31P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and Fasciola hepatica

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 248, No. 1, July, pp. 200-209, 1986

31P-NMR Studies of the Metabolisms of the Parasitic Helminths Ascaris suum and Fasciola hepatica SUSAN PIETRZAK Departments

of *Chemistry

ROHRER,*,’ and TBiological

HOWARD

J. SAZ,t AND THOMAS

Sciences, University

Received February

NOWAK**2

of Notre Dame, Notre Dame, Indiana

66556

20, 1986

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism’s internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism. o 1~86 Academic PM, I~C.

The applications of 31P-NMR spectroscopy have become increasingly important for metabolic studies of intact biological systems since the first demonstrations of its usefulness for such studies (1, 2). Although the method has been widely used for studies of bacterial and mammalian metabolism, it has only recently been applied to examinations of parasitic helminths (3,4). As a consequence of their size, many parasitic helminths are prime candidates for the applications of NMR spectroscopy. The advantages of the NMR approach over more traditional methods include the ability to simultaneously monitor several phosphorus-containing metabolites and the elimination of manipulative arti-

facts. The 31P spectra of intact parasites can be obtained in a noninvasive manner. A primary goal of these studies was to establish whether the 31P NMR methd can be applied to studies of the metabolism of the parasitic helminths Ascaris and Fasciola. The feasibility of using 31P NMR to examine the effects and possible modes of action of drugs on the energy metabolism of the parasites was tested. MATERIALS

Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

METHODS

Adult Ascaris suum was kindly supplied by the Wilson Meat Packing Plant, Logansport, Indiana. Fasciola hepatica metacercaria were supplied by Baldwin Aquatics, Inc., Monmouth, Oregon. Closantel was a kind gift from Dr. R. Fetterer of the United States Department of Agriculture, Beltsville, Maryland. Enzymes used for the determinations of ATP and of glycolytic intermediates were purchased from

i Present address: Department of Biochemistry, University of Alabama at Birmingham, Birmingham, Ala. 35294. * To whom correspondence should be addressed.

0003-9861/86 $3.00

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IN

VIVO

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OF

Ascaris

Boehringer-Mannheim. Mest glucose 6-phosphate, glycerophosphorylcholine, fructose diphosphate, Penolpyruvate, adenosine diphosphate, reduced nicotinamide adenine dinucleotide, creatine phosphate, and arginine phosphate were supplied by Sigma, Inc. (St. Louis, MO.). Streptomycin and penicillin were products of Eli Lilly, Inc. (Indianapolis, Ind.) and E. R. Squibb & Sons, Inc. (New York, N. Y.), respectively. Ascaris suum adults were transported from the abbatoir and maintained in a balanced salt solution (5). Approximately 8 h prior to beginning NMR experiments they were transferred to the NMR buffer. In experiments where pH determinations were made, intact parasites were maintained for 4 h in the stated buffer. The pH of the buffer was adjusted by varying the proportions of Mes and Tris. No additional salt was added beyond that of the basic medium (5) to compensate for any minor change in ionic strength as the pH was adjusted. The pH values stated are meter readings calibrated with standard buffers. No adjustment for 10% DzO was made. Inorganic phosphate was omitted from the buffer to avoid confusion of the Pi resonance with the internal Pi resonance. When muscle or muscle extracts were required, strips of body wall muscle were obtained by dissection (6). Intact Fasciola hepatica adults were dissected from bile ducts of rats which had been infected with 30 metacercariae at least 4 months prior to harvest. Upon removal from the bile ducts, worms were maintained for several hours in the NMR experimental buffer to allow for emptying of gut contents and to establish a steady state of metabolism. NMR experiments were performed on a Nicolet 300 Multinuclear NMR spectrometer using quadrature phase detection in the Fourier transform mode. In the experiments with Ascati reported in this paper, female worms only were used. The samples were placed directly into lo-mm NMR tubes. Each spectrum represents a total of 5000 acquisitions which were accumulated using 16K data points and a spectral width of 6000 Hz unless otherwise indicated. The radio frequency pulse employed had a duration of 11 ps (60’ pulse angle) and the recycle time was 0.7 s. A longer recycle time (4 s) does not substantially change the quality of the spectra or the relative peak intensities. The total time to acquire a single spectrum of these parasites with a good signal-to-noise ratio was 56 min for the experiments reported here. In all experiments

a Abbreviations used: Mes, Z(N-morpholino)ethanesulfonic acid, P-enolpyruvate, phosphoenolpyruvate; NTP, nucleoside triphosphate; NDP, nucleoside diphosphate; FDP, fructose diphosphate; closantel, N-{5-chloro-4-[(4-chlorophenyl) cyanomethyll2-methylphenyl}-2-hydroxy-3,5-diiodobenzamide.

swum

AND

Fasciola

hepatica

201

reported, a continuously changing buffer was maintained in the NMR tube by perfusing the system with the use of a peristaltic pump. Perfusion insured a constant supply of glucose and salts to the worms as well as removal of excreted acidic end products which would influence the pH of the medium. When perfusion was not performed a marked decrease in the pH of the medium occurred. Where noted, the reservoir containing fresh buffer was bubbled with either 5% CO.J95% N2 or 5% CO.J95% air. The flow rate of the buffer through the NMR tube was 12 ml/h. The buffer for NMR experiments was that reported by Tielens et al (3) and contained the following: 39 mM NaHCOa; 78 mM NaCI; 5.4 mM KCI; 1 mM NaH,P04; 0.8 mM MgSOa; 1.8 mM CaC12; 11 mM glucose; 12.5 mM Mes; 12.5 mhi Tris; streptomycin 75 mg/liter; penicillin 71 international units/ml. The temperature inside the magnet was normally maintained at 22°C. Spectra of Ascati were also taken at 37”C, but the quality of the spectra was much poorer. This poorer quality was caused by the increased motility of the worms. The Fasciola hepatica and Ascati suum were held at room temperature in the above buffer before experiments commenced. An external standard of 85% H3PO1 was sealed in a capillary tube and placed inside the NMR tubes which contained the parasites and used as the reference standard for setting zero ppm. Ten percent D20 was routinely added to the buffer for setting the field/ frequency lock. All spectra were obtained in the presence of 10% Da0 since, in its absence, there was occasionally sufficient drift to obscure the usually welldefined resonances observed with intact worms. There was no obvious detrimental effect on the worms by the 10% DzO. To determine the effects of closantel on glycolytic intermediates and on ATP levels independently, incubations of the parasites were performed at room temperature and they were perfused with the same buffer employed in the corresponding NMR experiments. Worms were incubated in Erlenmeyer flasks and were removed at the designated times. They were immediately quick-frozen in liquid nitrogen, weighed, and homogenized in cold 3% perchloric acid. Homogenates were centrifuged to removed particulate matter and the pH was adjusted to 6.0 before freezing to await further analysis. The determination of ATP and glycolytic intermediates was performed by enzymatic analysis (7-9). The ATP was determined using hexokinase and glucose-6-phosphate dehydrogenase, glucose g-phosphate by glucose-6-phosphate dehydrogenase, P-enolpyruvate by pyruvate kinase and lactate dehydrogenase, FDP by aldolase, triose phosphate isomerase, and glycerol-a-phosphate dehydrogenase, and dihydroxyacetone phosphate by glycerol-a-phosphate dehydrogenase.

202

ROHRER,

SAZ,

RESULTS

Identi)icaticm of Phosphorus-Containing Metabolites in a Cell-Free Extract Prepared from Ascaris Muscle Strips A spectrum of a standard solution of known phosphate compounds at pH 7.0 was compared with that of a cell free extract of Ascaris muscle (Fig. 1). The resonances which were observed were sharp and most standards were resolvable. Note that the resonances from FDP and AMP were superimposed and virtually indistinguishable. Not all sugar phosphates were resolvable and are designated as SP.

‘lo FIG. 1. alP-NMR spectra of metabolites of glycolysis and of a cell-free Ascuti extract. A solution of known standards (A) was run in 10% DaO, pH 7.0, and assignments were made from published spectra and from spectra of individual samples. The cell-free extract (B) was prepared by homogenizing 10 g of muscle in 20 ml cold 3% perchloric acid followed by centrifugation for 15 min at 13,000 rpm. The solution was neutralized with NaOH and 1.8 ml was added to 0.2 ml DzO directly in the NMR tubes. The following abbreviations are employed: G-6-P, glucose 6-phosphate; Pi, inorganic phosphate; PEP, phosphoenolpyruvate; NADH, nicotinamide adenine dinucleotide (reduced); SP, sugar phosphates; GPC, glycerophosphorylcholine; NTP (NDP), nucleoside tri(di)phosphate, the (Y, 8, and y designating the phosphorus position; FDP, fructose 1,6-diphosphate.

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Glycerophosphorylcholine was found to be present in the muscle homogenates. This metabolite was identified by examination of the NMR spectrum of the same Ascaris homogenate upon the addition of known Glycerophosglycerophosphorylcholine. phorylcholine was previously observed in Fasciola (4). Neither creatine phosphate, arginine phosphate, nor an analogous phosphate-containing metabolite was observed in the extract from the muscle of these parasites. Comparison of Spectra from Intact Ascaris suum Adults and Intact Fasciola hepatica Adults When spectra from the adult nematode Ascaris swum were compared with those from the adult trematode Fasciola hepatica, many similarities and a few differences were observed (Fig. 2). In Ascaris, a high NTP:NDP ratio (3.85:1) for “free nucleoticle” was apparent compared with that of Fasciola (NTP:NDP = 2.32:1). This ratio was measured by integrating the resonance for the p NTP and the (Yresonances of NTP and NDP and the p NDP and y NTP resonances. The nucleotides are probably almost exclusively aclenine. Both organisms contained relatively large amounts of glycerophosphorylcholine, the role of which remains obscure here as well as in other systems (4, 10,ll). There were differences in the sugar phosphate distributions between the two parasites as observed in their respective spectra. Fasciola possessed relatively high levels of glucose 6-phosphate while in Ascaris, this sugar phosphate was present in considerably lower proportions. In Ascaris, however, the FDP and/or fructose 6-phosphate levels were higher. Unfortunately, the two fructose phosphate esters could not be distinguished in these intact systems. P-enolpyruvate was barely distinguishable in the spectrum from Ascaris but can be seen as a small peak upfield from glycerophosphorylcholine in Fasciola hepatica. Phosphate &esters also have resonances in this region of the spectrum as do phospholipids. Immobilized phospholipids yield

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alP-NMR

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Fasciola

Determination of the Internal Intact Ascaris swum

GPC

A

GPC

w 3PPm

I B Fosciolo

hrpatico

FIG. 2. 3’P-NMR spectra of (A) intact Ascati suum and (B) intact Fasciola hepatica incubated in the balanced salt solution containing 11 mM glucose. The GPC resonance is labeled for reference. The resonances are assigned as follows: (1) glucose 6-phosphate; (2) fructose 1,6-diphosphate; (3) Pi; (4) glycerophosphorylcholine; (5) P-enolpyruvate; (6) y phosphate of ATP and /3 phosphate of ADP; (7) a phosphates of ATP and of ADP; and (8) p phosphate of ATP. The peak upfield from ‘7 is tentatively assigned to NADH.

very broad lines and are often “NMR invisible.” It is not clear why the /3-NDP resonance was resolvable in F. hepatica but not in Ascuria It has been suggested (4) that the NDP in F. hepatica is not bound to Mgf, thus affecting its chemical shift, but this remains subject to question. A recent study with Fasciola gave estimates of Me levels of 1.6-2.9 mM (12). In neither intact organism was creatine phosphate, arginine phosphate, or another analogous phosphagen detectable.

203

hepatica

pH of

To determine the internal pH of adult Ascaris worms under resting conditions and the response of the internal pH to external fluctuations, a calibration of the chemical shift of the Pi resonance with pH was first performed (Fig. 3). This was accomplished by measuring the chemical shift of a Pi buffer and the Pi resonance from an Ascaris cell-free extract over a range of pH values. The pH was adjusted with the addition of either HCl or KOH and the pH was independently measured potentiometrically. Since the chemical shift of the 31P of Pi is sensitive to a variety of external variables the use of the cellfree extract rather than Pi alone should yield a more reliable calibration curve to measure internal pH within the organism (13). The curve generated by titration of the Ascaris homogenate was used to determine the internal pH of intact helminths in subsequent experiments. The internal pH of adult Ascaris appeared to be constant at 7.1 regardless of the pH of the external medium over a range from 5.5 to 8.5. Any signal due to external

FIG. 3. Standard curve of the alP chemical shift of Pi as a function of pH. The titrations were performed by measuring the ‘rP chemical shift of a 50 mM KPOl buffer in 10% Da0 (A) relative to a phosphoric acid standard (0 ppm) or the 31P resonance of Pi in 3 ml of an Ascati muscle homogenate (0). The bulk pH was measured via a pH meter and the pH was adjusted with microliter additions of either KOH or HCl. The Pi standard required four spectral accumulations.

204

ROHRER.

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31P-NMR Spectra of Intact Fasciola hepatica under Aerobic and Anaerobic Conditions Spectra from intact F. hepatica adults incubated in air + 5% COB and in 95% Nz + 5% COz, respectively were measured (Figs. 4 and 5). Differences in the relative concentrations of the observed metabolites were compared under the two experimental conditions. Under aerobic conditions, both glucose 6-phosphate and glycerophosphorylcholine exhibited a decrease over the 8h observation period. The nucleoside triphosphate levels did not change over the course of the experiment. The negligible chemical shift of the Pi resonance indicated

5 ppm’ FIG. 4. Aerobic incubation of Fasciola hepatica adults in a perfused NMR tube. The buffer reservoir was continuously bubbled with a gaseous mixture of 95% balanced air and 5% COa. The times labeled for each spectrum indicate the length of perfusion time. The assignments labeled in the first spectrum are the same as in Fig. 2.

Pi could be eliminated by the addition of 1 mM MnClz to the external medium. The addition of up to 1 mM MnClz in the external medium did not perturb any of the 31P resonances within the organism over the course of 24 h (spectra not shown). In contrast to these pH effects, the internal pH of F. hepatica appeared to decrease slightly at external pH values below 7.0, but above this value the internal pH remained constant at 7.0 (3). Our estimate of the internal pH (7.0) of Fasciola agrees with Tielens’ estimate (3) and is close to the value recently reported (pH 6.87) by Mansour’s group (12).

0 hrs

5 Pm FIG. 5. Anaerobic incubation of Fasciola hepatica adults in a perfused NMR tube. The buffer reservoir was continuously bubbled with a gaseous mixture of 95% Na/5% COa. The times labeled for each spectrum indicate the length of perfusion time. The assignments which are labeled are the same as in Fig. 2.

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that the average internal pH of the worms remained constant. The results raise the possibility of an oxygen toxicity which lowers the glucose 6-phosphate and glycerophosphorylcholine levels. These observations would be consistent with the anaerobic environment of the adult Fasciola hepatica in the bile duct of the host. Anaerobically, nucleoside triphosphate as well as glucose 6-phosphate and glycerophosphorylcholine did not change significantly during the 8 h observation period. In addition, the pH monitored by the chemical shift of the Pi resonance did not change during the experiment. The Efects of the Anthelmintic on the “P-NMR Spectra of Fasciola hepatica

Closantel

Worms were removed from infected rats and placed in the appropriate medium prior to the beginning of spectrum accumulation. Spectra of F. hepatica adults were recorded over a period of 14 h (Fig. 6). NMR tubes were perfused during this time at a rate of 12 ml/h with a buffered solution as described under Materials and Methods. Several noticeable changes occurred over time in the spectra from worms that were kept in the standard buffer. The amount of inorganic phosphate increased and, of particular interest, glycerophosphorylcholine decreased approximately 47% over 14 h. The metabolites ATP, Penolpyruvate, and glucose 6-phosphate remained essentially unchanged over the time course of this experiment. There is some indication of more than one pool of Pi with different pH values, suggested by a downfield shoulder on the Pi resonance. These results were compared with those obtained from identical incubations except for the addition of the anthelmintic closantel (Fig. 7). In the presence of 5 X lop6 M closantel, the relative changes in phosphorus-containing metabolites were different from those seen in the controls (Fig. 6). The nucleoside triphosphate pool was not affected by the presence of the drug. The initial effect of the drug observed under our experimental conditions appears to

5wm FIG. 6. Incubation of Fasciola hepatica adults in a perfused NMR tube. Times given indicate the total time that worms have been removed from bile ducts and held in NMR buffer. The assignments which are labeled are the same as in Fig. 2.

be a significant decrease in glucose 6-phosphate levels. By 4 to 5 h of exposure to the drug (spectra not shown), glucose 6-phosphate levels were noticeably depressed by approximately 10%. An increase in Pi over this period is minor. By 10 to 13 h in the presence of the drug, internal glucose 6phosphate concentrations dropped approximately 37%. Although the physiological significance remains to be determined, closantel appears to slow the rate of disappearance of glycerophosphorylcholine. In the absence of the drug, glycerophosphorylcholine levels dropped 47% in 12 h compared with a 23% drop in 14 h in the presence of closantel.

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15 FIG. 7. Effects of closantel on the long-term incubation of Fasciolu kqootica adults in a perfused NMR tube. Closantel was added to the perfusion tube to a final concentration of 5 X 10m6 M. Times given are the time of exposure to the drug. Worms were removed from bile ducts and allowed to incubate in the NMR buffer for 9 h prior to the addition of closantel. The assignments which are labeled are the same as in Fig. 2.

In the presence of closantel, both Penolpyruvate and ATP levels remained unchanged. ATP concentrations did drop in experiments where higher dosages (5 X lop5 M) or longer periods of drug exposure were used. In such experiments the drop in ATP was concomitant with the irreversible loss of motility and muscle tone by the worms which were then assumed to be deceased. The levels of ATP had not dropped to zero, however. These results appeared to conflict with previous reports which indicated that the

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primary mode of action of the drug was on ATP levels, presumably a consequence of the uncoupling of ATP synthesis by oxidative phosphorylation (14,15). Therefore, to examine this further, essentially the same incubation as above was performed in the presence of 5 X lop6 M closantel at room temperature employing a perfusion system. The incubation was followed by rapid freezing of the parasites, perchloric acid extraction, and an independent measurement of tissue levels of ATP and of several glycolytic intermediates using enzymatic methods (7-9) (Table I). After a short period following exposure to closantel, the worms took up glucose from the medium and increased their levels of ATP and most of the glycolytic intermediates examined. This also was observed by 31PNMR (data not shown) when Fasciola were not allowed to equilibrate with the glucosecontaining buffer before the first spectrum was acquired. A similar initial uptake of glucose and increase in glucose 6-phosphate within 10 min was reported by Mansour et al. (4). Through 12 h of incubation in the presence of 5 X 10m6M closantel, ATP levels did not drop compared with controls (Figs. 6 and 7). In fact, relative ATP levels were even higher when closantel was present. At 20 h, ATP levels were depressed in both incubations. Glucose 6-phosphate was the metabolite most dramatically affected by the presence of the drug. Even at 4 h in the presence of closantel, glucose 6-phosphate levels did not rise appreciably as was the case in the absence of the drug. By 8 h, in the presence of the drug, glucose 6-phosphate levels dropped below the zero time level and continued to drop over the next 12 h. After 20 h, worms were still alive, but motility was sluggish. In addition, closantel appeared to have a sparing effect on P-enolpyruvate levels, since incubation in the presence of the drug resulted in concentrations of P-enolpyruvate higher than found in corresponding incubations without closantel. No significant effect of closantel was noted on FDP or triose phosphate accumulations. Most interesting, at higher doses (5 X 10m5M),

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TABLE1 EFFECTS OF CLOSANTEL Closantel (+ or -)

ON GLYCOLYTIC

INTERMEDIATES”

IN Fasciola

hepatica

Incubation (h)

ATP

G6Pb

PEP

FDP

DHAP

-

0

0.495 f 0.008c

2.690 k 0.033

0.332 f 0.024

0.421 2 0.037

0.188 + 0.051

+d

4 4

0.605 f 0.048 0.718 f 0.035

3.758 + 0.149 2.746 t 0.042

0.305 f 0.054 0.329 f 0.030

0.525 t 0.026 0.522 k 0.017

0.387 f 0.065 0.339 + 0.037

+

8 8

0.603 + 0.040 0.820 f 0.056

3.411 + 0.039 2.532 f 0.028

0.286 f 0.024 0.738 f 0.049

0.467 f 0.005 0.526 f 0.042

0.267 f 0.019 0.253 f 0.053

+

12 12

0.458 f 0.015 0.654 f 0.031

3.522 f 0.041 2.161 f 0.057

0.372 f 0.023 0.498 -c 0.013

0.475 f 0.012 0.456 f 0.012

0.246 + 0.042 0.206 + 0.023

-

20 20

0.396 f 0.014 0.372 f 0.014

3.569 f 0.052 1.540 + 0.102

0.324 f 0.004 0.388 f 0.003

0.319 f 0.050

0.157 f 0.032

0.344 + 0.011

0.160 + 0.032

1 4

0.573 f 0.022 0.394 2 0.020

1.718 + 0.044 1.640 + 0.098

0.681 + 0.033 0.587 f 0.057

0.493 + 0.052 0.509 + 0.040

0.333 IL 0.026 0.374 f 0.046

+

++e ++

a Metabolite levels in the tissue extracts were measured by enzymatic analyses using previously described methods (7, 8, 9). These determinations were performed at least in triplicate and the standard errors were calculated from this data. *Abbreviations used here are G6P; glucose 6-phosphate; PEP, P-enolpyruvate; DHAP, dihydroxyacetone phosphate. ’ Values are expressed in micromoles per gram wet weight of worm. ’ +, 5 X loe6 M ClOSantel. e+f,

5 X lo-* M ClOSantd.

similar but much more rapid effects were noted. Again, glucose 6-phosphate exhibited the first noticeable change. After only one hour the glucose 6-phosphate had dropped to low levels. In contrast, effects on ATP levels were not noticeable until the 4 h incubation. However, at 4 h incubation in 5 X 10e5 M closantel the worms were nonmotile and devoid of muscle tone, suggesting death. The positive effect of the drug on P-enolpyruvate accumulation at 8 h in the presence of 5 X 10e6 M closantel was expressed more rapidly at 1 and 4 h in the presence of 5 X 10m5M closantel. This increase in P-enolpyruvate may have arisen secondarily from an increased utilization of glucose 6-phosphate which then accumulated at the P-enolpyruvate carboxykinase branch point of metabolism. This effect may be a consequence of fluctuating ATP levels since it is possible that ATP acts as an inhibitor of the helminth P-enolpyruvate carboxykinase (16).

These findings suggest that glucose 6phosphate accumulation is an earlier target for closantel than that of ATP. Both NMR and analytical data indicate a decrease in glucose 6-phosphate levels which precedes any drop in ATP levels. Furthermore, the decrease in ATP, where observed, seemed to correlate with the death of the worms. DISCUSSION

31P-NMR spectroscopy has provided a noninvasive method with which to study the metabolism of intact parasitic helminths. This method permits the in situ measurement of metabolites which could not be determined previously without disrupting the parasites. Most phosphate resonances for both intact Ascaris suum and intact Fasciola hepatica have been identified. Our assignments, in general, agree with those recently published for Fasciola

208

ROHRER,

SAZ,

where 31P spectra of intact flukes and isolated tissue were studied (12). In both organisms, glycerophosphorylcholine was present in large amounts, and ATP, Pi, and sugar phosphates were readily observed. The sugar phosphate portions of the spectra were different in the two worms, indicating differences in distribution. Fasciola hepatica exhibited high glucose 6-phosphate concentrations while Ascaris exhibited high fructose diphosphate and/or fructose 6-phosphate levels. The control steps of glycolysis may differ between these two parasites, resulting in different steady state levels of sugar phosphates. Large changes in the pH of the external environment of Ascaris caused no significant alteration in the internal pH of this nematode. The internal pH of intact Ascaris females was estimated to be 7.1 from a standard titration curve. The effects of aerobic and anaerobic environments on the metabolic profile of Fasciola hepatica were examined. The adult of this trematode lives in the essentially anaerobic bile duct of its mammalian host, and might be expected to be uninfluenced by the presence of air in the environment. Spectra obtained from parasites incubated in either air + 5% COa or 95% Nz + 5% COZ confirmed the consistency of the nucleoside triphosphate pool regardless of the gas phase, indicating the anaerobic nature of Fasciola. However, decreased levels of glucose 6-phosphate and glycerophosphorylcholine were noted in response to the presence of air. These decreases suggest either a change in the metabolism, or, more likely, a toxic effect of oxygen on the parasite. Glycerophosphorylcholine levels in cardiac skeletal muscle tissue are related to stress and 31P NMR methods have recently been refined to quantitatively measure the levels of this metabolite in intact tissue (1’7). Most important, these studies bear out the initial postulation that NMR spectroscopy may be employed to examine the effects of possible anthelmintic agents on the energy-yielding metabolisms of parasites. The findings with 31P-NMR, obtained in response to closantel, have been confirmed by analytical procedures using enzymatic

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analyses. These observations strongly indicate that the initial effects of this drug are not on oxidative phosphorylation which is responsible for ATP production, as previously reported, (14,15), since ATP levels remained constant for long periods regardless of the presence or absence of closantel. Glucose 6-phosphate and glycerophosphorylcholine accumulation decreased as the initial response to clostantel. The effects on ATP levels appear to be secondary to the death of the worm. Although additional studies are required before a specific site of closantel action can be suggested with any degree of confidence, closantel’s effects on the metabolism of Fasciola could be observed with relative ease by employing 31P-NMR spectroscopy. This method observes “NMR-visible” levels of metabolite phosphates. NMR methods and enzymatic analyses have indicated that 60% of the total ADP and 50% of the total Pi are “NMR invisible” in the intact liver fluke (12). The dynamic effects of aerobiosis and closantel on glycerophosphorylcholine are of particular interest, since the physiological role of this intermediate remains obscure. Other systems in which this component has been reported include frog gastrocnemius, rabbit heart, dystrophic chicken pectoralis, and semen (10, 11). Although it has been suggested that it arises as a phospholipid breakdown product, the possibility of its involvement in energyproducing pathways also has been suggested (10). It appears that both Ascaris and Fasciola would provide good experimental materials for further investigations of the physiological function of glycerophosphorylcholine. ACKNOWLEDGMENTS This investigation was supported in part by Grants AI-09483 (H.J.S.) and AM 17049 (T.N.) from the National Institutes of Health, United States Public Health Service, and by the Merck Institute for Therapeutic Research. The research was performed while T.N. was supported as Research Career Development Awardee (AM00486) of the National Institutes of Health. The authors thank Dr. William C. Campbell

IN and

Dr.

Robert

S. Rew

of the

help and cooperation Thanks to D. Schifferl NMR our

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experiments

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of Ascaris

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“P-NMR

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