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The formation of biliary mud in chinese liver fluke infections
CLINICA
THE
CHIMICA
7
ACTA
FORMATION
OF BILIARY
MUD
IN CHINESE
United College,
The Chinese
LIVER
FLUKE
INFECTIONS*
L. MA**
of Chemistry,
DeFartment (Received
August
University
of’ Hong Kong
gth, 1967)
SUMMARY
The
chemical
constituents
of 16 specimens
of biliary
mud
collected
from
human cases of clonorchiasis are analysed. The formation of such mud is investigated and discussed. Polyphenol oxidase is believed to play an important part in the mud formation.
The Chinese liver fluke, Clonorchis
sinensis,
is a common
parasite
of man
and
the cat. It is largely distributed in Southeast Asia, the South China coast, in Japan and in Korea. In heavy infections the fluke causes blocking of the biliary passages and frequently gives rise to complications, such as cholangitis and formation of biliary mud1-5. The complications are usually initiated by obstruction of the main hepatic ducts or the common bile duct, aggravated by bacterial infection, such as
E. coli=. Hou3 showed that there is a close relationship
between carcinoma
of the liver
and infection by the flukes. This has been reviewed by Willis6 and Steiner’. The incidence of this infection is high among the Chinese in Hong Kong2+. Workers in Hong Kong have pointed out that the formation of biliary mud presents difficulty in medical or surgical treatment because it is recurrent. Hence the importance of studying the mechanism of such mud formation needs no emphasis. In the present study the chemical constituents of the biliary mud collected from human cases were ascertained. As a result of the analysis, the protein content was found to be high and the material gave a positive reaction in tests for scleroprotein, a quinone-tanned product. This has led us to investigate whether the mud formation would be associated with certain enzymes, such as polyphenol oxidase which is rich in eggs and which catalyses the reaction of biological tannings9s. It was subsequently found that when fresh eggs of the flukes OY purified polyphenol oxidase were incubated with bile containing E. coli in a test tube, black “mud” could be formed. The following is a report based on these investigations. * A part of this work was presented at the I rth Pacific Science Congress held in Tokyo, Japan, on September 2, 1966. ** Hon. Consultant in Clinical Chemistry, Caritas Medical Centre, Kowloon, Hong Kong. Clin. Chim. Acta, rg (1968) 7-10
8
MA
EXPERIMENTAL
(I)
Chemical ~~uZysis of ~iZia~y .mud Colleetiogzof specimens. 16 specimens were obtained : 6 were collected at surgical operations and were kindly supplied by Mr. James Cook, formerly Senior Surgical Specialist at the Queen Mary Hospital; IO were collected at post-mortems and were kindly supplied by Dr. Lillian SC. Pang and Dr. M. H. Chen. The specimens were dried at least for 4 weeks in a desiccator containing phosphorus pentoxide, at room temperature. They were then pooled, well mixed and stored until subjected to the following analyses. Organic analysis. Total nitrogen and protein nitrogen were determined by the micro-Kjeldahl methodlo; protein nitrogen was estimated after bile pigments and cholesterol were removed by chloroform in a Soxhlet extractor. The chloroform extract was then concentrated by heat evaporation in a boiling water bath; the resulting concentrate was diluted to 50 ml (EI) in a volumetric flask, 40 ml were accurately measured and further concentrated to approximately IO ml; they were then well mixed with IO g of gypsum and transferred quantitatively into a Whatman thimble for Soxhlet extraction with acetone (Ez). Gypsum was used to remove bile pigment+. EI was used for estimation of bile pigmentslO and Ez, after removal of acetone by evaporation, was used for estimation of cholesterol”. The results are shown in Table I. TABLE
I
ORGANIC
AND
Chemical
constit%4ents
INORGANIC
CONSTITUENTS
OF THE
BILIARY
MUD
g/r00 g dry sam@e
Total nitrogen Protein nitrogen Rile pigments Cholesterol Moisture and volatile substances at IIOO i: 9 Ash at 550” & 5” Sodium Potassium Copper Calcium Magnesium Phosphorus
24.32 8.15 39.88 16.22 I.15
I 8.74 j.TZ 0.85 0.78 1.65 1.04 3.05
It should be mentioned that the residue after chloroform extraction becomes greyish black in colour, suggesting the presence of inorganic substances. Inorganic alzalysis. The following inorganic ions were determined after the mud was incinerated at 550”: sodium and potassium by flame-photometry; copper, by diethyldithiocarbamate method12; calcium and phosphorus by methods described by King and Woottonlo; magnesium by estimating the colour complex formed with Titan yellow13. The results are shown in Table I. (2)
Formatiom of mud in vitro It has been observed that the mud formed in clonorchiasis is usually brownish
C&n. Chim.
Acta,
19 (1968)
pro
BILIARY
MUD IN CLONORCHIASIS
9
black in colour and that the worm becomes darkened when it is dead. We were interested in the colour change of the worm and the following experiments were carried out in the dark to investigate this problem: (a) Approximately 25 mg of the eggs collected from fresh worms in cats were mixed in a zo-ml test tube containing 5 ml of human bile (pH 8.04) and I ml of E. coli suspension in 0.9% NaCl, the protein concentration of which was 20 mg/roo ml. The mixture was incubated at 37’ and dark precipitates were observed after a period of 3-j days. When the mixture was incubated in the absence of the eggs, neither precipitates nor colour changes were observed. (b) By purified polyphenol oxidase. Since the eggs contain a high level of polyphenol oxidase, we used the purified enzyme instead of the eggs in the following experiment: IO mg of purified polyphenol oxidase, prepared from mushrooms obtained from L. Light and Co. Ltd., England, were mixed with 5 ml of human bile and I ml of E. coli suspension of the same protein concentration as used in {a). The mixture was incubated at 37” and dark precipitates were observed after a period of 5-7 days; more precipitates were formed when the mixture was allowed to incubate for a longer period. The amount of precipitate was, however, less than that obtained in the former experiment, because in the former, the eggs themselves formed part of the precipitate. (31 Tests f& sclero~~otei~imelaniptpigments The qualitative tests, which Hackman’* employed for detecting scleroproteins, are as follows: (a) Boil with 4 N ethanolic KOH ; a deep red colour indicates a positive test; (b) Heat with concentrated aqueous solution of KOH; disappearance of the pigmented colour of the sample indicates a positive test. Both the biliary mud, after chIoroform extraction, and the precipitates formed by polyphenol oxidase, bile, and l?‘. coli gave positive results by these tests. DISCUSSION
In previous studies*“?‘@on the chemical composition of the worm, it was found that the copper content was as high as 40 mgfroo g in the dry worm tissue. Investigations were made to see whether this metal was associated with certain enzymes, and subsequently, polyphenol oxidase was detected. Further study showed that this enzyme played an important part in the formation of the shell of the egg of the clonorchis. This enzyme was found to be distributed in the vitellaria, the uterus and in the eggs. The enzyme activity in the eggs was found to be particularly high. The hardening and darkening of the eggshell in the cockroach and in helminths have been shown by Pryor and Smyth et a1.8 to be due to the tanning properties of oxidized phenols: when phenol is acted on by polyphenol oxidase, the corresponding quinone is formed and reacts with the basic groups of the protein, introducing crosslinks between the protein chains. As a result, tanning of the eggshell takes place. These findings by Pryor are confirmed by Hackman who further shows that the phenolic group of tyrosine residues in the protein becomes oxidized to an o-quinone which also acts as a tanning agent.
Clin. Chim.
A&,
19 (1968)
7-m
IO
MA
The causes of gall-stones formation has recently been reviewed by Rains17 who concluded that : (I) the primary cause may be found by studies of bile-pigment metabolism and its physicochemical properties; (z) stasis of bile is of importance as it favours the aggregation of minute stones and subsequent additional layering of stones in an environment containing proteinous material or layers of bile of different specific gravity. Stasis also prevents the natural discharge of early minute stones into the duodenum; (3) infection plays its part as a cause of stasis, and it provides the ~Y~~~~~~~~s envirolzment in which stones will grow. It should be mentioned that stones formed in experimental rabbits by ligating the common bile duct after introducing E. coli were found to be pale-yellow in colour, However, in clonorchiasis the mud formed is brownish black. This mud gives a positive reaction in tests for scleroprotein, which strongly suggests that a biological tanning process is taking place. As polyphenol oxidase is involved in biological tanning of insect cuticles and helminth eggshell, it is believed that this enzyme plays an important part in the formation of biliary mud in heavy infections of Clonorchtis sine&s.
I am grateful to the Director of the Research Institute of Science and Technology of this University for a grant for these studies. REFERENCES I fi. H. DIGBY, Bait. J. Swg., 17 (1930) 578. 2 J. COOK, P. C. Hou, H. C. Ho ANI) A. J. S. MCFADZEAN, Byit, J. Swg., 42 (1954) 188. 3 P. C. Hou, J. Pathol. Bacterial., 72 (1956) 239. 4 J. FUNG, &it. J. Surg., 48 (1961) 404. 5 G. B. ONG. Arch. Surg., 84 (1962) 199. 6 R. A, WILLIS. Pathology oj Tzamours, 3rd ed., Butterworth, London, 1960, p. 241. 7 P. E. STEINER, in D. H. COLLIPZS (Ed.),Nlodwti Trends ilz PathoEogy, Butterworth, London,
1959, p- 3or. 8 ful. G. i%. PRYOR, PYOC. Roy. SOc. London R, 128 (Igz$o) 378, 393. g J. D. SMYTH AND J. A. CLEGG, ExptE. Parasitology, 8 (1959) 286. IO E. J. KING AND I. D. P. WOOTTON, Microanalysis in Medical Biochemistry, II
IZ 13
14 rg
London, 1956, PP. 39? 75, 102. G. A. HARRISON, Chemical Methods in Clinical Medicine, 376. A. EDEN AND H. H. GREEN, Biochem. J.3 34 (1940) 1202. V. G. G. HZWRY, J. Lab. C&z. Med., 23 (1937) 1079. R. H. HACKMAN, Biochem. J.. 54 (1953) 371. L. MA, J. Parasitol., 49 (1963) 197.
16 L. IMA, 1. Parasilol., 50 (1964) 235. 17 rl.J. H. RAINS, Bvit. Med. J., oo (1962) 685. CEin. Chim. Ada,
Ig (1968)
7-10
3rd ed., Churchill,
4th cd., Churchill, London,
1957. p.
THE
CHIMICA
7
ACTA
FORMATION
OF BILIARY
MUD
IN CHINESE
United College,
The Chinese
LIVER
FLUKE
INFECTIONS*
L. MA**
of Chemistry,
DeFartment (Received
August
University
of’ Hong Kong
gth, 1967)
SUMMARY
The
chemical
constituents
of 16 specimens
of biliary
mud
collected
from
human cases of clonorchiasis are analysed. The formation of such mud is investigated and discussed. Polyphenol oxidase is believed to play an important part in the mud formation.
The Chinese liver fluke, Clonorchis
sinensis,
is a common
parasite
of man
and
the cat. It is largely distributed in Southeast Asia, the South China coast, in Japan and in Korea. In heavy infections the fluke causes blocking of the biliary passages and frequently gives rise to complications, such as cholangitis and formation of biliary mud1-5. The complications are usually initiated by obstruction of the main hepatic ducts or the common bile duct, aggravated by bacterial infection, such as
E. coli=. Hou3 showed that there is a close relationship
between carcinoma
of the liver
and infection by the flukes. This has been reviewed by Willis6 and Steiner’. The incidence of this infection is high among the Chinese in Hong Kong2+. Workers in Hong Kong have pointed out that the formation of biliary mud presents difficulty in medical or surgical treatment because it is recurrent. Hence the importance of studying the mechanism of such mud formation needs no emphasis. In the present study the chemical constituents of the biliary mud collected from human cases were ascertained. As a result of the analysis, the protein content was found to be high and the material gave a positive reaction in tests for scleroprotein, a quinone-tanned product. This has led us to investigate whether the mud formation would be associated with certain enzymes, such as polyphenol oxidase which is rich in eggs and which catalyses the reaction of biological tannings9s. It was subsequently found that when fresh eggs of the flukes OY purified polyphenol oxidase were incubated with bile containing E. coli in a test tube, black “mud” could be formed. The following is a report based on these investigations. * A part of this work was presented at the I rth Pacific Science Congress held in Tokyo, Japan, on September 2, 1966. ** Hon. Consultant in Clinical Chemistry, Caritas Medical Centre, Kowloon, Hong Kong. Clin. Chim. Acta, rg (1968) 7-10
8
MA
EXPERIMENTAL
(I)
Chemical ~~uZysis of ~iZia~y .mud Colleetiogzof specimens. 16 specimens were obtained : 6 were collected at surgical operations and were kindly supplied by Mr. James Cook, formerly Senior Surgical Specialist at the Queen Mary Hospital; IO were collected at post-mortems and were kindly supplied by Dr. Lillian SC. Pang and Dr. M. H. Chen. The specimens were dried at least for 4 weeks in a desiccator containing phosphorus pentoxide, at room temperature. They were then pooled, well mixed and stored until subjected to the following analyses. Organic analysis. Total nitrogen and protein nitrogen were determined by the micro-Kjeldahl methodlo; protein nitrogen was estimated after bile pigments and cholesterol were removed by chloroform in a Soxhlet extractor. The chloroform extract was then concentrated by heat evaporation in a boiling water bath; the resulting concentrate was diluted to 50 ml (EI) in a volumetric flask, 40 ml were accurately measured and further concentrated to approximately IO ml; they were then well mixed with IO g of gypsum and transferred quantitatively into a Whatman thimble for Soxhlet extraction with acetone (Ez). Gypsum was used to remove bile pigment+. EI was used for estimation of bile pigmentslO and Ez, after removal of acetone by evaporation, was used for estimation of cholesterol”. The results are shown in Table I. TABLE
I
ORGANIC
AND
Chemical
constit%4ents
INORGANIC
CONSTITUENTS
OF THE
BILIARY
MUD
g/r00 g dry sam@e
Total nitrogen Protein nitrogen Rile pigments Cholesterol Moisture and volatile substances at IIOO i: 9 Ash at 550” & 5” Sodium Potassium Copper Calcium Magnesium Phosphorus
24.32 8.15 39.88 16.22 I.15
I 8.74 j.TZ 0.85 0.78 1.65 1.04 3.05
It should be mentioned that the residue after chloroform extraction becomes greyish black in colour, suggesting the presence of inorganic substances. Inorganic alzalysis. The following inorganic ions were determined after the mud was incinerated at 550”: sodium and potassium by flame-photometry; copper, by diethyldithiocarbamate method12; calcium and phosphorus by methods described by King and Woottonlo; magnesium by estimating the colour complex formed with Titan yellow13. The results are shown in Table I. (2)
Formatiom of mud in vitro It has been observed that the mud formed in clonorchiasis is usually brownish
C&n. Chim.
Acta,
19 (1968)
pro
BILIARY
MUD IN CLONORCHIASIS
9
black in colour and that the worm becomes darkened when it is dead. We were interested in the colour change of the worm and the following experiments were carried out in the dark to investigate this problem: (a) Approximately 25 mg of the eggs collected from fresh worms in cats were mixed in a zo-ml test tube containing 5 ml of human bile (pH 8.04) and I ml of E. coli suspension in 0.9% NaCl, the protein concentration of which was 20 mg/roo ml. The mixture was incubated at 37’ and dark precipitates were observed after a period of 3-j days. When the mixture was incubated in the absence of the eggs, neither precipitates nor colour changes were observed. (b) By purified polyphenol oxidase. Since the eggs contain a high level of polyphenol oxidase, we used the purified enzyme instead of the eggs in the following experiment: IO mg of purified polyphenol oxidase, prepared from mushrooms obtained from L. Light and Co. Ltd., England, were mixed with 5 ml of human bile and I ml of E. coli suspension of the same protein concentration as used in {a). The mixture was incubated at 37” and dark precipitates were observed after a period of 5-7 days; more precipitates were formed when the mixture was allowed to incubate for a longer period. The amount of precipitate was, however, less than that obtained in the former experiment, because in the former, the eggs themselves formed part of the precipitate. (31 Tests f& sclero~~otei~imelaniptpigments The qualitative tests, which Hackman’* employed for detecting scleroproteins, are as follows: (a) Boil with 4 N ethanolic KOH ; a deep red colour indicates a positive test; (b) Heat with concentrated aqueous solution of KOH; disappearance of the pigmented colour of the sample indicates a positive test. Both the biliary mud, after chIoroform extraction, and the precipitates formed by polyphenol oxidase, bile, and l?‘. coli gave positive results by these tests. DISCUSSION
In previous studies*“?‘@on the chemical composition of the worm, it was found that the copper content was as high as 40 mgfroo g in the dry worm tissue. Investigations were made to see whether this metal was associated with certain enzymes, and subsequently, polyphenol oxidase was detected. Further study showed that this enzyme played an important part in the formation of the shell of the egg of the clonorchis. This enzyme was found to be distributed in the vitellaria, the uterus and in the eggs. The enzyme activity in the eggs was found to be particularly high. The hardening and darkening of the eggshell in the cockroach and in helminths have been shown by Pryor and Smyth et a1.8 to be due to the tanning properties of oxidized phenols: when phenol is acted on by polyphenol oxidase, the corresponding quinone is formed and reacts with the basic groups of the protein, introducing crosslinks between the protein chains. As a result, tanning of the eggshell takes place. These findings by Pryor are confirmed by Hackman who further shows that the phenolic group of tyrosine residues in the protein becomes oxidized to an o-quinone which also acts as a tanning agent.
Clin. Chim.
A&,
19 (1968)
7-m
IO
MA
The causes of gall-stones formation has recently been reviewed by Rains17 who concluded that : (I) the primary cause may be found by studies of bile-pigment metabolism and its physicochemical properties; (z) stasis of bile is of importance as it favours the aggregation of minute stones and subsequent additional layering of stones in an environment containing proteinous material or layers of bile of different specific gravity. Stasis also prevents the natural discharge of early minute stones into the duodenum; (3) infection plays its part as a cause of stasis, and it provides the ~Y~~~~~~~~s envirolzment in which stones will grow. It should be mentioned that stones formed in experimental rabbits by ligating the common bile duct after introducing E. coli were found to be pale-yellow in colour, However, in clonorchiasis the mud formed is brownish black. This mud gives a positive reaction in tests for scleroprotein, which strongly suggests that a biological tanning process is taking place. As polyphenol oxidase is involved in biological tanning of insect cuticles and helminth eggshell, it is believed that this enzyme plays an important part in the formation of biliary mud in heavy infections of Clonorchtis sine&s.
I am grateful to the Director of the Research Institute of Science and Technology of this University for a grant for these studies. REFERENCES I fi. H. DIGBY, Bait. J. Swg., 17 (1930) 578. 2 J. COOK, P. C. Hou, H. C. Ho ANI) A. J. S. MCFADZEAN, Byit, J. Swg., 42 (1954) 188. 3 P. C. Hou, J. Pathol. Bacterial., 72 (1956) 239. 4 J. FUNG, &it. J. Surg., 48 (1961) 404. 5 G. B. ONG. Arch. Surg., 84 (1962) 199. 6 R. A, WILLIS. Pathology oj Tzamours, 3rd ed., Butterworth, London, 1960, p. 241. 7 P. E. STEINER, in D. H. COLLIPZS (Ed.),Nlodwti Trends ilz PathoEogy, Butterworth, London,
1959, p- 3or. 8 ful. G. i%. PRYOR, PYOC. Roy. SOc. London R, 128 (Igz$o) 378, 393. g J. D. SMYTH AND J. A. CLEGG, ExptE. Parasitology, 8 (1959) 286. IO E. J. KING AND I. D. P. WOOTTON, Microanalysis in Medical Biochemistry, II
IZ 13
14 rg
London, 1956, PP. 39? 75, 102. G. A. HARRISON, Chemical Methods in Clinical Medicine, 376. A. EDEN AND H. H. GREEN, Biochem. J.3 34 (1940) 1202. V. G. G. HZWRY, J. Lab. C&z. Med., 23 (1937) 1079. R. H. HACKMAN, Biochem. J.. 54 (1953) 371. L. MA, J. Parasitol., 49 (1963) 197.
16 L. IMA, 1. Parasilol., 50 (1964) 235. 17 rl.J. H. RAINS, Bvit. Med. J., oo (1962) 685. CEin. Chim. Ada,
Ig (1968)
7-10
3rd ed., Churchill,
4th cd., Churchill, London,
1957. p.