Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities

A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400-MHz wide-bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation-integrated cross-flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients k(L)a of more than 1.0 s(-1) are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate >15,000 mg O(2) .(L h)(-1)] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo (31)P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP-sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo (13)C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 g(cell dry weight) . L(-1), the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L-lactate, L-glutamate, succinate, and L-lysine with a time resolution of 10 min after impressing a [1-(13)C]-glucose pulse.     

Free cytosolic Ca2+ measurements with fluorine labelled indicators using 19FNMR

Characterisation by 19F NMR of fluorine-labelled indicators of cytosolic free Ca2+ concentration (by 5FBAPTA) and pH (by Fquene) is described, together with the techniques used to load the cell suspensions with the indicators for NMR spectroscopy. Useful features of the 19F NMR indicators include direct identification of the intracellular cation bound to the indicators, internal calibration of [Ca]i and pHi from the spectra, and simultaneous measurements of two or more indicators in the same cell suspension. Perturbations of cellular functions by 5FBAPTA and quin 2 are very similar, but vary widely in different cell systems. The [Ca]i and pHi responses of normal and transformed cells to mitogens and growth factors in serum are compared with data from similar experiments using fluorescence indicators. The only major discrepancy in [Ca]i measurements using the two independent assays was observed in Ehrlich ascites tumour cells. These cells have a high intracellular Zn2+ content which substantially quenches the quin 2 fluorescence, but does not affect [Ca]i measurements by 5FBAPTA. The Zn2+ present in the cells is detected as a separate response in the 5FBAPTA spectrum. The time course of the Ca signal in 2H3 cells stimulated by antigen to release histamine by exocytosis has been defined using 5FBAPTA and quin 2. Extension of the 19F NMR technique to [Ca] i and pHi measurements in perfused organs is illustrated in rat heart and responses to pharmacological agents are demonstrated. Developments in prospect to improve sensitivity and to measure [Na]i with a new family of indicators are outlined.     

Mapping of oxygen tension and cell distribution in a hollow-fiber bioreactor using magnetic resonance imaging

We mapped the distribution of dissolved oxygen and mammalian cells in a hollow-fiber bioreactor (HFBR) using (19)F NMR T(1) relaxation time imaging measurements on an infused perfluorocarbon probe molecule and diffusion-weighted (1)H NMR imaging of water. This study shows how cell density influences dissolved oxygen concentration in the reactor and demonstrates that NMR can play an important role in defining the biochemical engineering parameters required for optimization of HFBR design and operation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 56-61, 1997.     

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.     

Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.     

Interaction of hexadecyltrimethylammonium bromide with yeast cells

The interaction of hexadecyltrimethylammonium bromide (CTAB) with two yeast cells, Kluyveromyces fragilis and Saccharomyces cerevisiae, has been studied. Strong binding of CTAB to the cell was inferred from 1H and 13C NMR studies, the probable site of binding being the cell-surface. 13C and 31P NMR studies have indicated facilitation of free passage of molecules from outside to inside the cell and vice versa on treatment with CTAB. 31P NMR studies showed that intracellular pH (pHi) was affected in presence of CTAB and the rate of exchange of H+ and PO4(-3) between outside and inside of the cell was 508 s-1. CTAB treatment of yeast cells also affected pH and conductance measurements of the cell-suspension. There was a marked difference in the pH changes around the critical micellar concentration (CMC) of CTAB. The observed pH changes were dependent on (i) CTAB concentration, (ii) pH of the cell-suspension and (iii) pK values of groups from molecules released from the cell. Also, it was shown that ionisation of phosphate diester from polar head groups of membranes constituting cell surface enhanced CTAB binding. Conductance measurements have shown that observed changes were independent of the concentration of yeast cells, but probably dependent on CMC of CTAB.     

Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas

Quantitative estimates of intracellular fluxes and measurements of intracellular concentrations were used to evaluate the effect of dissolved oxygen (DO) concentration on CRL 1606 hybridoma cells in batch culture. The estimates of intracellular fluxes were generated by combining material balances with measurements of extracellular metabolite rates of change. Experiments were performed at DO levels of 60% and 1% air saturation, as well as under oxygen-limited conditions. Cell extracts were analyzed to evaluate the effect of DO on the intracellular concentrations of the glutamate dehydrogenase reactants, as well as the redox state of the pyridine nucleotides in the cytosol and mitochondria. The relationship between cell density and pyridine nucleotide redox state was also investigated. Dissolved oxygen concentration had a significant effect on nitrogen metabolism and the flux through glutamate dehydrogenase was found to reverse at low DO, favoring glutamate formation. The NAD in the cytosol and mitochondria was more reduced under low DO conditions while the cytosolic NAD was more oxidized at low DO. Cytosolic NAD was reduced at higher cell densities while the redox states of cytosolic NADP and mitochondrial NAD did not exhibit significant variation with cell density. These results point to the fundamental role of the intracellular oxidation/reduction state in cell physiology and the possibility of controlling physiological processes through modulation of the dissolved oxygen level or the oxidation/reduction potential of the culture.     

In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis.

The metabolism of glucose by nongrowing cells of L. lactis strain MG5267 was studied under controlled conditions of pH, temperature, and gas atmosphere (anaerobic and aerobic) using a circulating system coupled to nuclear magnetic resonance (NMR) detection that allowed a noninvasive determination of intracellular pools of intermediate metabolites by 13C-NMR with a time resolution of 30 seconds. In addition, intracellular parameters, such as pH, NTP levels, and concentration of inorganic phosphate in the cytoplasm, could be monitored on-line by 31P-NMR with a time resolution of approx. 3 min. The time course for the concentrations of intracellular fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP), together with kinetic measurements of substrate consumption and endproducts formation, were used as a basis for the construction of a mechanistic model for glycolysis. In vivo measurements were complemented with determinations of phosphorylated metabolites in perchloric acid extracts. A top-down model was developed by simplifying the metabolism to the resolution allowed by the experimental data collected by in vivo NMR (grouped in seven metabolic steps). This simplified mechanistic model was adjusted to the metabolite concentrations determined by in vivo NMR. The results obtained led to the rationalization of the dynamics of glucose metabolism as being driven largely by ATP surplus. This excess causes accumulation of FBP due to NAD+ limitation, whose regeneration is dependent on downstream pyruvate reduction. The model was capable of predicting qualitative shifts in the metabolism of glucose when changing from anaerobic to aerobic conditions.     

A noninvasive technique for the measurement of the energetic state of free‐suspension mammalian cells

A perfusion small‐scale bioreactor allowing on‐line monitoring of the cell energetic state was developed for free‐suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10‐mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min^?1 for high‐density cell suspensions (10⁸ cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo ³¹P NMR was performed and demonstrated the small‐scale bioreactor platform ability to monitor the cell physiological behavior for 30‐min experiments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Measurement of intracellular (compartmental) pH by 31P NMR in Aspergillus niger

31P nuclear magnetic resonance (31P NMR) was used to monitor cytoplasmic and vacuolar pH values in the filamentous fungus Aspergillus niger. To obtain a homogeneous cell sample and to be able to perform long term in vivo NMR measurements A. niger mycelium was kept in a setup that allows perfusion of the cell plug within the NMR tube. Mycelial samples, however, became rapidly clogged during perfusion leading to (partial) anaerobiosis of the plug with subsequent acidification of the cytoplasm. As a result, only short-term NMR measurements (5-10 min) were possible using free mycelium. To increase and to prolong perfusion, A. niger was immobilized in Ca(2+)-alginate beads. Deteriorated spectra recorded under hypoxia could be completely restored in the presence of oxygen. With this system perfusion in the presence of citrate could be maintained for at least 18 h at much higher rates (15 ml min-1 compared with 4 ml min-1 for free mycelium). During this period 31P NMR spectra were highly invariable, indicating approximate steady-state intracellular conditions during long term measurements. Perfusion in the presence of glucose resulted in complete depletion of the vacuolar inorganic phosphate pool within 45 min and yielded a higher pH gradient over the tonoplast than when citrate was used (delta pH = 1.6 and 1.4, respectively).     

In vivo 31P NMR study of early cellular responses to hyperosmotic shock in cultured glioma cells.

Cell volume regulation in the face of osmotic stress is a fundamental homeostatic activity, and is most critical in brain, which is spatially constrained. Despite the importance of this phenomenon, little is known about volume regulation in the brain, primarily because of the cellular heterogeneity in the tissue. We describe here simultaneous in vivo 31P nuclear magnetic resonance (NMR) measurements of cell volume, intracellular pH and phosphate metabolites during early responses to hyperosmotic stress in C6 glioma cells perfused in NMR-compatible bioreactors. Cell volume was measured using dimethyl methylphosphonate (DMMP) as a probe which has an intracellular NMR resonance shifted upfield from the extracellular resonance. The sensitivity of these measurements allowed 31P NMR spectra to be collected every 30 s. Following an increase in osmolarity from 320 to 480 mOsm by addition of NaCl to the perfusate, C6 glioma cells shrank to 67% of their original volume. We also observed a simultaneous increase of intracellular pH coincident with the decrease in cell volume. The signals from ATP decreased by 10%, but those from phosphocreatine (PCr) increased by 31% after hyperosmotic shock. However, correcting the ATP signals for the decrease in cell volume indicated that its intracellular concentrations increased after treatment. Signals from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were not changed significantly. This is the first in vivo report of early cellular responses monitored by NMR spectroscopy following hyperosmotic shock in cultured cells.     

Cigarette smoke exposure increases the cell water organization and membrane order of cultured T cells.

In order to determine the effects of cigarette smoke (CS) exposure on the physical properties of cells, NMR water-proton relaxation time (which measures the intracellular water organization) and ESR spin labeling (which measures membrane order) measurements were performed on cultured Jurkat T cells exposed to CS. NMR spin-lattice relaxation time (T1) decreased with CS exposure in a dose-dependent fashion. A significantly depressed T1 value was obtained even when CS was delivered through a filter. Cell viability was not affected in this condition. Superoxide dismutase (SOD) prevented the depression of T1 value. These results suggest that superoxide radicals or subsequently generated species contained in the gas phase of CS increase the intracellular water organization in viable cells. CS exposure also increased the ESR membrane order parameter of nitroxide spin label. These physical characteristic changes may be important in CS-induced cell responses and cytopathology.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

Monitoring of dissolved oxygen and cellular bioenergetics within a pancreatic substitute

This work investigated the use of nuclear magnetic resonance (NMR) spectroscopy in combination with a mathematical model of an encapsulated cell system as a method for rapidly assessing the status of a pancreatic substitute. To validate this method, an in vitro experiment was performed in which the encapsulated cells were perfused in an NMR-compatible system and the dissolved oxygen (DO) concentration of the perfusing medium was lowered from 0.20 to 0.05 mM, then returned to 0.20 mM in a stepwise fashion. The cellular metabolic activity and bioenergetics were evaluated by measuring the oxygen consumption rate (via DO sensors) and nucleotide triphosphate levels (via (31)P NMR). By incorporating a perfluorocarbon emulsion into the alginate beads, the cellular oxygenation state was monitored by measuring the average intrabead DO (AIDO) concentration by (19)F NMR. The in vitro measurements were then compared with model predictions based on the measured external DO concentration and time. Model-predicted cell growth and AIDO closely matched the experimentally acquired data. As the DO concentrations both external to and within the pancreatic substitute are needed to apply this methodology in vivo, the feasibility of measuring the DO concentration from two distinct bead populations implanted in the peritoneal cavity of mice was established. It is concluded that PFC incorporation and (19)F NMR measurements, in combination with a mechanistic model of the encapsulated system, allow the tracking of the state of a pancreatic substitute in vitro and potentially in vivo.     

^19FNMR在生物医学研究中的应用

核磁共振（ＮＭＲ）是一种无创伤的物理测试方法,它可以直接用于体内和体外的生物样品测定,提供分子水平的信息。正常体内含氟成分很少,测定进无本底信号干扰,因此在体内研究中引进氟代指示剂进行＾１９ＦＮＭＲ研究是目前普遍采用的方法。＾１９ＦＮＭＲ可可以用来测定药物在体内代谢过程、胸内游离的离子如Ｃａ＾２＋和Ｍｇ＾２＋、胞内ｐＨ、氧浓度或氧压力（ｐＯ２）、膜电位、组织温度、血液容积和细胞容积等多项生理生化指     

Method of high-precision microsample blood and plasma mass densitometry

The reliability of the mechanical oscillator technique (MOT) for blood and plasma mass density measurements on small samples is quantified in this paper. Sources of measurement errors that can reduce both the accuracy and precision of density determinations include storage of plasma samples, inhomogeneity of blood samples, and density reading before adequate temperature equilibration. Measurements on fractions from identical samples and repeated samplings from test subjects under steady-state conditions revealed a 10(-2) g/l reproducibility of density readings. The mean plasma density (PD) readings did not change significantly after up to 1-wk storage at +4 degrees C or up to 2 mo storage at -20 degrees C. The variability of the PD findings increased with storage time and were generally higher with storage at -20 degrees C, compared with +4 degrees C. Densitometers of different sizes were used to evaluate rheological influences on blood density (BD) readings. Linear correlations between PD and plasma protein concentration, between BD and blood hemoglobin concentration, and between erythrocyte density and mean corpuscular hemoglobin concentration were significant (P less than 0.001). Rapid density measurements with up to 10(-2) g/l reliability on small (less than 0.1 ml) volumes of biological fluids and continuous blood densitometry can be performed with use of the MOT.     

23Na and flame photometric studies of the NMR visibility of sodium in rat muscle.

23Na nuclear magnetic resonance spectroscopy (NMR) is increasingly being used to study Na+ gradients and fluxes in biological tissues. However, the quantitative aspects of 23Na NMR applied to living systems remain controversial. This paper compares sodium concentrations determined by 23Na NMR in intact rat hindlimb (n = 8) and excised rat gastrocnemius muscle (n = 4) with those obtained by flame photometric methods. In both types of samples, 90% of the sodium measured by flame photometry was found to be NMR-visible. This is much higher than previously reported values. The NMR measurements for intact hindlimb correlated linearly with the flame photometric measurements, implying that one pool of sodium, predominantly extracellular, is 100% visible. From measurements on excised muscle, in which extracellular space is more clearly defined, the NMR visibility of intracellular Na+ was calculated to be 70%, assuming an extracellular space of 12% of the total tissue water volume and an extracellular NMR visibility of 100%. 23Na transverse relaxation measurements were carried out using a Hahn spin echo on both intact hindlimb (n = 1) and excised muscle (n = 2) samples. These showed relaxation curves that could each be described adequately using two relaxation times. The rapidly relaxing component showed a T2 value of 3-4 ms and the slowly relaxing component a T2 of 21-37 ms. A spin lattice relaxation (T1) measurement on intact hindlimb yielded a value of 51 ms. These relatively long relaxation times show that the quadrupolar relaxation effect of Na+ complexing to large macromolecules or being otherwise motionally restricted is relatively weak. This is consistent with the high NMR visibilities reported here.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.     

23Na NMR study of intracellular sodium ions in Dictyostelium discoideum amoeba

The intracellular sodium concentration in the amoebae from the slime mold Dictyostelium discoideum has been studied using 23Na NMR. The 23Na resonances from intracellular and extracellular compartments could be observed separately in the presence of the anionic shift reagent Dy(PPPi)7-2 which does not enter into the amoebae and thus selectively affects Na+ in the extracellular space. 31P NMR was used to control the absence of cellular toxicity of the shift reagent. The intracellular Na+ content was calculated by comparison of the intensities of the two distinct peaks arising from the intra- and extracellular spaces. It remained low (0.6 to 3 mM) in the presence of external Na+ (20 to 70 mM), and a large Na+ gradient (20- to 40-fold) was maintained. A rapid reloading of cells previously depleted of Na+ was readily measured by 23Na NMR. Nystatin, an antibiotic known to perturb the ion permeability of membranes, increased the intracellular Na+ concentration. The time dependence of the 23Na and 31P NMR spectra showed a rapid degradation of Dy(PPPi)7-2 which may be catalyzed by an acid phosphatase.     

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell–cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. ³¹P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in ³¹P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid ³¹P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible ³¹P NMR spectra of phospholipids.