Biochemical analyses of the cyst wall of Echinococcus granulosus batsch

Comp. Biochem. Physiol., 1971, Vol. 40Bt pp. 797 to 805. Pergamon Press. Printed in Great Britain

BIOCHEMICAL ANALYSES OF THE CYST WALL OF ECHINOCOCCUS G R A N U L O S U S BATSCH* A. V. S A D A N A N D t Department of Biological Sciences, Madurai University, Madurai, South India (Received 17 March 1971) Abstract--1. The organic and inorganic constituents of the laminated mem-

brane of the cyst wall of Echinococcus granulosus are protein 7.5 per cent, carbohydrates 2 per cent, lipids 1"3 per cent and ash 1.6 per cent in terms of fresh wt. Water content is 82.4 per cent. 2. There is no significant variation in the chemical composition of this membrane collected from human and bovine hosts. INTRODUCTION

VARIOUS WORKERShave studied the composition of the hydatid fluid and germinal membrane of the hydatid cyst, scolices and hooks of the adult Echinococcus granulosus (Mazzocco, 1923; Lemaire & Ribere, 1935; Agosin et al., 1957; Kilejian et al., 1961; Gallagher, 1964). Previous studies carried out on a quantitative basis of certain organic components of the cyst wall related to tissues which were a combination of the laminated and germinal layers (Cmelik, 1952; Cmelik & Briski, 1953). On the basis of qualitative studies it was reported that mucopolysaccharide forms the principal structural element of the laminated membrane of the hydatid cyst and that amino acids formed only a small fraction of this membrane (Kilejian et al., 1962). This membrane was shown to consist of a network of fibres on which dense granular particles were scattered (Morseth, 1967). Practically no quantitative data are available on the biochemical composition of the laminated membrane as distinct from the germinal membrane. The present investigations were therefore carried out in order to obtain quantitative data on the organic and inorganic constituents of the laminated membranes of fertile hydatid cysts of Echinococcus granulosus from bovine and human hosts. A comparative study of the amino acids present in this membrane from fertile and sterile bovine cysts was also attempted. MATERIALS AND METHODS

Hydatid cysts of Echinococcus granulosus were collected from patients who underwent surgery at the Government Erskine Hospital, Madurai and from freshly slaughtered cattle at the Municipal abattoir. Analyses of carbohydrates, lipids and proteins were made on the * This paper formed part of the dissertation submitted to Madurai University for the award of the degree of Master of Philosophy in Zoology during April 1969. t Present address: Department of Social and Preventive Medicine, Madurai Medical College, Madurai-13, South India. 797

798

A . V . SADANAND

laminated membranes of three fertile cysts from the lung of a female patient and four fertile cysts from the lungs of an ox. Analyses of amino acids were carried out on the laminated membranes of three fertile and three sterile cysts taken from the liver of cattle. Ash, inorganic contents and water content were estimated in the laminated membranes of fertile cysts, seven from the lungs of cattle and four from the peritoneal cavity of a patient. F o r all biochemical studies, the cysts collected were immediately brought to the laboratory, the cyst fluid drained off and the cyst wall separated from the surrounding adventitious tissues of the hosts. T h e germinal layer was scraped off with a scalpel. The laminated membrane thus isolated was rinsed in distilled water repeatedly, homogenized and carbohydrates, lipids and proteins were estimated. Amino acids, water content, ash and inorganic constituents of freshly collected tissues were also determined. Carbohydrate was estimated by the phenol-sulphuric acid method (Dubois et aI., 1956). Lipids and protein were estimated by standard methods (Raymont et al., 1964). Analyses of amino acids were carried out by means of unidimensional, two-dimensional and thin-layer chromatograms. F o r each analysis a weighed quantity of tissue was hydrolysed by heating with 6 N HC1 over a sand-bath under reflux for 18 hr. The hydrolysate was neutralized and evaporated to dryness over a water-bath. The n-butanol extract of the residue was spotted either on filter paper (Whatman No. 1) or on a glass plate coated with silica gel-G following the standard methods. For unidimensional chromatography the solvent used was a mixture of n-butanol, acetic acid and water (4 : 1 : 1). For two-dimensional runs, a mixture of pyridine, isoamylalcohol and water (10 : 10 : 7) was used. Spots were detected by spraying 0-2% ninhydrin in butanol. Amino acids present were identified by comparison with standards run simultaneously. The individual spots were assessed densitometrically. Ash was determined by heating weighed samples in silica crucibles at 600°C for 6 hr in a muffle furnace. Water content was estimated by drying weighed quantities of the membrane in a hot air oven at 60°C to constant weight. The difference between the wet and dry weights was considered as being the water content of the tissue. Mineral constituents of the ash were determined by standard qualitative methods. Quantitative determinations of calcium, magnesium and phosphate were made following the methods of Fiske & Subbarow (1925). T h e other elements and radicals found in traces were not estimated quantitatively. After eliminating the phosphate, calcium was precipitated as oxalate, washed and dissolved in 2 N sulphuric acid and titrated against standard permanganate solution. Magnesium was estimated by precipitation as oxinate in 2 N hydrochloric acid by adding pure potassium bromide and titrating against 0"1 N KBro3 with methyl red as the indicator until a slight excess of bromate solution was added. This excess was accurately determined using 0"1 N thiosulphate solution using starch as an indicator and the excess titre deducted from the individual titre value. RESULTS R e s u l t s o f d e t e r m i n a t i o n s o f c a r b o h y d r a t e s , l i p i d s a n d p r o t e i n in t h e l a m i n a t e d m e m b r a n e s o f fertile cysts f r o m h u m a n a n d b o v i n e h o s t s are p r e s e n t e d in T a b l e s 1 a n d 2 r e s p e c t i v e l y . A s m a y b e seen f r o m T a b l e 1, t h e c a r b o h y d r a t e c o n t e n t r a n g e d f r o m 1.6 to 2.38 p e r c e n t w i t h a m e a n v a l u e o f 2-01 p e r c e n t in h u m a n p a r a s i t i c tissue. L i p i d s , o n t h e o t h e r h a n d , r a n g e d f r o m as low as 0-7 to 1"96 p e r c e n t w i t h a m e a n o f 1.31 p e r cent. T h e p r o t e i n c o n t e n t was c o n s t a n t l y h i g h r a n g i n g f r o m 6 to 8.55 p e r c e n t w i t h a m e a n o f 7.53 p e r cent. T h e c o m p o s i t i o n o f t h e l a m i n a t e d m e m b r a n e f r o m fertile cysts o f t h e b o v i n e h o s t is m o r e o r less t h e s a m e as t h a t f r o m t h e h u m a n host. I n t h e case o f t h e cyst m e m b r a n e f r o m t h e h u m a n h o s t t h e w a t e r c o n t e n t r a n g e d b e t w e e n 81.5 a n d 83.8 p e r c e n t w i t h a m e a n o f 82.6 p e r cent w h e r e a s in t h e m a t e r i a l f r o m t h e b o v i n e h o s t it v a r i e d f r o m 75.8 to 88.9 p e r c e n t

10-50 20-22 20-05 20"65 15"00 15-75 12"50 18-20

1 2 3 4 5 6 7 8

0.25 0-48 0-45 0"33 0-26 0-29 0"26 0"34 Mean

2"01

2-38 2-38 2"24 1"60 1"73 1 "84 2"08 1'86

Percentage carbohydrate 50.00 63.00 60.50 58"65 50.00 75-00 72"50 62'45

Tissue taken (rag) 0,50 1"01 0"76 0"43 0-70 0"64 1"12 1"22 1-31

1-10 1-60 1"25 0"73 1.40 0-85 1"55 1-96

Percentage lipid content

Mean

Lipid content (rag)

Sample No.

Carbohydrate content (nag)

Tissue taken (rag wet wt.)

Lipids

Carbohydrates

7.6 8.3 10"0 10-75 13"62 15.55 16'50 18-25

Tissue taken (nag)

0.65 0-71 0-65 0.84 0"98 1 "26 0-99 1"37

7.53

8.55 8.55 6"50 7"81 7"20 8"10 6.00 7'50

content

Percentage protein

Mean

Protein content (rag)

Protein

T A B L E 1 - - B I o c H E M I C A L STUDIES ON THE LAMINATED MEMBRANE OF FERTILE HYDATID CYST WALL FROM HUMAN HOST

o

r~

13.8 11.5 9.8 9.1 15.5 10.5 12.6 12.5

1 2 3 4 5 6 7 8

0.23 0.19 O.19 0.24 0.28 0.22 0"23 0.25 Mean

1"93

1.63 1.65 1.89 2-58 1.81 2"09 1.82 2-00

Percentage carbohydrate 198.0 130.5 115.5 142-0 150.0 120.0 136.25 140.0

Tissue taken (mg) 2.1 1.8 1.9 1.6 2.2 1.9 1-7 1.7 1'34

1.06 1.37 1.64 1.l 2 1.46 1-58 1.24 1.21

Percentage lipid content

Mean

Lipid content (mg)

Sample No.

Carbohydrate content (rag)

Tissue taken (rag wet wt.)

Lipids

12.8 10.2 7.8 18.0 16.0 19-4 20.2 15.0

Tissue taken (mg)

1.05 0-65 0.65 1.45 1.25 1.45 1.25 1-13

7"48

8.20 6.40 8.33 8"05 7.81 7-47 6"18 7.53

Percentage protein content

Mean

Protein content (mg)

Protein

STUDIES ON THE L A M I N A T E D M E M B R A N E OF F E R T I L E H Y D A T I D CYST W A L L F R O M BOVINE HOST

Carbohydrates

TABLE 2--BIOCHEMICAL

>

>

U~ > ~7

.<

5185 2350 3500

2650 1575 1200

Human

Bovine

Mean

Mean

82"20

75.80 81"96 88"85

82"63

38"76 81-50 82"62

Percentage of water

Water content

2-3524 1.5235 1"7520

2"2431 5.1235 3.5000

Weight of tissue taken (fresh wt. gm)

AND B O V I N E I N T E R M E D I A T E H O S T S

0"0468 0"0268 0.0231

0"0332 0.0661 0"0602

Weight of ash obtained (gin)

Mean

Mean

1"686

1.98 1 "76 1"32

1-496

I "48 1 "29 1 "72

Percentage of ash

Ash content

OF E S T I M A T I O N S OF W A T E R A N D ASH C O N T E N T S I N T H E L A M I N A T E D M E M B R A N E S OF F E R T I L E H Y D A T I D CYSTS F R O M H U M A N

Intermediate Weight of tissue taken host (rag)

TABLE 3--RESULTS

oo

0

0

H rrJ ¢3

0

0¢)

47-42

Bovine

23 "66

19"36

Calcium

1 "05

1-07

Magnesium

Sodium Potassium Chloride

Sodium Potassium Chloride

Other constituents found (not estimated)

Iron Aluminium

Iron Aluminium

Traces found

2"01 1 "93

Carbohydrates 1'31 1 '34

Lipids

7"53 7'48

Protein

82.6 82"2

Water

1"50 1 "69

Ash

* For carbohydrates, lipids and protein each value represents mean of eight estimations and for water and ash mean of three estimations.

Human Bovine

Intermediate host

AND BOVINE HOSTS I N P E R C E N T A G E OF FRESH W E I G H T *

T A B L E 5 - - C O M P A R A T I V E DATA ON THE B I O C H E M I C A L C O N S T I T U E N T S OF THE L A M I N A T E D M E M B R A N E OF H Y D A T I D CYST W A L L F R O M H U ~ I A N

46"69

Human

Phosphate

In percentage of ash

D A T A ON T H E ANALYSES OF M I N E R A L C O N S T I T U E N T S OF T H E ASH C O N T E N T OF THE L A M I N A T E D M E M B R A N E OF H Y D A T I D CYST W A L L

Intermediate host

TABLE 4

oo

z

t~

BIOCHEMICAL ANALYSES OF THE CYST WALL OF ECHINOCOCCUS GRANULOSUS

803

with a mean of 82.2 per cent. Similarly, the ash content also showed a very slight variation in the cyst membranes from the two hosts (Table 3). Analyses of the various mineral constituents of the laminated membranes of cysts collected from the two hosts showed a very close resemblance (Table 4). Phosphate and calcium form the major constituents and along with magnesium they form 67 and 72 per cent of the ash in human and bovine parasitic tissue respectively. The calcium level is noticeably higher in the case of material from the bovine host. Sodium and potassium, chlorides and traces of iron and aluminium are also present in both cases. The ratio of calcium to phosphate was 1 : 2 in the bovine cyst wall and 1 : 2.4 in the human cyst wall. A comparative account of the various constituents in this parasitic material taken from the two hosts is tabulated (Table 5).

Amino acids Paper and thin layer chromatography of the amino acids present in the protein hydrolysates of the laminated membranes of fertile hydatid cysts of E. granulosus from bovine hosts revealed ten distinct spots. A densitometric reading of the chromatograph is depicted in Fig. 1. In contrast, the chromatographic pattern obtained with the laminated membranes of sterile cysts showed twelve distinct spots (Fig. 2).

0'16

_

iI

cm I

® O't2

--

0'08

®

o

0'04

®

°A)4

®® Amino acids

FIC. 1. Showing the densitometric readings of the chromatographic pattern of amino acids present in the laminated membrane of the fertile hydatid cyst wall. The peaks A-J correspond to the following amino acids: A, Histidine; B, arginine; C, glycine; D, hydroxyproline/serine/aspartic acid; E, alanine; F, proline; G, tyrosine; H, methionine/valine; I, tryptophane; J, leucine/isoleucine. The Alizarin Red S method for the occurrence of calcium in the cyst wall resulted in a strong positive reaction on the laminated membrane but the germinal layer gave only a weak reaction. Presence of heavy deposits of calcium on the 27

804

A.V. SADANAND

laminated membrane was unmistakable and this finding is corroborated by the result of the estimation of calcium content in ash.

0.1~

g o

0 l~

I

I

J

J

F

f

A m i n o acids

FIG. 2. Showing the densitometric readings of the chromatographic pattern of amino acids present in the laminated membrane of the sterile hydatid cyst wall. The peaks A-L correspond to the following amino acids: A, Lysine; B, histidine; C, arginine; D, glycine; E, hydroxyproline/serine/asparfic acid; F, alanine; G, proline; H, tyrosine; I, methionine/valine; J, tryptophane; K, phenylalanine; L, leucine/isoleucine. DISCUSSION The biochemical investigations on the laminated membranes of fertile hydatid cysts from human and bovine hosts reveal no significant variation in organic or inorganic components. A study of the carbohydrates showed that it consisted of about 2 per cent of the wet wt. of the tissue. The lipid content of the laminated membrane consisted of about 1-3 per cent of the wet wt. Results of analyses of inorganic constituents showed the presence of Na, K, C1, PO4, Mg and Ca besides traces of aluminium and iron in the laminated membranes of the cysts. Staining with Alizarin Red S also indicated the presence of calcium in appreciable quantities in the laminated membrane and as scattered granules in the germinal membrane. The analyses of ash showed that 19.4 and 23.7 per cent of the ash to be calcium in the laminated membranes of cysts from human and bovine hosts respectively. Among the organic constituents analysed by biochemical techniques, protein was found to be the major component of the laminated membrane. It constituted 7.53 per cent of the wet wt. Kilejian et al. (1967) found evidence for the presence of an appreciable amount of protein in the laminated membrane and larval scolices, but based on their infrared spectra and chemical analyses concluded that mucopolysaccharides constitute the principal structural material of the laminated membrane. An interesting observation was the presence of two additional amino acids namely, lysine and phenylalanine in the sterile cyst wall. Further work is in progress to elicit the significance of this difference. The presence of strongly basic

BIOCHEMICAL ANALYSES OF THE CYST WALL OF E C H I N O C O C C U S G R A N U L O S U S

805

amino acids such as histidine, arginine and lysine indicated the presence of basic proteins in the tissue. Other significant features were the absence of cystine, moderate glycine, alanine and tryptophane contents and a high tyrosine content. T h e high tyrosine and moderate tryptophane contents were suggestive of elastoidin. T h e absence of cystine, moderately high glycine and alanine contents and the possible presence of hydroxyproline was suggestive of a resemblance to collagen. Preliminary X-ray diffraction studies also indicated that the membrane consists of some form of collagen-like protein but was not typical of collagen. (Ramachandran, 1969, personal communication). Histochemical studies also confirm the presence of collagenous and elastinous substances in the membrane (Singh & Rao, 1967; Sadanand, 1970, unpublished). Acknowledgements--The author is grateful to Dr. S. Krishnaswamy, Head of the Department of Biological Sciences, Madurai University for suggesting the problem and also for his guidance and help during the course of the work. The valuable help of Mr. V. V. Raman, Professor of Chemistry, Madurai Medical College and Dr. S. Krishnan of the Department of Biological Sciences, Madurai University is also gratefully acknowledged.

REFERENCES AoOSIN M., VoN B~ND T., RIWRA G. F. & MCMAKONP. (1957) Studies on the metabolism of Echinococcus granulosus. Expl. Parasit. 6, 37-51. CMELIKS. (1952) Ein antigenes polysaccharid aus den Eehinococcus cysten. Biochem. Z. 322, 456-462. CMELIK S. & BRISKIB. (1953) Untersuchugen. l~ber Eiweissfraktio neu von Taenia echino coccus. Biochem. Z. 324, 104-114. Dunols M., GILIS K. A., HAMILTONT. K., ROBERSP. A. & BENNSTJ. (1956) Calorimetric methods of sugar and related substances. Anal. Chem. 28, 350-356. FISKE C. H. & SUBBAROWY. (1925) Colorimetric determination of phosphorus. `7. biol. Chem. 66, 375-400. GALLACHERI. M. C. (1964) Expl. Parasit. 15, 110-117. KILp.JIANA., SAWR K. & SCHWABEC. W. (1962) Infra red spectra and chemical composition of the hydatid cyst. Expl. Parasit. 12, 377-392. KILEJIAN A., SCHINAZI L. A. & SCHWABEC. W. (1961) Histo-chemical observations on Echinococcus granulosus. .7. Parasitol. 47, 181-188. LEMAI~ G. & RIBERe R. (1935) Sur la composition chimique de la liquide hydatique. Comp. Rend. Soc. Biol. 118, 1578-1579. MAzzocco P. (1923) Composition du liquide hydatique. Camp. Rend. Soc. Biol. 88, 342343. MORSETH D. J. (1967) Fine structure of hydatid cyst and protoscolex of Echinococcus granulosus. `7. Parasitol. 53, 312-325. RAVMONT J. E. G., AUSTIN J. & LINFORD E. (1964) Biochemical studies on marine Zooplankton. `7. Cons. Int. Explor. Mar. 28, 354-363. SINCH H. & RAo B. V. (1967) Some observations on the histochemistry of daughter cysts from hydatids of buffaloes. Ind. Vet.?. 44, 933-938. Key Word Index---Hydatid cysts--human and bovine; laminated membrane; chemical composition ; amino acids ; collagen; elastoidin; Echinococcus granulosus.