In vivo flow 31P FT-NMR spectroscopy of a nematode parasite

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MAGNETIC RESONANCE I N MEDICINE 16,350-356 ( 1990)

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In Vivo Flow 31PFT-NMR Spectroscopy of a Nematode Parasite S. N. THOMPSON,E. G. PLATZER,A N D R. W.-K. LEE Analytical Chemistry Instrumentation Facility and Deyarcmencs oj'Entornology, Nernatology and Chemistry, University ofCal$ornia. Riverside, California, 92521 Received May 9, 1990; revised August 13, 1990

3'PNMR spectroscopy of a parasitic nematode, Sfeinernema caryocaysue,is described. In vivn spectra were generated from nematode suspensions continuously circulatingthrough the spectrometer. By flowing the organism, saturation effects were avoided and short interpulse delays significantly reduced spectral generation time. To maximize sensitivity, 90" pulses were employed. Changes in energetic status in response to oxygen availability were easily and rapidly monitored in circulating nematodes. 0 1991 Academic Press, Inc. INTRODUCTION

Research in this laboratory involves metabolic investigation on nematodes and other parasitic helminths of medical importance. Such studies are aimed at the identification of parasite-specific metabolic targets for new chemotherapeutic agents: the "magic bullet" concept ( I ) . The 31PNMR spectrum has been demonstrated as a valuable indicator of energy status for monitoring the effects of anthelmintic compounds in intact parasites ( 2 ) . NMR investigations under controlled conditions, however, have been restricted to a few large parasite species, where several individuals can be retained and perfused within the spectrometer in the traditional manner ( 3 ) .The tiny size of many helminth parasites prohibits direct perfusion. In this case, NMR analysis is usually camed out on large numbers packed in the spectrometer under static conditions. The protracted time required to generate spectra results in the inability to regulate oxygen tension and other factors, and the 3'P NMR spectrum may not reflect that of the organism in a normal and viable state. While methods have been described for perfusing cells attached to inert media or embedded in gel threads ( 4 ) ,adapting such methods would have limited success in studies on many small helminths, as well as many other invertebrates, because of the extreme mobility of these organisms. In this article we describe conditions for rapidly generating the in vivo 31PNMR spectra of the infective stage of an insect parasitic nematode, Steinernema carpocapsae, by continuous recirculation of the organism in suspension. MATERIALS AND METHODS

Nematode Suspensions Large numbers of third stage S. carpocapsae were obtained from Dr. Ramon Georgis of Biosys, Inc., Palo Alto, CA. Individual nematodes are approximately 500 pm long 0740-3 I94/90 $3.00 Copynght 0 1990 by Academic Press. Inc. All nghts ofreproduction In any form reserved.

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and 25 pm in width. Approximately 20 million nematodes, representing a dry weight of 1.8 g, were suspended in 50 ml of 0.05 M NaCl buffered with 10 m M HEPES, pH 7. With the aid of a peristaltic pump the nematode suspension was continuously circulated through a Nicolet NT 300 superconducting spectrometer from a 75-ml conical shaped reservoir outside the magnet (Fig. 1 ). The suspension was gassed in the reservoir with a fine stream of 95% 0 2 / 5 % COZ.Hypoxic conditions were established by gassing with 95% N2/5% C 0 2 . Nematodes entered the probe in a 12-mm NMR tube, and passed through a 2.5-cm premagnetization region prior to entering the observation region. The tube was supplied through the bottom of the spectrometer by &,-in.-i.d. vinyl tubing and exited from the top of the NMR tube and into the reservoir through a similar length of vinyl tubing. The nematode mass contained within the radiofrequency coil at any time was approximately 70 mg dry weight. Static analysis with the entire observation region filled with nematodes was also camed out.

FT-NMR Analysis 3'P NMR spectra of nematode suspensions were generated at 121.5 MHz using a 50-ps (90") pulse, 0.25s acquisition time, and 30-Hz line broadening. Interpulse

delay was varied from 0.25 to 15 s. Spectral width was f4 kHz.

FIG. 1 . Simplified diagram of the flow system employed for in vivo NMR spectroscopy of 3einernerna carpocupsue suspensions (components not shown to scale).

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Spectral assignments were based on chemical shift values previously reported in the literature ( 5 ) as well as 31PNMR analysis of freeze-extracted nematodes.

Freeze-Extract ion oJNematodes Extracts were prepared by freezing nematodes in liquid N2 and powdering the frozen nematodes with a mortar and pestle. The frozen powder was extracted with 7% perchloric acid and, after centrifugation, the supernatant was neutralized. A titration of the extract was also carried out to determine the relationship between the chemical shift of inorganic phosphate (Pi)and pH (6). RESULTS AND DISCUSSION

In Vivo Spectrum The 3'P NMR spectrum of S. carpocapsae suspensions was composed of numerous peaks (Fig. 2a). /3 ATP and phosphoarginine were clearly discernable. By flowing the nematodes through the spectrometer, saturation effects were avoided and short interpulse delays could be employed to reduce spectral generation time. Satisfactory spectra were obtained under static conditions when greater tissue mass was analyzed by filling the entire observation region (Fig. 2b). Despite the increased mass, however, 45 O pulses were necessary to obtain satisfactory sensitivity. Phosphoarginine was not observed in spectra of nematodes maintained under static conditions. Nematodes ana-

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FIG. 2. ( a ) In vivo "P NMR spectrum of a suspension of Steinernemu curpocupsue flowing at 60 ml/min under aerobic conditions. Spectrum was generated from 1200 data acquisitions using a 90" pulse, 0.25s acquisition time, and 3-s interpulse delay. Nematode mass contained within the observation region of the probe at any time was approximately 70 mg. ( b ) In vivo spectrum of 250 mg of nematodes maintained under static conditions. Spectrum was generated from 1200data acquisitions using a 45" pulse, 0.5-s acquisition time, and interpulse delay of 2.5 s. Spectral assignments were as follows: ( 1 ) 3.57 k 0.12 ppm (Xk S.D., n = 5 nematode suspensions), phosphomonoesters, ( 2 ) 1.80 -t 0.1 I ppm, inorganic phosphate, ( 3 ) -0.33 k 0.04 ppm, glycerophosphorylcholine glycerophosphorylethanolamine (present as a minor downfield shoulder), ( 4 ) -3.81 ? 0.05 ppm, phosphoarginine, ( 5 ) -5.83 f 0.06 ppm, y ATP 0ADP, ( 6 ) -10.75 f 0.24 ppm, 01 ADP ATP, ( 7 ) - 19.53 C 0.1 1 ppm, (3 ATP. (c) Difference spectrum obtained by subtraction from ( b ) of a spectrum generated during the interval 4 and 5 h later.

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lyzed under static conditions also had higher relative levels of sugar phosphates and inorganic phosphate (Pi) and lower ATP. The assignment of glycerophosphorylcholine together with a minor contribution of glycerophosphorylethanolamine as the sole components of peak 3 in the in vivo spectrum of S. carpocapsae (Fig. 2 ) is tentative at this time. Wadsworth and Riddle ( 7) recently reported the presence of a highly labile uridine-containing phosphodiester at approximately -0.6 ppm in the in vivo 31PNMR spectrum of the free-living nematode Caenorhabditis elegans. Although a spectral component with that chemical shift was not observed in S. carpocapsae it may be a minor component of in vivo peak 3.

Effects of Delay Time, Flow Rate, and Premagnetization Interpulse delay had a major impact on spectral intensity (Fig. 3 ) . The effect on individual spectral components was dependent on flow rate and was particularly evident with phosphoarginine and glycerophosphorylcholine,which have relatively long T 1's (Fig. 4). The effects of flow rate on Pi were similar but could not be accurately estimated because of its low level in the in vivo spectrum obtained under aerobic conditions. At a flow rate of 50 mllrnin, interpulse delays of at least 3 s were required to ensure maximal intensities for all spectral components. Intensity corrections were necessary when shorter delays were used. Although previous studies (8) have demonstrated that signal enhancement in flowing solutions is adversely affected by the finite premagnetization time available for sample equilibration, increasing the size of the premagnetization region from 1 to 5 cm during in vivo analysis of S. carpocapsae had no effect on spectral intensity at the flow rates employed in the present study. During the experiments described below a flow rate of 60 ml/min and premagnetization region

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FIG. 3. Effect of interpulse delay time on the in vivo "P NMR spectrum of Steinernema carpocapsae. Spectra were generated from 1000 data acquisitions using a 90" pulse and 0.25-s acquisition time. Nematodes were flowing at a rate of 50 ml/min.

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FIG.4. Effect of flow rate and interpulse delay on the relative intensities of ( a) phosphoarginine and ( b ) glycerophosphorylcholine in the in vivo "P NMR spectrum of Steinernemu carpocapsae flowing at 50 rnl/ rnin. ( 0 )50 rnlimin, (0)25 ml/min, ( A ) 12.5 rnlfmin. NMR parameters as in Fig. 3.

of 2.5 cm were employed. At 60 ml/min, nematodes passed through the probe at a velocity of approximately 1 cm/s. When short interpulse delays were employed, spectral adjustments were made using intensity correction curves (e.g., Fig. 4).

Eflects of Gas Phase on the in Vivo Spectrum When nematode suspensions were exposed to hypoxic conditions in vivo, alterations in phosphoarginine and p ATP were quickly observed (Fig. 5). Indeed, changes in these metabolites were detected within 5 min from spectra generated from 500 data acquisitions using a 0.5-s interpulse delay. In that case S / N ratios (2 k S.E., n = 20) under aerobic conditions for phosphoarginine and fl ATP were 14.0 & 3.4 and 12.6 t 2.8, respectively. It should be noted that exchange rate constants, and thus the observed T 1, may be affected by hypoxic conditions. Although such effects would necessitate minor adjustments in the correction factor used for estimating spectral components during hypoxia, such adjustments were not considered during the present study. Accurate estimation of changes in minor spectral components, including P,, required longer delays and increased data accumulation. Picould be accurately estimated during longer experiments on nematodes exposed to hypoxia. In that case decreases in the relative intensity of phosphoarginine were exactly offset by increases in Pi. The NMR index [ ATP phosphoarginine/ATP phosphoarginine Pi]( 9 )was used to evaluate the effects of long-term exposure to hypoxia on the energy status of S. carpocarpsae (Fig. 6 ) . The index decreased under hypoxia and was quickly restored upon return of aerobic conditions. Phosphoarginine usually decreased to below detectable levels during long-term exposure to hypoxia. The effect of hypoxia on the chemical shift of Pi was closely correlated with the observed changes in the NMR index. The NMR index, therefore, is a valuable in vivo indicator of both the metabolic and the energetic status of the nematodes during oxygen stress. Based on the titration curve obtained from nematode extracts, the upfield shift in Pi during the above experiment represented a pH change from approximately 7.5 under aerobic conditions to 6.9 during hypoxia (Fig. 6).

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FIG.5. Effect of short-term hypoxia on the relative intensity of phosphoarginine and p ATP in the in vivo "P NMR spectrum of Steinernemu curpocupsue, ( 0 )/3 ATP, ( 0 )phosphoarginine. Spectra were generated at 10-min intervals from 600 data acquisitions using a 90" pulse, 0 . 2 5 s acquisition time, and 1-s interpulse delay. The intensity of phosphoarginine was corrected. Nematodes were flowing at a rate of 60 ml/min.

FIG.6 . Effect of long-term hypoxia on intracellular pH and in vivo energetic status of Steinernemu curpocupsue. NMR index = p ATP phosphoargininelp ATP phosphoarginine p,. During long-term exposure to hypoxia, phosphoarginine usually decreased below observable levels and the NMR index was not calculated. Spectra were generated at approximately 1 h intervals from 1200 data acquisitions using a 90" pulse, 0.25-s acquisition time, and 3-s interpulse delay. A flow rate of 60 ml/min was employed.

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Spectral changes observed over time in packed nematodes analyzed under static conditions were similar to those in flowing nematode suspensions exposed to hypoxia and included an upfield shift and increase in the relative level of Pi (Fig. 1b-inset) . A decrease in phosphomonoesters was also noted. ATP was probably not depleted because of the capacity of the nematode to produce ATP by anaerobic fermentation through the action fumarate reductase (10). The same spectal changes, as well as a depletion of ATP, were previously reported in 31PNMR spectra of densely packed hamster cell suspensions ( 1I ). CONCLUSION

Continuous circulation of S. carpocapsae in suspension proved very successful for in vivo NMR analysis under controlled conditions. High quality spectra with satisfactory S / N were generated in a fraction of the time required with noncirculating animals. The method enabled rapid detection of changes in high energy phosphorus metabolites. The results suggest that this method may be satisfactory for whole-animal NMR studies of microscopic invertebrates, including nematode helminths of medical importance, that can be obtained in sufficient quantity.

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1. C. BRYANTA N D C. A. BEHM,in “Biochemical Adaptation in Parasites,” pp. 117-177, Chapman &

Hall, New York, 1989. 2. S. N. THOMPSON, E. G. PLATZER,AND R. W.-K. LEE,Mol. Biochem. Parasitol. 22,45 ( 1987). 3. W. J. ~ S U L L I V AM. N ,R. EDWARDS, AND R. S. NORTON,Parasitol. Today 5,19 ( 1989). 4. W. M. EGAN,in “NMR Spectroscopy of Cells and Organisms” (R. K. Gupta, Ed.), pp. 136-168, CRC Press, Boca Raton, FL 1987. 5. D. G. GADIAN, G. K. RADDA,G. K. RICHARDS, AND P. J. SEELEY,in “Biological Applications of Magnetic Resonance” (R. G. Shulman, Ed.), pp. 463-535, Academic Press, New York, 1979. 6. J. K. M. ROBERTS,N. WADE-JARDETZKY, AND 0.JARDETZKY, BiochemisIry 20, 5389 ( 1981). 7. W. G. WADSWORTH, AND D. L. RIDDLE, Dev. B i d . 132, 167 (1989). 8. D. A. LAUDE,R. W.-K. LEE,AND C. L. WILKINS,Anal. Chem. 51, 1281 (1985). 9. N. LAVANCHY, J. MARTIN,AND A. ROW, J. Physiol. Paris 80, 196 ( 1985). 10. J. BARRETT,“Biochemistry of Parasites,” University Park Press, Baltimore, MD, 1980. 11. D. L. FOXALL, J. S. COHEN,AND J. B. MITCHELL, Exp. Cell Res. 154, 521 (1984).

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