hydatidosis, a zoonitic parasitic disease, is one of the most important economic and health problem in Iraq and many other countries in the Middle East. It is caused by Echinococcus Multilocularis and Echinococcus granulosus, the larval stage of which is localized in the liver, lungs and other visceral organs of intermediate hosts such as sheep, goats and cattle.Human are considered to be an accidental intermediate host, either by eating improperly disinfected vegetables or by close contact with dogs. The mature form lives in the ileum of candies as its  definitive hosts, where it leads to an asymptomatic infection .Serology or immunodiagnostic tests are the most useful techniques for the detection of hydatid cysts in human patients.Objectives:One of the reasons why Echinococcus granulosus cysts are described as a public health problem is the lack of effective chemotherapy.Reviewing the literature bears in mind the possibility of an effectual and successful chemotherapeutic treatment in the early stages of cyst development, as indicated by many reported studies. Thus, the major problem in the management of Hydatid disease seems to be related to the lack of an efficient routine diagnostic test capable of identifying asymptomatic patients at early stages of the disease. Consequently, the need for a consistent diagnostic test, applicable for mass screening in hyperendemic regions, has become mandatory.
Much attention has been focused on the multilocularis species which represent the major type affecting western countries .These cumulative efforts have recently evolved a commercially available and reliable
immunodiagnostic test for the specific diagnosis of Alveolar ehinococcus.Unfortunately, the test fails to identify most of the patients suffering from cystic echinococcosis, caused by the closely related echinococcus granulosus.Hence, this will detract from the valuablity of the test in districts,where echinococcus granulosus is well known as the main endemic type,including Iraq, and necessitating the requirement for further studies with regard to echinococcus granulosus.The present study was conducted to estimate the kinetic parameters and biochemical characteristics of alkaline phosphatase in fertile and sterile cyst membranes from patients with hepatic cystic echinococcosis. The raised alkaline phosphatase serums of patients suffering cholestasis caused by gallstones were used as a representative alternate to Human liver- type alkaline phosphatase. Comparisons were aimed to reveal whether these two cyst membranes enzymes are different from each other and from the Human liver- type alkaline phosphatase.Subject and methods:In this study two samples were used, the cyst and human serum.Hydatid cysts were obtained from six hepatic cystic echinococcosis patients attending The Red Crescent Private Hospital, Baghdad, for surgical intervention from  January 2007 through August 2008. Based on ultrasonographic examination, only two were identified as fertile, three were sterile and one patient with calcified cyst.Fasting venous blood samples were acquired from patients with professional diagnosis of gallstones, attending the above hospital for surgical treatment. Serum was separated, 5'-nucleotidase activity was measured to confirm that the raised alkaline phosphatase activity is mainly due to liver isoenzyme Collected serum samples were pooled and divided into aliquots,  stored at -2 C  until further analysis.The present study was measurement of alkaline phosphatase activity in serum and cyst homogenate according to the method of kind and king.Kinetic studies will involve the affinity of these enzymes to their substrate, as well as the effect of other factors including hydrogen ion concentration (pH) and temperature. Heat inactivation and the effect of specific inhibitor, namely L-phenylalanine, are also to be examined on the activity of the three alkaline phosphatase enzymes.
Results:
 
1. Different alkaline phosphatase activity were observed in fertile cysts while there was no alkaline phosphatase activity in hydatid cyst homogenated from sterile cysts and still not able to be detected even
when larger volume of the hydatid cyst homogenated sample were used for the estimation and can be explained that by the presence or the absence of fresh protoscoleces in fertile or sterile hydatid cyst,
respectively. Because of this statement it can be differentiate between the sterile & fertile cyst.  
2. By the results of the biochemical characterization of alkaline phosphatase, there were different Km values, optimum pH and optimum temperature for each hydatid cyst homogenated & humanliver type.
3. The heat-sensitive specifically showed that the echinococcus granulosus alkaline phosphatase was more heat resistance than echinococcus multilocularis & liver-type enzyme.
4. The respond of different human alkaline phosphatse isoenzymes to L-phenylalanine have been shown to varied from low sensitive (liver and bone) to highly sensitive (placental and intestinal). Thus, the wide
range of IC observed herein, can be ascribed to the presence of isoenzymes other than liver type in the serum samples enrolled in this study. No inhibitory effect was observed on alkaline phosphatase from hydatid cyst homogenate.