Z. Parasitenk. 48, 271 273 (1976) © by Springer-Verlag 1976

Trace Elements in

Isoparorchis hypselobagri

Meenakshi Srivastava Upgraded Depart~ment of Paghology ~nd Bacteriology, lVfediea!College, Lucknow, India. S. P. G u p t a Department of Zoology, University of Lucknow, India Received October l, 1975

Summary. The trace elements contents of the tissue of Isoparorchis hypselobagri were ascertained by quantitative analyses. The iron content was found to be particularly high in comparison of copper and zinc. Introduction L i t t l e i n f o r m a t i o n is a v a i l a b l e on t h e t r a c e elements of H e h n i n t h p a r a s i t e s despite of t h e fact t h a t t h e y a r e quite significant for n u t r i t i o n as well as cons t i t u e n t s of r e s p i r a t o r y pigments. The most c o m p l e t e analyses a r e those of F l u r y (1912) who d e m o n s t r a t e d t h e presence of t h e m e t a l s Na, K, Ca, Mg, F e a n d A1 in Asearids b u t was u n a b l e to d e m o n s t r a t e t h e presence of Cu a n d Mn. S a l i s b u r y a n d A n d e r s o n (1939) d e m o n s t r a t e d t h e presence of Na, K , Ca, Mg a n d F e in t h e tissues of Cysticercus ]asciolaris. Von B r a n d (1939) d e m o n s t r a t e d q u a l i t a t i v e l y t h e presence of Na, K , Mg, Ca, Fe, PO4, SO 4, CO s a n d C1 in t h e tissues of an Aeant h o c e p h M a n p a r a s i t e Macracanthorhynchus hirudinaceus. Von B r a n d a n d Saurwein (1942) d e m o n s t r a t e d in Macracanthorhynchus hirudinaceus t h e presence of Na, K , Ca, Mg, Mn, A1, F e a n d Cu b y s p e c t r o g r a p h i c analysis. Ill a d d i t i o n to t h e metals, t h e s p e c t r o g r a p h r e v e a l e d t h e presence of P a n d Si. Cu was quantit a t i v e l y d e t e r m i n e d a n d f o u n d to be less t h a n 0.01% a n d F e 0.29 to 0.30% (dry basis). Von B r a n d (1952) r e v i e w e d t h e inorganic substances of some h e l m i n t h s a n d of some p a r a s i t i c fluids. Ma (1963) i n v e s t i g a t e d t h e inorganic substances as well as t r a c e e l e m e n t s in t h e tissues of Clonorchis sinensis b y e o l o r i m e t r y a n d emission s p e e t r o g r a p h y a n d f o u n d t h a t t h e c o p p e r c o n t e n t was c o m p a r a t i v e l y high. I n view of t h e growing r e a l i z a t i o n of t h e i m p o r t a n c e of t h e " t r a c e elements"~ in m a n y biological fields, i t was d e c i d e d to d e t e r m i n e eolorimetricMly some of t h e t r a c e e l e m e n t s in t h e tissues of Isoparorchis hypsdobagri. Materials and Methods

Collection o/Worms. Adult worms, Isoparorchis hypselobagri were collected from the air bladder of Wallagoniaattu obtained locally from a fish market. The instruments used for cutting the parasites were made of stainless steel. The worms were thoroughly washed with physiological saline prepared with deionized water. They were then drained for 5 hours at 4 ° C. These worms were wiped dry with Whatman filter paper, weighed and immediately used for the determinations. Preparation o/Ash. The weighed parasites were dried and ashed in a crucible at 105 ~= 5°C and 600 ~ 10°C respectively until constant weight was attained. Two 1-ml portions of con-

272

M. Srivastava and S. t ). Gupta

centrated t t N O a were added twice and evaporated by heating. The ash was weighed and dissolved in 5 ml of 6 n HC1, diluted with deionized water to a known volume and stored in a polyethylene bottle until analysis and was kept a t 4°C.

Quantitative Assay Copper was determined according to the method of Gubler et al. (1952). Zinc was determined according to the method of Valee and Gibson (1948). Iron was determined b y using Bathophenanthroline as given b y Varley (1969). Glassware. Pyrex b r a n d glassware was used throughout. A 1:1 mixture of concentrated tt2SO 4 a n d HNO s was used for cleaning the glassware which were then washed thoroughly with this mixture as well as with tap water and deionized water respectively (Thiers, 1957). The glassware was stored in polyethylene bags until use. Reagents. All the chemicals were of B . D . H . An l a R or microanalytical grade unless specified otherwise. The necessary precautions as suggested b y Thiers (1957) and Sande]l (1959), were observed.

Results Table 1. Percentage of total solids, moisture/volatile substances and ash in lsoparorchis

hypselobagri Sample No. 1 (%) Total solids moisture a n d volatile substances a t 105 :£ 5 ° C Ash a t 6 0 0 i 10 ° C

Sample No. 2 (%)

Sample No. 3 (%)

Average (%)

9.2

9.0

10.0

9.9

90.8

91.0

90.0

90.6

1.5

1.32

2.1

1.64

The results of the total solids, moisture/volatile substances a n d ash are expressed in g/100 g fresh tissue.

Table 2. Trace elements detected in the ash of Isoparorchis hyp,~elobagri Sample No.

Cu

Zn

Fe

1. 2. 3.

10.27 11.0 10.6

13.2 12.5 14.1

28.0 29.0 30.1

Average

10.6

13.26

29.03

The results, expressed in mg/100 g dry tissue, were average values of triplicate determinations and are given as above-mentioned.

Discussion T h e t r a c e e l e m e n t s Cu, F e a n d Z n w e r e f o u n d i n t h r e e s a m p l e s . M o l y b d e n u m a n d l e a d w e r e n o t i n v e s t i g a t e d b e c a u s e t h e y a r e n o t a n e s s e n t i a l c o n s t i t u e n t of a n y l i v i n g o r g a n i s m a n d l e a d is o n e of t h e m o s t t o x i c t r a c e e l e m e n t s o c c u r r i n g i n food.

Trace Elements in Isoparorchis hypselobagri

273

The trace elements of Isoparorchis hypselobagri, Cu, Z n a n d Fe a m o u n t to be 10.6, 13.26 a n d 29.03 rag/100 g respectively (dry tissue). Our findings differ from those of Ma (1963) who f o u n d a copper c o n t e n t of 38 rag/100 g of d r y tissue, a zinc c o n t e n t of 8 rag/100 g of d r y tissue a n d a n iron c o n t e n t of 48 rag/100 g of d r y tissue i n Clonorchis sinensis. H e also observed t h a t 30% of the total copper is a t t r i b u t e d largely to polyphenol oxidase which contains 0.3 % of copper. The a m o u n t of iron in [soparorchis hypselobaffri was high in comparison to copper a n d zinc. This high q u a n t i t y of iron m a y be due to t h e fact t h a t the present worm feeds on blood. Von B r a n d (1952) m e n t i o n s iron as inorganic deposits in the i n t e s t i n a l cell of m a n y nematodes. Thus it can be p r e s u m e d t h a t iron gets deposited in the i n t e s t i n a l cell d u r i n g feeding.

References Brand, T. yon: Chemical and morphological observations upon the composition of Macrasanthorl~ynehus hirudi~aceus (AcanthocephMa). J. Parasit. 25, 329-342 (1939) Brand, T. yon: ChemicM physiology of endoparasitic animals. New York, N.Y.: Academic Press Inc. 1952 Brand, T. yon, Saurwein, J. : Further studies upon the chemistry of Macracanthorhynchus hirudinaceus. J. Parasit. 28, 315-318 (1942) Flury, F.: Zur Chemie und Toxikologie der Ascariden. Arch. exp. Path. Pharmakol. 67, 275-392 (1912) Gubler, C. J., Lahey, M. E., Ashenbrucker, H., Cartwright, G. E., Wintrobe, M. M.: Studies on copper metabolism. J. biol. Chem. 196, 209 (1952) Ma, L. : Trace elements and polyphenol oxidase in Clonorchis sinensis. J. Parasit. 49, 197-203 (1963) Salisbury, F. L., Anderson, 1%. J. : Concerning the chemical composition of Cysticercus [asciolaris. J. biol. Chem. 129,505-517 (1939) Sandell, E.B.: Colorimetric determination of traces of metals, 3rd ed. New York, N.Y.: Interscience Publishers, Ltd. 1959 Thiers, R. E. : Contamination in trace elements analysis and its control. In: Glick, D., Method of biochemical analysis, vol. 5. New York, N.Y. : Interscience Publishers, Inc. 1957 VMee, B. L., Gibson, J. G. : An improved Dithiozone method for the determination of small quantities o~ zinc in blood and tissue samples. J. biol. Chem. 176, 435 (1948). Varley, H. : Practical clinical biochemistry. 4th edition. New Delhi: Arnold Heinemann Publishers, 1969 Dr. S. P. Gupta Department of Zoology Luclmow University Lucknow, India