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Metabolic fate of histidine in the parasitic worm Fasciola hepatica L.
Comp. Biochem. Physiol., 1969, Vol. 29, pp. 885 to 887. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION METABOLIC FATE OF H I S T I D I N E IN THE PARASITIC WORM F A S C I O L A H E P A T I C A L. B. K U R E L E C , * M A R I J A R I J A V E C and R. K L E P A C Department of Biochemistry and Physiology, Institute of Biology, University Zagreb, Yugoslavia and Institute of Physiology, School of Veterinary Medicine, Hannover, West Germany
(Received 14 October 1968) Abstract--1. Liver flukes were incubated in a medium with added histidine l'C-2(ring). The same labelled molecule was added to the liver fluke's homogenate. Both the medium and the homogenate were essayed for histamine content either by column chromatography separation or by radioactivity measurement. 2. These findings are discussed in regard to the presence of large amount of histidine in the liver fluke's free amino acid pool INTRODUCTION
RECENTLY it has been shown that histidine, proline and alanine represent 50 per cent of the amino acid pool in the liver fluke (Kurelec & Rijavec, 1966). It is known that in many free-living invertebrates one-third or more of the amino acid nitrogen is provided by only a few amino acids (Awapara, 1962); however, among these histidine was never observed. An important metabolic reaction of histidine is its deearboxylation to histamine, which is known to exert profound pharmacological effects. T h e activity of histidine decarboxylase is very low in animal tissues and the pathway via histamine represents quantitatively only a small proportion of the degraded histidine (Tabor, 1955). T h e large amount of histidine in Fasciola hepatica is rather surprising in view of its parasitic mode of life. W e therefore turned our attention to investigating the degradation path of histidine with respect to the histamine-forming capacity in Fasciola hepatica. MATERIALS AND METHODS
The liver flukes (3 g wet wt.) were incubated in 6 ml of Ringer solution for a period of 60 rain; 100 i.u. of penicillin and 0.2 mg/ml of streptomycin were added to the medium. At the end of the incubation time the medium was stored in the cold (-40°C), and the flukes were homogenized, deproteinized with alcohol, the filtrate evaporated and the residue taken up in water and stored. Another group of flukes (1 g wet wt.) was homogenized in 2 ml of 1% KC1 in an all-glass Potter-Elvehjem homogenizer in ice for 30 sec. * Present address: Institute of Biology, University Zagreb, Zagreb, Heinzelova 55, Yugoslavia. 885
B. KUl~LSC, MARIJARIJAVECAND R. KLEPAC
886
One ml of the homogenized material was incubated for 30 min with 5/~C of histidine 14C2(ring) (Amersham, England) and then deproteinized as before (0.39 mg histidine/0.01 mc). All the materials were analysed by column chromatography for amino acids according to the method of Moore et al. (1958) using an automatic amino acid analyser (Locarte Co., London). In the analysis of basic amino acids after appearance of the arginine peak we pumped through the column 0.2 N NaOH. With the first 5 ml of the alkaline eluate histamine was quantitatively eluted. The radioactivity of the eluate was measured continuously by a two-channel scintillation counter (Packard Instruments, Inc.) connected directly to the amino acid analyser. A 2-ml flow cell with anthracene powder was used. Counting efficiency was about 25 per cent. The areas under the activity curves were measured by a planimeter and the activity expressed in m m ~. A control run of CZ4-1abelled histidine showed radioactive impurities at points 1, 2 and traces at the position of histamine. The same results were obtained when '4C-histidine was incubated with boiled homogenate. RESULTS After incubation of the flukes in Ringer solution, 5.75 per cent of added activity was found in the flukes. O f that activity 5.9 per cent is in the f o r m of histidine and 14.1 per cent in the metabolites. F r o m metabolized histidine 14.7 per cent is in the f o r m of histamine, or 2.07 per cent of added histidine. T h e analysis of the m e d i u m shows a similar distribution of the radioactivity (see Fig. 1). cpm 20000.
Histidine
(
16000160001400012000"
NH~
L~ .8 .7 .6
I
I
.5 luO00-
.,~
BOO0-
.3
6000.
.2
I lI
i
4000" 2000.
L
i
.I
1.2 II
;
Jl
I t I
I I |
Histamine
I I i
I I
Lysine ]
•
30
60
NaOH
N 90
,'~o
~'so
f
t
tlo~,
Activity - - - - - - t~uontity
Fio. 1. A typical chromatogram-actigram of the medium with added histidine 14C after incubation of the liver flukes. I n the m e d i u m 16.8 per cent of the added histidine is metabolized, 15.5 per cent of this having been converted to histamine (2.25 per cent of added). Specific activity of histamine in the fluke's b o d y was 77.7 m m 2 [ l ~ M and 1571 mm2//~M in the m e d i u m . I t is difficult to interpret this result, b u t it is possible to draw conclusions about the excretion tendency of histamine.
FATE OF HISTIDINE
IN
FASCIOLA
HEPATICA
L.
887
The homogenized tissue of Fasciola hepatica metabolizes 16.5 per cent of added histidine, a quarter of which was found in the form of histamine (25.3 per cent) or 4-2 per cent from added histidine. The activity peak 2, (see Fig. 1) corresponds to ghitamic acid, as is shown by the column chromatography analysis of acidic and neutral amino acids. The identity of the substances numbered 1, 3, 4 and 5 was not established. DISCUSSION The results show that the liver flukes tissue has an active histidine decarboxylase. This is in conflict with the findings of Mettrick & Telford (1963), who were not able to demonstrate the activity of that enzyme in the liver fluke's tissue (using a different technique). The results also show that histamine is freely diffusible in a physiological medium, showing the same characteristics as other investigated amino acids (Kurelec & Ehrlich, 1963). Bearing in mind the great concentration of histidine in the liver fluke's tissue (Kurelec & Rijavec, 1966), the present findings support the hypothesis that certain aspects of toxicology of Fasciola hepatica are due to the release of histamine. REFERENCES AWAPARAJ. (1962) Free amino acids in invertebrates: a comparative study of their distribution and metabolism. In Amino Acid Pools (Edited by HOLD~ J. T.), pp. 158-175. Elsevier, Amsterdam. KURELECB. & EHRLICHI. (1963) ~ber die Natur der von Fasciola hepatica (L.) in vitro ausgeschiedenen Amino- und Ketos~uren. ExpI Parasit. 13, 113-117. KURELECB. & RUAVECM. (1966) Amino acid pool of the liver fluke (Fasciola hepatica L.). Comp. Biochem. Physiol. 19, 525-531. METTRICK D. F. & TELFORDJ. M. (1963) Histamine in the phylum Platyhelminthes. s~. Parasit. 49, 653-656. MooRs S., SPACKMAND. H. & STEIN W. H. (1958) Chromatography of amino acids on sulphonated polystyrene resins..4nalyt. Chem. 30, 1185-1189. TABORH. (1955) Degradation of histidine. In Metabolism of the Amino Acids (Edited by MeELROY W, D.), pp. 373-390. Johns Hopkins Press, Baltimore. Key Word Index---Histidine; histamine; liver fluke; Fasciola hepatica.
SHORT COMMUNICATION METABOLIC FATE OF H I S T I D I N E IN THE PARASITIC WORM F A S C I O L A H E P A T I C A L. B. K U R E L E C , * M A R I J A R I J A V E C and R. K L E P A C Department of Biochemistry and Physiology, Institute of Biology, University Zagreb, Yugoslavia and Institute of Physiology, School of Veterinary Medicine, Hannover, West Germany
(Received 14 October 1968) Abstract--1. Liver flukes were incubated in a medium with added histidine l'C-2(ring). The same labelled molecule was added to the liver fluke's homogenate. Both the medium and the homogenate were essayed for histamine content either by column chromatography separation or by radioactivity measurement. 2. These findings are discussed in regard to the presence of large amount of histidine in the liver fluke's free amino acid pool INTRODUCTION
RECENTLY it has been shown that histidine, proline and alanine represent 50 per cent of the amino acid pool in the liver fluke (Kurelec & Rijavec, 1966). It is known that in many free-living invertebrates one-third or more of the amino acid nitrogen is provided by only a few amino acids (Awapara, 1962); however, among these histidine was never observed. An important metabolic reaction of histidine is its deearboxylation to histamine, which is known to exert profound pharmacological effects. T h e activity of histidine decarboxylase is very low in animal tissues and the pathway via histamine represents quantitatively only a small proportion of the degraded histidine (Tabor, 1955). T h e large amount of histidine in Fasciola hepatica is rather surprising in view of its parasitic mode of life. W e therefore turned our attention to investigating the degradation path of histidine with respect to the histamine-forming capacity in Fasciola hepatica. MATERIALS AND METHODS
The liver flukes (3 g wet wt.) were incubated in 6 ml of Ringer solution for a period of 60 rain; 100 i.u. of penicillin and 0.2 mg/ml of streptomycin were added to the medium. At the end of the incubation time the medium was stored in the cold (-40°C), and the flukes were homogenized, deproteinized with alcohol, the filtrate evaporated and the residue taken up in water and stored. Another group of flukes (1 g wet wt.) was homogenized in 2 ml of 1% KC1 in an all-glass Potter-Elvehjem homogenizer in ice for 30 sec. * Present address: Institute of Biology, University Zagreb, Zagreb, Heinzelova 55, Yugoslavia. 885
B. KUl~LSC, MARIJARIJAVECAND R. KLEPAC
886
One ml of the homogenized material was incubated for 30 min with 5/~C of histidine 14C2(ring) (Amersham, England) and then deproteinized as before (0.39 mg histidine/0.01 mc). All the materials were analysed by column chromatography for amino acids according to the method of Moore et al. (1958) using an automatic amino acid analyser (Locarte Co., London). In the analysis of basic amino acids after appearance of the arginine peak we pumped through the column 0.2 N NaOH. With the first 5 ml of the alkaline eluate histamine was quantitatively eluted. The radioactivity of the eluate was measured continuously by a two-channel scintillation counter (Packard Instruments, Inc.) connected directly to the amino acid analyser. A 2-ml flow cell with anthracene powder was used. Counting efficiency was about 25 per cent. The areas under the activity curves were measured by a planimeter and the activity expressed in m m ~. A control run of CZ4-1abelled histidine showed radioactive impurities at points 1, 2 and traces at the position of histamine. The same results were obtained when '4C-histidine was incubated with boiled homogenate. RESULTS After incubation of the flukes in Ringer solution, 5.75 per cent of added activity was found in the flukes. O f that activity 5.9 per cent is in the f o r m of histidine and 14.1 per cent in the metabolites. F r o m metabolized histidine 14.7 per cent is in the f o r m of histamine, or 2.07 per cent of added histidine. T h e analysis of the m e d i u m shows a similar distribution of the radioactivity (see Fig. 1). cpm 20000.
Histidine
(
16000160001400012000"
NH~
L~ .8 .7 .6
I
I
.5 luO00-
.,~
BOO0-
.3
6000.
.2
I lI
i
4000" 2000.
L
i
.I
1.2 II
;
Jl
I t I
I I |
Histamine
I I i
I I
Lysine ]
•
30
60
NaOH
N 90
,'~o
~'so
f
t
tlo~,
Activity - - - - - - t~uontity
Fio. 1. A typical chromatogram-actigram of the medium with added histidine 14C after incubation of the liver flukes. I n the m e d i u m 16.8 per cent of the added histidine is metabolized, 15.5 per cent of this having been converted to histamine (2.25 per cent of added). Specific activity of histamine in the fluke's b o d y was 77.7 m m 2 [ l ~ M and 1571 mm2//~M in the m e d i u m . I t is difficult to interpret this result, b u t it is possible to draw conclusions about the excretion tendency of histamine.
FATE OF HISTIDINE
IN
FASCIOLA
HEPATICA
L.
887
The homogenized tissue of Fasciola hepatica metabolizes 16.5 per cent of added histidine, a quarter of which was found in the form of histamine (25.3 per cent) or 4-2 per cent from added histidine. The activity peak 2, (see Fig. 1) corresponds to ghitamic acid, as is shown by the column chromatography analysis of acidic and neutral amino acids. The identity of the substances numbered 1, 3, 4 and 5 was not established. DISCUSSION The results show that the liver flukes tissue has an active histidine decarboxylase. This is in conflict with the findings of Mettrick & Telford (1963), who were not able to demonstrate the activity of that enzyme in the liver fluke's tissue (using a different technique). The results also show that histamine is freely diffusible in a physiological medium, showing the same characteristics as other investigated amino acids (Kurelec & Ehrlich, 1963). Bearing in mind the great concentration of histidine in the liver fluke's tissue (Kurelec & Rijavec, 1966), the present findings support the hypothesis that certain aspects of toxicology of Fasciola hepatica are due to the release of histamine. REFERENCES AWAPARAJ. (1962) Free amino acids in invertebrates: a comparative study of their distribution and metabolism. In Amino Acid Pools (Edited by HOLD~ J. T.), pp. 158-175. Elsevier, Amsterdam. KURELECB. & EHRLICHI. (1963) ~ber die Natur der von Fasciola hepatica (L.) in vitro ausgeschiedenen Amino- und Ketos~uren. ExpI Parasit. 13, 113-117. KURELECB. & RUAVECM. (1966) Amino acid pool of the liver fluke (Fasciola hepatica L.). Comp. Biochem. Physiol. 19, 525-531. METTRICK D. F. & TELFORDJ. M. (1963) Histamine in the phylum Platyhelminthes. s~. Parasit. 49, 653-656. MooRs S., SPACKMAND. H. & STEIN W. H. (1958) Chromatography of amino acids on sulphonated polystyrene resins..4nalyt. Chem. 30, 1185-1189. TABORH. (1955) Degradation of histidine. In Metabolism of the Amino Acids (Edited by MeELROY W, D.), pp. 373-390. Johns Hopkins Press, Baltimore. Key Word Index---Histidine; histamine; liver fluke; Fasciola hepatica.