Study on the cholesterol metabolism of fasciola hepatica
The mechanism of the lipids metabolism of parasites is still not completely known. Warren (1957) observed variation in the total amount of ether-soluble material present in Hymenolepis diminuta. Shorb and Shorb (1962) reported that swine nematodes (Oesophagostomum quadrispinulatum, O. dentatum, ascaris lumbricoides, Trichuis suis) contained palmitic, stearic, oleic and linoleic acids. Harrington (1965) demonstrated that Hymenolepis diminuta had a mean lipid content of 5.2% of the fresh worm weight, 21.2% of the dry weight. Beames (1964) and Beames (1965) analyzed the volatile and non-volatile acids in the neutral fats from Ascaris lumbricoides and found phospholipids. Ginger and Fairbairn (1966) reported the total lipids of Hymenolipis diminuta comprised 5.8% of the fresh body weight, and cholesterol was the only sterol found.
With regard to the lipid metabolism of rarasites, Han (1963) observed that the converison of (14)**C-acetate was not incorporated into the cholesterol fractionin Clomorchis sinensis. Fairbairn et al. (11961) found that lipids were not meatbolized during starvation in Hymenolipis diminuta. Meyer et al. (1966) found that Spirometra mansonoides lacked the mechanisms required for the synthesis de novo of its sterols. Jacobson et al. (1967) observed a significance in vitro rate of incorporation of 1-(14)**C-acetate into the lipids of Hymenolepis diminuta.
In regard to the interrelation between the growth rate of the parasite and lipids especially cholesterol, Wyss et al. (1960) found that oleic acid showed better growth effect on Trichomonas foctus in the absence of cholesterol, but this effect was not observed in the presence of it. He also observed that linoleic and linolenic acids were toxic to the trichomonads in the absence of cholesterol, but the reverse was experienced in the presence of cholesterol.
Fasciola hepatica inhabits the bile ducts and it leads to te suggestion that a bile duct is the bset site to obtain bile containing large amounts of cholesterol. The present study analyzes the cholesterol content of viliary and hepatic parasites and compares this content with that of other parasites, and also the cholesterol metabolism in the liver fluke.
Materials and Methods
1. Parasites used
1) Fasciola hepatica:
Parasites were collected from cattle bile ducts at a local slaughter house. They were washed repeatedly with saline to measure cholesterol.
2) Parasites for comparative studies
a. Hepatic parasites
The metacercariae of Clonorchis sinensis were collected from raw fish (Pseudorasbora parva) by digestion with artificial gastric juice, and were introduced directly into the rabbit stomach.
After 2 months the adult worm of Clonorchis sinensis were collected from the bile ducts and used for this experiment after repeated washing with saline.
A piece of liver tissue of non-infected (control) and infected with Capillaria hepatica were used for the measurement of cholesterol.
b. Pancreatic parasite
These were collected from the pancreatic duct of infected cattle and used for the experiment after repeated washing with saline.
c. Intestinal parasites
Ascaris lumbricoides and Taenia saginata:
These worms were obtained following the administration of anthelminthics, and were prepared as above.
2. Lyophilization of parasites
Fasciola hepatica, Clonorchis sinensis, Eurytrema pancreaticum and Ascaris lumbricoides were frozen at -70℃ for 20 hours. Each of the worm was powdered after desiccation and kept in an ampoule.
3. Incubation of fasciola hepatica and the survival time in the medium fasciola hepatica was removed from the bile ducts of cattle at a local slaughter house and immediately transferred to laboratory. After repeated washing the active worms were incubated at 35℃ in following media.
1) Tyrode solution 15mml,
2) Tyrode solution 7.5ml. with horse serum 7.5ml.
Tyrode solution 10ml. with cattle serum 5ml.
3) Tyrode solution 15ml. with cattle bile 0.15ml.
4) Tyrode solution 7.5ml. with horse serum 7.5ml. and cattle bile 0.15ml.
After incubation for 12∼72 hours, the cholesterol content of the worm and media were measured. Also the survival time fo Fasciola hepatica in the above media was checked.
4. Measurement of the cholesterol content
1) Serum and bile
Cholesterol content of serum measured by the method of Kingsley and Schaffert (1949) and of bile by Eriksson's method (1957).
The method described by Oser (1965) was used to measure cholesterol content.
1. The cholesterol content of the parasites varied according to the site of parasitism.
Fasciola hepatica had a mean cholesterol content of 4.19mg/gm at fresh state and 22.35mg/gm in the lyophilized state. In contrast, Clonorchis sinensis contained 3.33mg/gm in the fresh state and 22.08mg/gm in the lyophilized with Capillaria hepatica was greater than that of the non-infected liver tissue in Rattus
The pancreatic fluke, Eurytrema pancreaticum, had a cholesterol content of 4.42mg/gm in the fresh state and 20.55mg/gm in the lyophilized state. The values correspinded to those found in Clonorchis sinensis and fasciola hepatica.
Among the intestinal parasites, Ascaris lumbricoides contained 0.31mg/gm of cholesterol in the fresh state and 1.67mg/gm in the lyophilized state. Especially in the female worm the cholesterol content of the anterior half was greater than that of the posterior half of the worm. This content was less than that of the hepatic parasites. Taenia saginate had a mean cholesterol content of 1.57mg/gm in gravid proglottides and 3.4mg/gm in the mature proglottides. The cholesterol amount in gravid proglottides was less than that of the mature or immature proglottides.
2. Survival time of Fasciloa hepatica in various media.
After incubation in Tyrode solution for 24 hours, most of the worms were dead. However the survival time was prolonged in the media containing cholisterol. The relation between the mean survival time and the cholesterol content of the media was significant.
3. Changes in cholesterol content in the worm and the media after incubation.
No change in the cholesterol content of the worm and the medium was observed after incubation in Tyrode solution for 72 hours. During incubation for 12∼72 hours the cholesterol content increased in the worm but decreased in various media containing cholesterol.
Summary and Conclusion
1. Hepatic parasites (Fasciola hepatica, Clonorchis sinensis and Capillaria hepatica) and pancreatic fluke (Eurytrema pancreaticum) contained a larger amount of cholesterol than did intestinal parasites (Ascaris lumbricoides and gravid proglottides of Taenia saginats.)
2. The survival time of Fasciola hepatica in the media containing cholesterol was longer than those in cholesterol free media.
3. The amount of cholesterol in Fasciola hepatica which were incubated in media containing cholesterol increased, whereas the content of cholesterol in the media were decreased. No change in cholesterol content was observed when Fasciola hepatica was incubated in Tyrode solution.
The results indicate that Fasciola hepatica needs cholesterol for its survival but has no capacity for cholesterol biosynthesis and merely absorbs chlesterol from the local environment.