Dose-dependent metabolic response of mammary carcinoma to photodynamic therapy

The metabolic response of mammary carcinoma in the C3H mouse to photodynamic therapy (PDT) was measured using in vivo 31P nuclear magnetic resonance (31P-NMR) spectroscopy and pH microelectrodes. Twenty-four hours after administration of Photofrin II (12.5 mg/kg), the tumor was subjected to photoactivation using an argon dye laser. Optical treatment doses were 200, 400, and 600 J/cm2 and corresponded to the following tumor control doses: TCD10/30, TCD50/30, and TCD90/30, respectively. In vivo 31P-NMR spectra and pH micro-electrode measurements were obtained prior to treatment and at 4, 24, 48, and 72 h and 1 week post-treatment. The data revealed a significant (P less than 0.0002) alkalosis as indicated by the pH measured by NMR compared to pH measured by microelectrodes at all treatment levels and time points. Spectral differences between treatment groups were apparent as early as 4 h after treatment. The ratio of beta-nucleoside triphosphate to inorganic phosphate at 4 h after treatment was significantly (P less than 0.01) smaller for 600 J/cm2 treatment than for 200 J/cm2 treatment. At curative (600 J/cm2) levels, from 48 h on, no phosphate resonances were detected in the spectra. The pH measured by NMR transiently decreased from pretreatment levels after 200 and 400 J/cm2 treatment (P less than 0.002, P less than 0.009, respectively), while no change in pH from pretreatment values was found after 600 J/cm2 treatment. The data suggest that the early metabolic response of mammary carcinoma to PDT, as indicated by 31P-NMR spectroscopy, is dose dependent, and may be a sensitive indicator of biological outcome to treatment.     

Phosphorus-31 nuclear magnetic resonance studies of intracellular pH,phosphate compartmentation and phosphate transport in yeasts

³¹P NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically on glucose. Direct introduction of substrate into the cell suspension, without interruption of the measurements, revealed rapid changes in pH upon addition of the energy source. All ³¹P NMR spectra of the yeasts studied indicated the presence of two major intracellular inorganic phosphate pools at different pH environments. The pool at the higher pH was assigned to cytoplasmic phosphate from its response to glucose addition and iodoacetate inhibition of glycolysis. After addition of substrate the pH in the compartment containing the second phosphate pool decreased. A parallel response was observed for a significant fraction of the terminal and penultimate phosphates of the polyphosphate observed by ³¹P NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same intracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic conditions. In the presence of energy source a pH gradient as large as 0.8 to 1.5 units could be generated across the vacuolar membrane. Under certain conditions net transport of inorganic phosphate across the vacuolar membrane was observed during glycolysis: to the cytoplasm when the cytoplasmic phosphate concentration had become very low due to sugar phosphorylation, and into the vacuole when the former concentration had become high again after glucose exhaustion.Non-Standard Abbreviations NMR nuclear magnetic resonance - ppm parts per million - PP polyphosphate - P_i,c cytoplasmic inorganic phosphate - P_i,v vacuolar inorganic phosphate - pH_in,c cytoplasmic pH - pH_in,v vacuolar pH - FCCP carbonyl p-trifluoromethoxyphenylhydrazone     

31P nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH.

31P nuclear magnetic resonance (NMR) was used to directly observe the binding of inorganic phosphate to alkaline phosphatase. Evidencq for the tight binding of 1.5-2.0 mol of inorganic phosphate per dimer of alkaline phosphatase is presented. Two distinct forms of bound phosphate are observed, one predominating above pH 7 and representing the non-covalent E-P1 complex and the other predominating below pH 5 and representing the covalent E-P1 complex. The 31P NMR line width of the E-P1 complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH.     

Elucidation of the cytoplasmic and vacuolar components in the inorganic phosphate region in the 31P NMR spectrum of yeast

Subcellular compartments, such as the vacuole in yeast, play important roles in cell metabolism and in cell response to external conditions. Concentrations of inorganic phosphate and pH values of the vacuole and cytoplasm were determined for anaerobic Saccharomyces cerevisiae cells based upon (31)P NMR spectroscopy. A new approach allows the determination of these values for the vacuole in cases when the resonance for inorganic phosphate in the cytoplasm overlaps with the resonance for inorganic phosphate in the vacuole. The intracellular inorganic phosphate resonance was first decomposed into two components by computer analysis. The assignments of the components were determined from in vivo correlations of P(i) chemical shift and the chemical shifts of the cytoplasmic sugar phosphates, and the pH dependency of the resonance of pyrophosphate and the terminal phosphate of poly-phosphate (PP(1)) which reside in the vacuole. An in vivo correlation relating PP(1) and P(i) (vac) chemical shifts was established from numerous evaluations of intracellular compositions for several strains of S. cerevisiae. This correlation will aid future analysis of (31)P NMR spectra of yeast and will extend NMR studies of compartmentation to cellular suspensions in phosphate-containing medium. Application of this method shows that both vacuolar and extracellular P(i) were phosphate reserves during glycolysis in anaerobic S. cerevisiae. Net transport of inorganic phosphate across the vacuolar membrane was not correlated with the pH gradient across the membrane.     

Effect of Hypoglycemic Encephalopathy upon Amino Acids, High-Energy Phosphates, and pHi in the Rat Brain In Vivo: Detection by Sequential 1H and 31P NMR Spectroscopy

Metabolic alterations in amino acids, high-energy phosphates, and intracellular pH during and after insulin hypoglycemia in the rat brain was studied in vivo by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Sequential accumulations of 1H and 31P spectra were obtained from a double-tuned surface coil positioned over the exposed skull of a rat while the electroencephalogram was recorded continuously. The transition to EEG silence was accompanied by rapid declines in phosphocreatine, nucleoside triphosphate, and an increase in inorganic orthophosphate in 31P spectra. In 1H spectra acquired during the same time interval, the resonances of glutamate and glutamine decreased in intensity while a progressive increase in aspartate was observed. Following glucose administration, glutamate and aspartate returned to control levels (recovery half-time, 8 min); recovery of glutamine was incomplete. An increase in lactate was detected in the 1H spectrum during recovery but it was not associated with any change in the intracellular pH as assessed in the corresponding 31P spectrum. Phosphocreatine returned to control levels following glucose administration, in contrast to nucleoside triphosphate and inorganic orthophosphate which recovered to only 80% and 200% of their control levels, respectively. These results show that the changes in cerebral amino acids and high-energy phosphates detected by alternating the collection of 1H and 31P spectra allow for a detailed assessment of the metabolic response of the hypoglycemic brain in vivo.     

Studies on cryoprotectant equilibration in the intact rat liver using nuclear magnetic resonance spectroscopy: a noninvasive method to assess distribution of dimethyl sulfoxide in tissues

Nuclear magnetic resonance (NMR) spectroscopy was used in the study of rat livers following flushing with a clinically used preservation solution containing either 12 or 30% (v/v) Me2SO. The extent of equilibration of Me2SO in the tissue after 10-15 min of perfusion with Me2SO and again after subsequent washout with Me2SO-free medium was assessed by 1H NMR spectroscopy. 31P NMR spectroscopy was used to follow the changes in ATP, ADP, inorganic phosphate, and tissue pH. The data show that 1H NMR spectroscopy can be used as a sensitive and rapid method of assessing the equilibration and concentration of compounds such as Me2SO, since these compounds are likely to be present at concentrations greatly in excess of other constituents of the medium and will therefore give rise to strong, easily detected signals. At the same time, 31P NMR spectroscopy can be used to monitor the metabolic status of the tissue reflected in the levels of ATP, ADP, and inorganic phosphate, as well as being a noninvasive monitor of intracellular pH. The possibility of determining the tissue pH in the presence of solutes such as Me2SO is discussed.     

23Na and 31P NMR studies of the effects of salt stress on the freshwater cyanobacterium Synechococcus 6311

We have used 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy to elucidate some of the bioenergetic changes that occur in the freshwater cyanobacterium Synechococcus 6311 after a transition from growth medium (Na concentration 0.01 M) to medium containing 0.5 M NaCl. 23Na NMR analysis showed Na rapidly penetrates the cells under dark aerobic conditions; cells grown for several days in high salt medium, however, reestablish a low internal sodium content, comparable to control cells. For 31P NMR analysis, a system was devised to aerate and illuminate cell suspensions during spectral acquisition. The NMR spectra showed that when cells are presented with 0.5 M NaCl (final concentration), nucleotide triphosphate peaks decrease, the inorganic phosphate peak increases, and the cytoplasmic pH transiently increases from 7.4 to 7.9. Pyrophosphate added to cell suspensions is hydrolyzed to inorganic phosphate apparently by an extracellular phosphatase, allowing external and internal pools of inorganic phosphate to be distinguished. Nucleotide triphosphate levels fall almost as much when cells are incubated in darkness as under anoxia, indicating that both respiration and photosynthesis contribute to the maintenance of intracellular ATP levels. Cells grown in high salt medium for several generations exhibited a pattern of 31P metabolites similar to control cells, except that they produced more (and more intense) peaks in the monoester phosphate region, presumably signals from sugar phosphates.     

A hollow-fiber reactor design for NMR studies of microbial cells

A hollow-fiber membrane reactor was designed and constructed to allow perfusion of entrapped, dense Escherichia coli cells with nutrient medium during examination of cell metabolism using nuclear magnetic resonance (NMR) spectroscopy. Phosphorus-31 NMR spectra of the perfused cells included peaks for nucleoside di- and triphosphates, sugar phosphates, and pH-sensitive peaks for inorganic phosphate. The observed intensity of the lumenal inorganic phosphate peak was found to depend on flow rate, ruling out the use of this peak as a concentration reference. Absolute intracellular pH values obtained from NMR measurements were found to be accurate to 0.2 pH units due to uncertainties in intracellular ionic concentrations. Relative pH values, however, were found to be sensitive to cell energetic status. The response of E. coli intracellular pH following a shift to carbon starvation medium was monitored with a resolution of 3 min. Use of a hollow-fiber reactor for cell containment and perfusion during NMR spectroscopy enables metabolic experiments of longer duration and of greater variety than is possible using standard, nonperfused sample tubes.     

Phosphorus-31 nuclear magnetic resonance study on the effects of endurance training in rat skeletal muscle

To evaluate changes in muscle energetics following endurance training, we measured phosphorus-31 nuclear magnetic resonance (31P NMR) spectra on rat muscle in vivo before and after training in the same animals. The endurance training lasted for 3 months. The 31P NMR spectra were obtained serially at rest, during exercise by electrical stimulation, and during recovery. Intramuscular phosphocreatine (PCr), inorganic phosphate (P(i)), adenosine 5'-triphosphate (ATP) and pH were determined from the NMR spectra. The ratio of PCr:(PCR + P(i) at rest showed no difference between the trained and control groups even after 3 months of training. During exercise, however, this ratio was significantly higher in the trained group than in the control group. The ratio also recovered more rapidly after exercise in the trained group. The intramuscular pH decreased slightly by approximately 0.1 pH unit during exercise but did not show a significant difference between the groups. These results indicated that endurance training of 3 months duration improved the ATP supply system in the muscle. They also demonstrated that 31P NMR is a potent method for evaluating the effects of training in the same individuals.     

Changes in 31P nuclear magnetic resonance with tumor growth in radioresistant and radiosensitive tumors

In vivo 31P nuclear magnetic resonance (31P NMR) spectroscopy has been used to compare metabolic profiles with tumor radiosensitivity. A radioresistant mammary carcinoma (MCa) and a radiosensitive methylcholanthrene-induced fibrosarcoma (Meth-A) were studied by 31P NMR spectroscopy in the tumor volume range of approximately 100-1200 mm3. The MCa showed a constant pH in this volume range; the ratio of phosphocreatine to inorganic phosphate (PCr/Pi) for 160-300 mm3 tumors was 0.33 +/- 0.11 (mean +/- standard deviation) and did not change (0.29 +/- .09) for tumors in the volume range of 600-1200 mm3. In comparison, the Meth-A showed a decrease in tumor pH as volume increased from 160-300 mm3 (pH 7.16 +/- 0.4) to 600-1200 mm3 (pH 6.94 +/- .07). Tumor PCr/Pi decreased from 0.70 +/- .16 (160-300 mm3) to 0.33 +/- .16 (600-1200 mm3). The radiation doses for control of MCa-induced tumors in 50% of the treated tumors ranged from 65 (150-250 mm3) to 71 Gy (1000-1300 mm3) and for the Meth-A-induced tumors ranged from 35 (150-250 mm3) to 38 Gy (1000-1300 mm3). These results suggest that 31P NMR spectra may be a qualitative predictor of tumor hypoxia, although further studies of human and rodent tumors are necessary to support this hypothesis.     

P-31 Nuclear Magnetic Resonance Analysis of Brain: The Perchloric Acid Extract Spectrum

Abstract: Perchloric acid (PCA) extracts were prepared from liquid-N₂-frozen guinea pig brains and their organophosphate profiles examined by P-31 nuclear magnetic resonance (NMR) spectroscopy. Thirty-two phosphorus-containing brain metabolites were characterized and quantitated. A distinctive feature of brain tissue metabolism relative to that of other tissues probed by P-31 NMR is its pronounced ribose 5-phosphate content. Comparison of brain metabolite levels following control or sublethal cyanide treatment (4 mg/kg) revealed specific cyanide-induced changes in brain metabolism. Brains from cyanidetreated animals were characterized by a reduced phosphocreatine content and elevated α-glycerolphosphate and inorganic orthophosphate contents relative to control. P-31 NMR spectra of brain PCA extracts at pH 7.2 were also obtained under conditions that approximate those used for in vivo and intact tissue in vitro P-31 spectroscopic analyses. The spectra reveal nine separate resonance bands corresponding to: sugar phosphates, principally ribose 5-phosphate (3.7δ); inorganic orthophosphate (2.2δ); glycerol 3-phosphorylethanolamine (0.3δ); glycerol 3-phosphorylcholine (−0.1δ); phosphocreatine (−3.2δ); adenosine tri-(β-ATP) and di-(β-ADP) phosphate ionized end-groups (−6.2δ); α-ATP, α-ADP, and nicotinamide adenine dinucleotides esterified end-groups (−11.1δ); uridine diphosphohexose, hexose esterified end-groups (−13.0δ); and β-ATP ionized middle group (−21.6δ). Knowledge of the phosphatic molecules that contribute resonances to the brain P-31 NMR spectrum as well as understanding their magnetic resonance properties is essential for the interpretation of in vivo brain spectroscopic data as well as brain extract data, since these same compounds contribute to the intact brain P-31 spectrum.     

31P nuclear-magnetic-resonance studies of pyridoxal and pyridoxamine phosphates. Interaction with cytoplasmic aspartate transaminase.

The 31P nuclear magnetic resonance (NMR) spectrum of the phosphate in free pyridoxal or pyridoxamine phosphate reveals a resonance signal that is coupled to the methylene protons of the 5-CH2 with JHP of 6.0 +/- 0.3 Hz. Proton noise decoupling results in a single signal with a pH-dependent chemical shift with deprotonation of the phosphate resulting in a shift of the 31P resonance to lower fields. A single 31P NMR signal at a frequency corresponding to fully ionized phosphate monoesters is observed in aspartate-transaminase-bound pyridoxal or pyridoxamine phosphate. The 31P resonance in the holotransaminase is pH-independent and is unaffected by saturating concentrations of substrates or inhibitors. Only denaturation with 6 M guanidine with HCl results in changes in the 31P of the holoenzyme. It appears that the phosphate group of pyridoxal phosphate is bound to a positive pocket in the holoenzyme and remains fully ionized in the pH range of 5.6 to 9.2. The phosphate-binding properties are present even in the apoenzyme which is able to bind inorganic phosphate which then can be displaced by pyridoxal or pyridoxamine phosphate in the process of holoenzyme formation.     

31P NMR measurements of myocardial pH in vivo

A 31P NMR magnetization transfer method for measuring myocardial pH in vivo is demonstrated in the lamb, dog and cat. The method involves measuring the difference in chemical shift between the resonances of phosphocreatine and inorganic phosphate in magnetization transfer difference spectra in which the gamma-phosphate resonance of ATP has been saturated. The method has been verified by measuring the chemical shift difference between the resonances of 2-deoxyglucose 6-phosphate and phosphocreatine following infusion of the animals with 2-deoxyglucose. The measured pH values are significantly lower than those obtained in previous studies on the heart in vivo.     

Estimation of intracellular sugar phosphate concentrations in Saccharomyces cerevisiae using 31P nuclear magnetic resonance spectroscopy

A systematic procedure has been formulated for estimating the relative intracellular concentrations of sugar phosphates in Saccharomyces cerevisiae based upon (31)P nuclear magnetic resonance (NMR) measurements. The sugar phosphate region of the (31)P NMR spectrum is first decomposed by computer analysis, and the decomposition consistency and identification of individual sugar phosphate resonances are established based on in vitro chemical shift calibrations determined in separate experiments. Numerous evaluations of intracellular S. cerevisiae compositions for different strains and different cell environments provide the basis for in vivocorrelations of inorganic phosphate chemical shift with the chemical shifts of 3-phosphoglycerate, beta;-fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6 phosphate. Relative intracellular sugar phosphate concentrations are obtained by correcting peak areas for partial saturation during transient in vivo experiments. In vivo concentrations estimated by this method agree well with estimates for similar systems based on other techniques. This approach does not require costly la belled compounds, and has the advantage that other important metabolic state variables such-as internal and external pH and intracellular levels of phosphate, ATP, ADP, NAD(H), and polyphosphate may be determined from the same (31)P spectrum. Extension of this strategy to other cellular systems should be straightforward.     

Chromium binding Bacillus cereus VITSH1—a promising candidate for heavy metal clean up

Bacteria survive metal stress by several mechanisms and metal binding is one such mechanism which has been screened in the present study to investigate the survival strategies of metal resistant bacteria. The production of siderophores, a metal chelating agent, was detected by chrome azurol S agar assay. The changes in cell wall studied by analysing the peptidoglycan and teichoic acid content indicated an increase in the cell wall content. Evaluation of morphological and physiological alterations like cell size, granularity analysed by SEM and flow cytometry analysis revealed an increase in cell size and granularity respectively. The transformation of phosphates monitored by ³¹P NMR analysis indicated the presence of inorganic phosphate. Based on the cell wall changes and the ³¹P NMR analysis, the surface charge of the organism was studied by zeta potential which displayed a difference at pH7.     

31P NMR studies of Clostridium thermocellum. Mechanism of end product inhibition by ethanol

31P NMR studies of intact cells and perchloric acid extracts are used to investigate the effect of ethanol on the bioenergetics and glycolysis of Clostridium thermocellum, an anaerobic bacterium potentially useful for the single step conversion of biomass to ethanol. Whole cells suspended in phosphate buffer and given a carbon source (cellobiose) at 60 degrees C rapidly establish a pH gradient across the membrane that can be monitored by the chemical shifts of inorganic phosphate in the exterior buffer and in the cytoplasm. Peak intensities can be related to phosphate active transport rates. Wild type bacteria and cells grown in inhibiting concentrations of ethanol establish similar pH gradients, but with slower kinetics and slower phosphate transport rates for the cells adapted to growth in ethanol. Direct addition of ethanol does not affect the rate of pH gradient formation or phosphate transport. Thus, while ethanol does not directly affect processes for energy conservation carried out by the membrane, adaptation to ethanol does alter membrane functions such as phosphate transport. 31P NMR spectra of perchloric acid extracts show that when wild type cells are adapted to grow in inhibiting concentrations of ethanol and then energized with cellobiose, sugar phosphate content is increased and the steady state distribution of glycolytic intermediates is altered. Nucleotide triphosphate/nucleotide diphosphate ratios are unaltered in these cells. These results strongly indicate that in C. thermocellum growth inhibition by ethanol is related to a blockage in glycolysis.     

Metabolic changes of the Antarctic green alga Prasiola crispa subjected to water stress investigated by in vivo 31P NMR

The energy status and the phosphate metabolism of Prasiola crispduring and after desiccation stress was investigated by in vivo³¹P NMR. The effect of desiccation was simulated by additionof the nonionic osmoticum PEG 200 (polyethylene glycol). Photosynthesisand respiration were effectively inhibited under these conditions.The most notable changes in the in vivo ³¹P NMR spectra werean increase in the cytoplasmic inorganic phosphate signal afterPEG stress, a decrease in the polyphosphates and a lowfieldshift of the core polyphosphate signal followed by an appearanceof extracellular inorganic phosphate. Cytoplasmic pH remainedalmost constant during stress. After a return to control conditions,photosynthesis and respiration recovered within 4 h as wellas the concentrations of the phosphorus metabolites. An as yetunassigned phosphate signal increased in the phosphodiesterregion of the NMR spectra. Simultaneousty, the polyphosphatesignal recovered in intensity and chemical shift. It is suggestedthat phosphate metabolism and complexation of cations to polyphosphatesmay play an important role in the distinct desiccation toleranceof P. crispa. Key words: In vivo ³¹P NMR, Prasiola crispa, desiccation tolerance, polyphosphates     

Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate [Jaenicke, L. v., & Koch, J. (1963) Justus Liebigs Ann. Chem. 663, 50-58], and the product was characterized by 31P, 1H, and 13C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by 31P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 degrees C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by 18O incorporation from H2(18)O into Pi, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N10-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k cat values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.     

Observation of Cerebral Metabolites in an Animal Model of Acute Liver Failure In Vivo: A 1H and 31P Nuclear Magnetic Resonance Study

Acute liver failure was induced in rats by a single intragastric dose of carbon tetrachloride. This causes hepatic centrilobular necrosis, as indicated by histological examinations, and produces a large increase in the activity of serum alanine aminotransferase. The plasma NH4+ level (mean +/- SEM) was 123 +/- 10 microM in the control group and 564 +/- 41 microM in animals with acute liver failure (each n = 5). 31P nuclear magnetic resonance (NMR) was used to monitor brain cortical high-energy phosphate compounds, Pi, and intracellular pH. 1H NMR spectroscopy was utilised to detect additional metabolites, including glutamate, glutamine, and lactate. The results show that the forebrain is capable of maintaining normal phosphorus energy metabolite ratios and intracellular pH despite the metabolic challenge by an elevated blood NH4+ level. There was a significant increase in the brain glutamine level and a concomitant decrease in the glutamate level during hyperammonaemia. The brain lactate level increased twofold in rats with acute liver failure. The results indicate that 1H NMR can be used to detect cerebral metabolic changes in this model of hyperammonaemia, and our observations are discussed in relation to compartmentation of NH4+ metabolism.     

Characterization of the 31P NMR spectrum of Manduca sexta and effects of antimetabolites

High resolution ³¹P nuclear magnetic resonance spectroscopy (NMR) was successfully applied to 5th instar larvae of Manduca sexta. Conditions for in vivo analysis under non-saturating conditions are described. The ³¹P NMR spectrum of intact larvae was composed of six peaks. Their resonance frequencies are reported relative to orthophosphoric acid. Analysis of tissue extracts demonstrated the in vivo peaks to be composed of the β phosphorus resonance of nucleotide triphosphates (NTP) at −19.36 ppm; α phosphorus of NTP and nucleotide diphosphates (NDP) at −10.51 ppm; β and γ phosphorus of NDP and NTP, respectively, at −5.42 ppm; phosphoarginine (PA) at −3.45 ppm; inorganic phosphate (Pi) at +2.76 ppm and sugar phosphates at +3.34 ppm. The major sugar phosphate present in fat body extracts was trehalose-6-phosphate and this was the major phosphorus component of the spectrum of hemolymph. The spin-lattice relaxation times for each in vivo peak were determined.Titration of aqueous fat body and hemolymph extracts was carried out and the relationship between the chemical shift of Pi and pH determined. On this basis the pH of the hemolymph was estimated at approx. 6.7.The metabolic inhibitors, iodoacetate and dinitrophenol, had significant effects on the ³¹P NMR spectrum of intact larvae. Administration of iodoacetate caused a rapid increase in the levels of sugar phosphates together with decreases in NTP and PA. Dinitrophenol also caused declines in the relative levels of NTP and PA but sugar phosphates decreased as well. The experiments demonstrated the potential of in vivo NMR analysis for metabolic studies on high energy phosphate metabolites in M. sexta.