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Carboxylic Acids and Their Metabolic Enzymes as New Novel Biomarkers of Susceptible, Resistant Strains of Biomphalaria alexandrina and Snails Infected with Schistosoma mansoni.




Mona M. Mantawy1, Naema Zayed Mohamed1, Aza Fathy Arfa1, Hanan F. Aly1*



Abstract— Carboxylic acids play an important role in both aerobic and anaerobic metabolic pathways of both the snail and the parasite. Monitoring the difference between susceptible, resistance and the effects of infection by schistosome on Biomphalaria alexandrina carboxylic acids metabolic profiles represents a promising additional source of information about the state of meta- bolic system. In the present study , separation and quantification of fumaric , ,malic, succinic , pyruvic , lactic and propionic acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to detect correlations between these acids in both hemolymph and digestive gland (DG) samples in a total of 100 B. alexandrina snails (40 infected and 30 for each susceptible and resistance) . In addition several enzymes representing different metabolic pathways were determined in these specimens. These enzymes included aspartate and alanine aminotransferases (AST and ALT), lactate dehydrogenase (LDH), glucose phosphate isomerase (GPI), phosphofructokinase enzyme (PFK), glucose-3-P dehydrogenase (G3PDH) ,glucose and glycogen . The results showed significant difference in organic acids ,enzyme activities ,glucose and glycogen level either in hemolymph or digestive glands of resistance and susceptible snails .In addition , significant difference in all the studied parameters in infected snail as compared to susceptible uninfected one . Some possible explanations of the underlying mecha- nisms were discussed.. These findings highlight the potential of metabolomics as a novel approach for fundamental investiga- tions of host-pathogen interactions as well as disease surveillance and control.

Index Terms — carboxylic acid, liver function enzymes ,glycolytic enzymes , Schistosoma mansoni,

1 INTRODUCTION

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Resh water pulmonate snails of the genus Biomphalaria are best known for their role as intermediate hosts of the widely distributed parasite, Schistosoma mansoni [1]. This parasite, one of the causal agents of the debilitating disease (intestinal schistosomiasis), infects 207 million people world- wide and puts an estimate of 779 million people at risk of ac- quiring infection [2 One of the keys to understand the present and future of Schistosoma mansoni infection in Egypt is to understand more about the snails that play an indispensable
role in its transmission [3].
Parasitic helminths have an absolute dependency on carbo-
hydrates for their energy source, and one of the key features of carbohydrate catabolism is the excretion of a wide range of carboxylic acids. However, most of them have low molecular absorptive and thus are poorly detectable compounds by pho- tometric detection [4]. The depletion of energy sources and alterations in the carbohydrate metabolism caused by the trematode larvae on their snail intermediate host was thought to be similar to that observed during estivation in which food uptake ceases, water loss occurs, and gas exchange may also be affected.

————————————————

1Therapeutical Chemistry Department , National Research Center , Dokki, Giza, Egypt. \

, Hanan F. Aly1* Therapeutical Chemistry Department , National

Research Center , Dokki, Giza, Egypt. E.mail: alyhanan25@yahoo.com



All of these alterations exert an influence on the snail’s me- tabolism which may be reflected in the concentration of me- tabolites resembling consumption of host metabolites by the developing parasites [5]. Becker [5] stated that starvation of Biomphalaria glabrata for 6 days was equivalent to 30 days’ infection by S. mansoni. Several studies have demonstrated that infected schistosome vectors grow faster and have a greater soft tissue mass than their unparasitized counterparts [6]. This appears inconsistent with the starvation hypothesis. Moreover, it was recently reported that the adenylate energy charge of the digestive gland-gonad tissue complex (DGG), unlike in estivation, is affected very little by infection. Thus, the infected snail appears to undergo some degree of physio- logical adaptation to the parasitized state. The finding that snail hosts have the potential for regulating hemolymph car- bohydrate level indicates that host metabolic responses to infection are compensatory and aimed at maintaining blood glucose in response to continuous utilization by developing parasite [7]. Biomarkers of diseases refer to cellular, biochemi- cal, or molecular alterations that occur during diseases and could be measured in biological matrices, such as tissue, cells, or fluids [8,9]. Over the last several years, researchers have started to explore the new array based technologies to map biomarkers of diseases and to identify targets for drug design that promise to provide signatures that are characteristics for each disease state by taking snap shot of metabolism [10]. En-

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hancement of our current understanding of a host’s metabolic response to a parasitic infection is a promising approach for biomarker identification, yet its potential for diagnosis and disease surveillance has been under used [11].


Combining several biomarkers have been shown to im- prove the discriminatory capability considerably. Recent de- velopments in the field of metabolomics now provide the tools to go one step further; identify profiles of metabolites that together serve as a biomarker [12]. The present study was initiated from this assumption and designed to identify a bi- omarker profile that could distinguish uninfected susceptible , resistant from infected snails . Organic acids are important component of parasite metabolism and participate in both catabolic (e.g glycolysis ) and anabolic (e.g gluconeogenesis) pathways . Pyruvate and lactate are indicators of glycolytic processes under aerobic conditions, while fumarate , succin- ate and malate are indicators of the tricarboxylic acid cycle . The presence of ketone bodies , such as β- hydroxybutrate and acetoacetate as well as of fatty acids , such as acetate and propionate , are indicative for lipid metabolism [13].Organic acid play a central role in parasite of S. mansoni metabolism which serve as indicators of various metabolic reactions since they represent important components of energy and parasites metabolism . Thus , they may indicate the use of carbohy- drates as an energy source in the flow of aerobic and anaero- bic transition , the replacement of glucose through gluconeo- genesis , and of protein via glucogenic amino acids or me- tabolism of lipids on a smaller scale via fatty acids and ketone bodies[14]. A profile of selected carboxylic acids was done using the malic, pyruvic, and fumaric acids as representatives of carbohydrate intermediary metabolism of both aerobic and anaerobic pathways which are directly linked to energy pro- duction and facultative metabolic ways in both the snail and the parasite [13-15]. Whereas, acetic and oxalic acids represent the end product of carbohydrate metabolism [14,15]. Studies on such acids as biomarkers in Biomphalaria snails after in- fection with S. mansoni may be important in developing ra- tional methods to diagnose infection, control, and even eradi- cate medically important snails that transmit parasitic diseas- es to humans and animals [16]. So, the present study aims to clarify the differences in carboxylic acids and their metabolic enzymes representing different pathways in hemolymph and digestive glands of resistance , susceptible and infected snails .modify the header or footer on subsequent pages.

2 . MATERIAL AND METHODS

Biological materials were obtained from the Medical Mala- cology Laboratory, Theodor Bilharz Research Institute, Imba- ba, Giza, Egypt. A total of 100 snails of an Egyptian strain, including 40 infected cases and 30 susceptible controls and 30 resistant were used in this study.

2.1 Snail maintenance

Snails were maintained in plastic trays, each con-

taining 10 snails and 1 L of aerated tap water (26±2˚C),

replaced twice a week, and fed boiled fresh lettuce and blue


green algae [17]. Aquaria were cleaned weekly for removal of feces and dead snails [18]. Laboratory reared B. alexandrina reaching 6-7 mm in shell diameter was exposed to S. mansoni miracidial infection according to Massa et al. [16]. Harvesting of the snails was done as follow: group 1 (G1) included 30 susceptible snails .Group 2 (G2) was 30 resistant snails; and group 3 (G3) was 40 snails of 2 weeks after infection. All snails have the same shell diameter and were maintained in the same manner and harvested at the same time .
2.2 Digestive gland extraction
The DG tissue extract samples were prepared by dissection free from the snail body then carboxylic acid extraction was done using 50 % Ringer`s solution (Carolina Biological Supply,USA). Centrifugation of the extract was done at 250 g for 15 min to collect the supernatants which were stored at -20
°C until use [16].

2.3 Extraction and separation of the organic acids

The organic acids were extracted immediately from the centri- fuged hemolymph using Bond – Elut ®columns (SAX – anion exchange-quartenary amine, manufactured by Analytichem International, Habor City,USA) as described by Lian et al. [19].Under vacuum, the columns were activated by consecu- tive washes with 1 ml of 0.5M HCL, 1 ml of methanol and 2 ml of HPLC-grade water. They were loaded with 200μl of hemolymph and 2ml water. The columns were disconnected from the vacuum pump and 250μl of 0.5 M sulphuric acid were applied to elute the organic acids retained on the matrix. The eluate was centrifuged at 1200g for 5 min and the super- natant stored at -70 °C until analysis by high performance liquid chromatography.
The liquid chromatography (HPLC-system Milton Roy-

Analyst 7800) was performed at room temperature using a BIORAD-Aminex ion exclusion HPX-78H column (300x7.8mm) designed specifically for the separation of organ- ic acids. The separation column was protected by a BIORAD- Aminex HPX-85 guard column. The mobile phase was sul- phuric acid (0.5mM) delivered at a flow rate of 0.8 ml/min. The elution profile was determined at 210nm. The elution profile was determined at 210nm. The injection volume of each sample was 100μl.
Pooling snails was used throughout the study, and the
mean of three trails for each HPLC analysis was done for each sample. The conversions to part per million concentrations (ppm).The concentration (ppm) of each acid in snail DG was calculated by multiplying the sample solution concentration interpolated from calibration curve (I) (ppm) by the original sample volume (V) (ml) and division of the product by the mass of the snail DG (M) (g) and for hemolymph, carboxylic acid concentration (μg/dl) was calculated by multiplying I times V times 100, and division of the product by the hemo- lymph volume (HV) (ml) according to Massa et al. [16].
.2.3. Enzyme assays

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Aspartate and alanine aminotransferases (AST and ALT):

AST and ALT were measured according to the method of Reitman and Frankel

[20],using dinitrophenylhydrazine as colour reagent. Lactate dehydrogenase (LDH):

Lactate dehydrogenase was measured according to the method of Babson and Babson [21], which based on the reduction of pyruvate to lactate by incubation with LDH in the presence of coenzyme NADH.

Glucose phosphate isomerase (GPI):

GPI was measured according to the method of King [22]. Phosphofructokinase (PFK):

PFK was measured according to the method of Zammit et al. [23]. Glucose-3-P dehydrogenase (G3PDH):

Was estimated according to the method of Voet and Voet [24].

Measurement of glucose:

Glucose was measured calorimetrically according to the method of Trinder [25]. Measurement of glycogen content:

glycogen content was estimated by the method of Nicholas et al. [26],

.

2.4. Statistical analysis

Statistical analysis is carried out using SPSS computer program ,post hoc and

Student –T-test , where significance level at p≤0.05

3 RESULTS


Table 1 and Fig 1 A represents the level of carboxylic acid in hemolymph of susceptible and resistance snails of Biompha- laria alexandrina . Significant increase in carboxylic acids ; Fumeric and Malic acids ,while significant decrease in suc- cinic acids in hemolymph of susceptible snail as compared to resistance one . However , Pyrouvic , lactic and Propionic acids showed insignificant change in hemolymph of both sus- ceptible and resistance snails. Concerning concentrations of organic acids in digestive gland , significant decrease was recorded in Fumeric , Succinic, Lactic and Propionic acids in digestive glands of susceptible snail as compared to re- sistance . While Malic and Pyrovic acids showed significant increase and insignificant change respectively.
Table 2 and Fig 1B demonstrate comparison between organic acids levels in hemolyph and digestive glands of infected snails . It was noticed that , insignificant change in Fumeric and Malic acids levels in both organs of infected snail, while
,significant decrease in Succinc and Propionic acids in he- molyph of infected snails as compared to digestive organ . On the contrary , significant increase was observed in Pyrouvic and Lactic acids of hemolyph of infected snail as compared to digestive organ(P≤0.05).

Table 3 declared glucose , glycogen and some hepatopancre- as enzymes representing different metabolic pathways in sus- ceptible , resistance and infected Biomphalaria alexandrina snail. Significant increase was recorded in amino acids path- way;AST and ALT in hepatopnacreas of susceptible snail as compared to resistance one . Infected snails showed signifi- cant inhibition of tranaminases enzymes either as compared to susceptible or resistance snails. Glycolytic enzymes ; PFK, GPI and G3PDH demonstrated significant increase in suscep-


tible snails than resistance . Although , infected snails showed significant increase in the all enzymes representing glycolytic pathway. In contradictory Kreb s cycle enzyme ; LDH showed significant inhibition in infected snail and sig- nificant increase in susceptible as compared to resistance Bi- omphalaria alexandrina snails. In addition, glucose level showed significant increase in susceptible as compared to resistance snails .However ,glycogen level exhibited signifi- cant decrease in susceptible as compared to resistance Bi- omphalaria alexandrina snails , while significant increase was recorded in infected snails.

Table 4 representing the Relative activities of lactate, py- ruvate, succinate, fumarate and malate in haemolymph and digestive gland of susceptible and resistant B. alxandrina snails. It was noticed that ,higher Lactate /Pyruvate
,Succinate/ Fumarate and Fumarate / Malate ratios in diges- tive gland and haemolymph of resistant snails as compared to susceptible one . In addition , AST / ALT and ALT / LDH exhibited higher ratio in digestive glands of resistant snails than susceptible one (Table 5).
.

4 DISCUSSION

Fresh water pulmonate snails of the genus Biomplalaria are best known for their role as intermediate hosts of the widely distributed parasite, Schistosoma man- soni. The major physiological and metabolic mechanisms of snail- chistosome relationships that are crucial for survival and growth[1].


Pooling of each 10 snails was used throughout the study and the mean of 3 trails for each HPLC analysis was done for each sample. Van Saun [27] encour- aged the use of pooled samples and stated that, though some variation may be masked, pooled samples may provide an economic alternative to traditional metabolic profiling, as most of the important measures of metabolic status showed minimal differences between pooled and individual samples and found them to be statistically equivalent. He stated that the real challenge of using pooled samples is interpretation. Empirically one can interpret pooled samples by determining how far they deviate from the midpoint of the reference range for the control [28]..

The importance of the measured carboxylic acids could be easily supported by the previous work of Massa et al. [16] who proved that infection with S.mansoni caused a significant reduction in the concentrations of acetic, fumaric, malic, and pyruvic acids in the digestive gland (DG) but not the hemolymph of Bimophalaria glabrata ( B. glabrata ) snails compared to uninfected snails. The significant reduction of certain carboxylic acids in the DG of B. glabrata patently infected with S .mansoni suggested that the infection stimulates reduced produc- tion or increased utilization by the snaisl tissue and /or the significant reduction of certain carboxylic acids in the DG patently infected with S. mansoni suggests that these acids are utilized by the sporocysts and cercariae in the snail tissue. in addition, infection stimulates reduced production or increased utilization by the snail tissue [16].

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Table 1 : Carboxlic acids concentrations in hemolymph and digestive gland of susceptible and resistance Biomphalaria alexandrina snails

) Organs 1) groups ) Mean±

) SD

) P value

) Hemolymph ) Fumeric

) S ) 3.4082 ) .46435

) 0.02

) R ) 1.5076 ) .80550 )

) Malic

) S ) 3.8196 ) 0.13265

) 0.000

) R ) 1.8767 ) 0.02255 )

) Succinic

) S ) 0.7506 ) 0.05305

) 0.000

) R ) 1.8303 ) 0.03445 )

) Pyrovic

) S ) 7.4669 ) 0.19895

) 0.23

0) R ) 6.8170 ) 0.76925 )

) Lactic

1) S

0) 6.2997 ) 0.78250

) 0.36

2) R

1) 7.3077

0) 1.49870

0)

) Propionic

3) S

2) 4.3878

1) .32831

1) 0.102

4) R

3) 2.9709

2) 1.11405 2)

) Digestive gland

) Fumeric

) Malic

0) Succinic

5) S

6) R

7) S

8) R

9) S

4) 2.5345

5) 3.5942

6) 7.1751

7) 1.3284

8) 3.5809

3) 0.35675

4) 0.01325

5) 0.09285

6) .29670

7) 1.35280

3) 0.007

4)

5) 0.000

6)

7) 0.037

tion technique was successfully adapted to be used on he- molymph and DG of tissue samples. Separation was achieved successfully, and it was possible to separate and quantify the 6 acids at the same time in one sample of he- molymph and in one sample of tissue extract with lesser retention time than those in other papers .Without need for
nique was a convenient, easier, faster, more accurate, and

1) Pyrovic

2) Lactic

0) R

1) S

2) R

3) S

9) 5.9799

0) 3.1791

1) 2.4350

2) 4.1512

8) 0.01098

9) 1.22415

0) 0.01590

1) 0.06630

8)

9) 0.35

0)

1) 0.000

29]. In the present study, the order of concentration of the carboxylic acids in the heamolymph of susceptible group was pyruvic ˃ lactic ˃ propionic˃ malic ˃ fumaric˃ succi n- ic, while those of resistant group was Lacti˃c pyruvic ˃ propionic ˃malic ˃ succinic ˃ fumaric. T he order is diffe r- ent from that found in the previous study of Massa et al.

3) Propionic

4) R

5) S

6) R

3) 5.1459

4) 5.8877

5) 8.2096

2) .00000

3) .50400

4) .01325

2)

3) 0.001

4)

Regarding the order of concentration of the studied acids in the DG of susceptible and resistant snails, the obtained date showed that an order of malic ˃propionic ˃ lactic ˃ succinic ˃

Organic acids concentrations in digestive glands are expressed

in mg/g tissue , while in hemolymph organic acids are expreesed in ug/dl.

In the present study, separation and quantitative determina- tion of the 6 carboxylic acids were done by the ion- suppression reversed-phase HPLC which has 2 significant features; the use of extremely diluted perchloric acid in water as the mobile phase decreases the total analysis time and pro- vides good separation of the samples that facilitates simulta

pyruvic ˃ malic respectively. These vari ations in the level of


the studied organic acids between susceptible and resistant snails could be helpful to suggest the critical importance of these acids for the success of host-parasite relationship This could be confirmed through considering the recent work of Abou El-Seoud et al. (2010) who found that carboxylic acids investigat- ed in both the haemolymph and tissue samples could be used as a potential diagnostic biomarkers to discriminate between infect- ed and non-infected Biomphalaria alexandrina (B.alexandrina)

snails. Moreover, the difference between

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resistance fumaric malic succinc

pyruvic lactic propionic

600

500

400

300

200

100

0

Hemolymph DG



Fig 1 : (A) Percentage change of carboxylic acids inhemolymph and digestive gland of resistance and susceptible Biomphalaria alexandrina snails

control fumaric malic succinc

pyruvic lactic propionic

180

160

140

120

100

80

60

40

20

0

Hemolymph DG



Fig 1: ( B) Percentage change of carboxylic acids in hemolymph and digestive gland of susceptible control and infected Biompha- laria alexandrina snails.
haemolymph and DGG concentrations of the measured acids within susceptible and resistant snails could be used to sug- gest that haemolymph and tissue metabolic rates are differ- ent. The change in the acid concentrations during the studied course of susceptibility, the following was reported[24]. Re- duction in the acid concentrations from that of the susceptible and during infection is explained by their possible use as me- tabolites by the developing schistosome sporocysts and cer- cariae [24]. These larval stages inhabit the intertubular spaces of the DG and cause mechanical and lytic damage to the di-

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gestive gland cells of the hepatopancreas [24]. The damage probably results in a leakage of the acids, which are in turn utilized by the larval schistosomes. Moreover, increased met- abolic activity associated with the presence of larval trema- todes may accelerate the use of the acids by host cells in the digestive gland as well [24], the site of active intermediary metabolism in these snails is associated with the mitochondria of the digestive gland cells [19].
The trial to test the carboxylic acids as a potential diagnos- tic biomarker was one of the aims of this study. HPLC analy- sis, which is less expensive and more rapid than the previous- ly mentioned techniques, succeeded in stating that these acids could be used as diagnostic biomarkers to detect whether the snail is susceptable or not . In addition, all of the studied acids in both the hemolymph and tissue samples could be used as a potential diagnostic biomarker except for the lactic acid in hemolymph. The key challenges in the anti-parasitic drug discovery are target selection and identification of appropri- ate small molecules as potential legends’ for these targets. The search for biomarkers that might be useful in the drug discov- ery and development is an active area of research, and the complexity of disease and drug effects means that biomarker combinations will be more accurate [28].


In the present work, malic acid recorded the most varied concentration between susceptible and resistant snails which is in good agreement with the previous work of Abou El- Seoud et al. [30] who reported that malic acid was the most sensitive acid that could discriminate between control and infected snails and they recommended further assessment on their validity for usage on field studies to diagnose schisto- somal infection of B.alexandrina snails and to state the stage of infection, and that assessment of these acids in earlier pre- potency diagnosis is required to be investigated.

Relative concentrations of the measured acids (Table 4) showed that haemo- lymph of susceptible snails recorded a lactate / pyruvate ratio of 0.84 compared to a ratio of 1.07 in the haemolymph of resistant snails which reflects the rate of glycolysis in susceptible snails. This could be supported through considering the work of El-Ansary [31] who reported different lactate / pyruvate in B.alexandrina snails parasitized by S.mansoni parasite. A lower ratio of lactate / pyruvate in susceptible snails could reflect a highly active lactate oxidase enzyme [31].


Mollusk-Parasite relationships represent a vast field of research. The dynamic interaction between mollusks and their trematode parasites leads to a state of co-existence or to in- compatibility, where the trematode is either destroyed and eliminated by the host snail defensive responses or fails to develop because the host is physiologically unsuitable ( Van der knaap and Loker , [32]. There is a high degree of specifici- ty of shistosomes to their intermediate snail hosts. Resistant snails possess a natural defense system which protects them against S.mansoni larvae, which generally die one to three days after penetrating the snail [33,34 and 35] . Although

susceptible snails are able to protect themselves against dis- ease transmitting agents, S.mansoni appears to be able to avoid the snail´s defense system, thus allowing its own devel- opment in the host. Several studies on snail-Schistosome in- teraction have shown that susceptibility to infection may be the result of genetic factors present in the genome of both the vector snail and the parasite [36,37 and 38].
Previous studies have demonstrated that the digestive gland
is the snail organ that provides the best conditions for the de-
velopment and multiplication of the parasites [39, 40 and 41] ,
where there is a very good food supply for the sporocysts [42,


43 and 44] .In case of Schistosoma mansoni , the larvae can multiply to reach a weight equivalent up to 50% of that of Biomphalaria glabrata digestive gland [44] .For a successful host-parasite complex, the host must have high levels of ener- gy metabolites to sustain normal metabolic activities during the developing of the parasites[44 and45] .
Many aspects of the host-parasite interaction remain to be clarified. When the parasites arrives the interfollicular con- nective tissue of the digestive gland, they are bathed in the haemolymph taking up large amount of free amino acids and carbohydrates for growth, energy metabolism and produc- tion of cercariae [46], and in exchange release products of their intermediate metabolism into the host´s body [17].The significant difference in carboxylic acids in susceptible and resistant snails , was explained by different reports , [18,19,21], as they that metabolite correlations do not neces- sarily correspond to proximity in the biochemical network as it is noted that neighboring metabolites and directly interact- ing metabolites in the metabolic network may have little or no correlation. It is not because they are not related, but because the variance in the enzymes that control them also affects them in equal amounts and different directions. This is what happens to most of the metabolite pairs and is the con- sequence of the systemic nature of metabolic control. Howev- er, most high correlations may be due to either stronger mu- tual control by a single enzyme or variation of a single en- zyme level much above others Abou Elseoud et al. [30]. Also, metabolites in chemical equilibrium will have nearly perfect positive correlation. As a consequence, metabolites with nega- tive correlation are not in equilibrium. In this case, the corre- lation does not originate from the enzyme that catalyzes the equilibrium reaction, as the metabolites have very small re- sponse towards it [16].
Consistent differences in both the hemolymph and tissue
carbohydrate metabolite correlation profiles of the studied
groups represent a promising additional source of infor-
mation about the state of a metabolic system. However, their
interpretation in terms of the underlying biochemical path- ways is not straight forward and largely defies an intuitive analysis. These findings highlight the potential of metabolom- ics as a novel approach for fundamental investigations of host-pathogen interactions as well as for disease surveillance and control.

Identifying enzymes involved in specific metabolic pathway detected only in S. mansoni-infected snails may allow the use

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of certain enzyme inhibitors as target chemotherapeutic con- trol agents. Alteration in the metabolism or metabolic status may also be used as a control measure without killing snails to keep balanced ecology [30].
The accumulation of organic acids as end products of energy metabolism is a common feature among parasitic helminthes [47]. Their concentrations in tissue and haemolymph were studied in infected snails and compared with those of non- infected snails (resistant & susceptible strains ). In this con- text, organic acids may serve as indicators of various metabol- ic reactions. Thus, they may indicate the use of carbohydrates as an energy source in the flow of aerobic and also during anaerobic because they can be correlated without the help of kreb’s cycle by a sequence of reactions and of protein via glu- cogenic amino acids [48], or metabolism of lipids on a small scale via fatty acids and ketone bodies [49]. Using such data, one can then evaluate in a broader manner the physiological processes that permit the snail to survive with or without pressure of infection.
One of the main methods of studying the evolutionary for- mation of metabolic forms is comparative analysis of activity and regulatory peculiarities of enzymes and polyenzymatic systems in the phylogenetic series of animals. Sufficiently numerous investigations of the adaptive changes in glycolytic enzymes in different types of invertebrates were reported [31]. It has been shown that the enzymatic activity of lactate dehydrogenase (LDH), an enzyme catalyzing the final step in glycolysis is reduced in the resistant snails to a considerable extent [31].
The LDH functions are partially fulfilled by alanine ami- notransferase (ALT) performing mutual transformation of pyruvate and alanine[50]. A second transamination enzyme, aspartate aminotransferase (AST) is connected to a great de- gree with oxidation metabolism additionally filling the pool of the Krebs cycle metabolites.
Whereas levels of LDH, ALT and AST have been measured in a number of marine invertebrates[31]. Little information is available on the activity levels or patterns of these enzymes in fresh water snails. The present study demonstrates that AST, ALT and LDH were detected in susceptible and resistant snails. AST activity show high significant difference between the susceptible and resistant snails. The distribution of ALT activity presents an other picture. Susceptible snails have the highest level than the resistant which has the lowest ALT ac- tivity.
It is seen from the presented data that AST and ALT activi- ties considerably changes within susceptible and resistant strains. Goromosova [51] compared the ratio of transaminas- es activities (AST& ALT) of invertebrates and with the view point of ecology, they showed that mollusks can be subdivid- ed into the groups. The first group includes the species living the attached life and adapted to anaerobic conditions. The AST/ALT ratio of which fluctuates within 0.1 – 0.3. Low
AST/ ALT values (0.4 – 1.0) are also characteristic of faculta- tive anaerobes. Some higher level of AST/ALT (1.0 – 1.7) is observed in a number of mollusks inhabiting a sufficient oxygenated environment ( the second group ). The third group unites oxyphilic and mobile molluscs with AST / ALT ratio of 2.0 and higher.




Based on these observations, activities of transaminases (AST&ALT) can serve as an index of the metabolic degree or a relative role of the aerobic and anaerobic pathways in the en- ergetic metabolism of the studied strains. Comparative analy- sis of the AST / ALT ratio of the studied strains showed that, the susceptible snails have the lowest AST / ALT ratio. They can be classified as aerobes but highly adapted to the anaero- bic conditions with AST / ALT ratio around 1.7. Resistant snails is extremely oxyphylic, unable to withstand under an- aerobic conditions (AST / ALT ratio of 4.5 ) ( Table 5). These results are in accordance with those of Nabih and El- Ansary [50],who proved through measuring the Michaelis constants (Km) of succinate oxidase and fumarate reductase of fresh water snails, susceptible and resistant to schistosoma infection that B. alexandrina and B. truncates as specific molluscan hosts for S. mansoni and S. haematobium respectively are aerobes and that lactate is the major end product of glycolysis. In the resistant snails Km (succinate) / Km (fumarate) Value led them to suggest that these snails species are facultative anaerobic adapted to hypoxia through succinate production ( Table 4) .
In our presented data, activity of AST in resistant strains and activity of ALT in the susceptible snails confirmed the previous observation of Nabih and El- Ansary [50], since AST can provide Krebs cycle intermediates which in turn favour the succinate production in resistant snails, while highly ac- tive ALT can be correlated to lactate production through transforming alanine to pyruvate. These results led us to ex- press a supposition about a regulatory role of AST of oxida- tive metabolism, while ALT participates in the regulation of glycolysis.
A comparison of the ALT activity with LDH level will illus-
trate their great role in the energetic metabolism during hy- poxia. Goromosova and Tamozhnyaya [52] observed a very low ALT/LD (0.04- 0.1) in sponges, hydroids. A somewhat higher ratio ( 0.2- 0.6 ) in a number of slow mobile gastro- pods and attached bivalve molluscs .ALT/ LDH ratios rec- orded for the susceptible snails confirmed the adaptive capa- bilities of the susceptible snails to anoxic conditions .
The present study clarify , significant increase in glycolytic
enzymes PFK, GPI and G3PDH were observed in infected
and susceptible snails , while low significant levels in resistant
snails of B. alexandrina . The enhancement in the activities of glycolytic enzymes in infected and susceptible snails could be attributed to increase metabolic activities of infected and sus- ceptible snails to compensate the inhibition of host Kreb s cycle caused by the parasitic infection (as indicated by lower- ing LDH level in infected snail ). The increase in anaerobic glycolysis might be attributed to activation of PFK due to de- creased citrate formation , provision of energy lead to inhibi-

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International Journal of Scientific & Engineering Research, Volume 4, Issue 10, October-2013 1046

ISSN 2229-5518

tion of Kreb s cycle , and decreased NAD/NADH ration due to inhibition of mitochondrial oxidation which favors conver- sion of pyruvate to lactate (Tielns , 1997 and Hamed et al.,
2004). Lower LDH activity in infected and resistance snails
revealed the aerobic –anaerobic switch induced in infected
state by developing parasite (Tielens et al., 1994) . Lower ac-
tivity in LDH in the direction could be easily correlated to the crabtree effect of schistosme (Tielens , 1997), through which lactate is accumulated and glycogen depleted confirming in- hibition of aerobic respiration and stimulation of anaerobic glycolysis (Kuser et al., 2000). These findings explained and confirmed the present results through the observed signifi- cant reduction in glycogen concentrations in infected and sus- ceptible snails as compared to resistant one due to their deg- radation into glucose to produce energy and enhanced gly- colysis which leads to significant increase in glucose level in infected and susceptible snails.

4 CONCLUSION



In conclusion, consistent differences in both the hemolymph and tissue carbohydrate metabolites profiles as well as some enzymes representing different pathways of resistant , sus- ceptible and B. alexandrina infected with S. mansoni were identified with a HPLC and colorimetric end point methods as these strategies allowed the prediction of infected and uninfected susceptible and resistance snails. These findings highlight the potential of metabolomics as a novel approach for fundamental investigations of host-pathogen interactions as well as disease surveillance and control.

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