NMR spectroscopy of cells.

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Annu. Rev. Physiol. 1992. 54:775-98 Copyright © 1992 by Annual Reviews 1nc. All rights reserved

Annu. Rev. Physiol. 1992.54:775-798. Downloaded from www.annualreviews.org Access provided by University of California - Davis on 01/25/15. For personal use only.

NMR SPECTROSCOPY OF CELLS Benjamin S. Szwergold Department of NMR and Medical Spectroscopy, Fox Chase Cancer Center, Burholme Ave., Philadelphia, Pennsylvania KEY WORDS:

7701

19111

cell culture, perfusion microcarriers, encapsulation, indicators

INTRODUCTION The

NMR experiments on isolated cell preparations (5, 23, 31)

afford control

over physiological and biochemical variables that are not under experimental control in intact animals, or in perfused organ systems. For intact tissues, the use of defined cell preparations solves (or circumvents) the problem of spatial

NMR spectroscopy since & Williams, this volume). However, this

localization, which has posed a challenge to in vivo its inception

(21, 96,

Koretsky

reductionist approach may make the models less relevant to what actually occurs in vivo. In certain cases, intermediate level model systems have been developed, such as the spheroid system, to approximate the tumor cells in vivo

(37)

and the renal tubule preparation

(1, 2, 14,52). NMR studies of cell

preparations have presented some unique challenges, many of which es sentially have been solved, and this relatively new technology is beginning to generate data not easily obtained by other means. With a few exceptions, this review focuses on studies of mammalian cells, which have been conducted over the past several years. Earlier reviews of

NMR spectroscopy of cells

and tissues are also noted

(3, 5, 18, 21, 31).

For

reviews on specific subjects, readers are referred to previous surveys on 'H

NMR

spectroscopy of erythrocytes

tracellular ions

(13, 46, 47, 107);

(94); NMR

spectroscopy studies of in

and studies of yeast cells

(19). 775

0066--4278/92/0315-0775$02.00

776

SZWERGOLD

METHODOLOGY


NMR Hardware Compared to optical and radio-isotope techniques, NMR is an insensitive method requiring substantially more sample material for analysis than these other methods. Overall, the signal to noise ratio of an NMR experiment depends on three sets of parameters: (a) fundamental constants such as Planck's constant and the gyromagnetic ratio of a specific nucleus; (b) characteristics of the spectrometer; and (c) properties of the sample. Since we do not have any control over the fundamental constants, the following discus sion deals with the instrumental and sample parameters. Because of space

limitations, the discussion of these parameters is general and qualitative. These issues are discussed in more detail elsewhere (36, 49). The instrumental/hardware parameters over which we have some control include strength of the magnetic field of the spectrometer, noise figure of the preamplifiers and amplifiers, temperature of th e probe magnetic field homogeneity factor, and the quality of the NMR detection coils. Optimized sensitivity depends on working at the highest possible magnetic field, using amplifiers with the lowest noise figure, optimizing the magnetic field homogeneity, and using the best possible detection coils. In most practical situations the field and noise parameters are set by the equipment. Magnetic field h omogeneity is, in most exp erim ents with cells, determined by proper ties of the sample (cells) rather than by instrumental characteristics. Thus the only instrumental parameter over which there is control is the quality of the detection circuitry (i.e. the NMR probe). Because of this situation a considerable amount of effort has been devoted to the optimization of NMR probe design, focusing in particular on the detection coil geometry. Hoult & Richards (49) concluded that a solenoidal ,

coil configuration offered much more sensitivity than an equivalent saddle or

Helmholtz coil configuration. However, extensive investigation has shown that these conclusions are not correct for biological samples for the following reasons: 1. Working with large samples at high magnetic fields necessitates the use of single-tum coils to which the simplifying assumptions used in the analysis of multi-tum coils do not apply. A full theoretical analysis of the use of single tum coils, (J. Murphy-Boesch, personal communication), using the Hoult & Richards approach without any simplifying assumptions, suggests t hat for such coils the expected sensitivity gain is reduced from 2.6 to 1.4, a ratio that was confirmed experimentally (81). 2. In an experimental setting this already reduced gain in sensitivity (mea sured as the integrated signal intensity) is more than offset by the loss in

NMR OF CELLS

777


magnetic field homogeneity across the sample. This is due to the fact that when the sample is placed transverse to the magnetic field axis, it encom passes a far less homogeneous region of the magnetic field than an equivalent coaxial sample. Consequently, the experimentally relevant peak signal-to noise ratio (as opposed to the integrated signal-to-noise ratio) is actually reduced with the solenoidal coil in comparison to an equivalent saddle coil (Figure 1) (80). On re-evaluation, it appears that for NMR experiments on cells and tissues at high magnetic fields (desirable because of increased sensitivity), the Helm holtz or saddle coil configuration is preferable to the solenoidal coil. Because of its geometry, the coaxial configuration also makes it easier to move the sample in and out of the coil. Another set of parameters that influences the signal-to-noise ratio depends PPA

X4

a

b

" I

20

Figure 1

"' I

to

""

""

1'

0 PPM

" "" '" I

-to

",I'

-20

31 P NMR spectra of sample NIH 3T3 cells on microcarrier beads obtained at 162 MHz

with a single-turn saddle coil (a) and a single-turn solenoidal coil (b). The sample used and

acquisition parameters were identical in both experiments (600 pulse, I sec. repetition rate, 1200 scans). In additional cells, the sample also contained 50 mM phenyl phosphonate as a reference. Peak identities are PPA-phenyl-phosphonic acid, Pi-inorganic phosphate, ATP-adenosine triphosphate.


778

SZWERGOLD

on sample characteristics: cell density, sample volume, and conductivity. Assuming that cell availability is not a problem, increasing either, or both, cell density or volume will increase the sensitivity. The upper limit on cell density in perfused systems appears to be about 70% (41), and sample volume can be increased to a point where losses from the attendant increase in conductivity and deterioration of magnetic field homogeneity offset sensitiv ity gains due from the increased sample volume. In spite of the importance of optimizing the sensitivity of NMR spectros copy of biological samples, to the best of our knowledge there has been no thorough analysis of this problem. Designs of probes for NMR spectroscopy of cells have been largely empirical, and it is likely that they are not optimal. Clearly, more research in this area is necessary. While the preceding discussion on sensitivity is applicable to all NMR nuclides, each NMR-detectable nucleus has specific properties and poses specific methodological challenges. The chapter by R. Gillies (this volume) addresses some of these challenges.

Cell Culture The problem of maintaining a high density of viable cells in the field of view of the NMR detection coil has been dealt with in a number of ways. The easiest approach is to work with simple suspensions of cells and perform the experiments rapidly (usually at a lowered temperature) so that no significant deterioration of cells occurs during the experiment. While this approach is adequate for some purposes and has been used extensively, especially in the study of erythrocytes (63, 94), it is clearly 1.lnsatisfactory for conducting long term experiments at a physiological temperature. An early modification of the cell suspension method addressed the problem of oxygenation by bubbling oxygen through the sample and/or stirring the suspension (5). In a recent elaboration of this approach, oxygen is supplied to the cell suspension through a coil of dialysis fibers immersed in the sample (1). Another infrequently used technique is to flow a dense suspension of cells through the NMR coil (29). The most widely used approach has been to confine or immobilize cells within the NMR sample and perfuse them (directly or indirectly) with medium. Several such methods that have been proposed and used to a varying extent over the past ten years include encasement of cells in a gel matrix, attachment of cells to microcarrier beads, cell entrapment between bundles of hollow dialysis fibers, and attachment of cells to mesh support systems. ENTRAPMENT IN GEL MATRIX This method entails mixing a cell prepara tion with a material such as melted agarose and then allowing the polymer and cell mixture to solidify into threads or beads (10, 16, 23, 25, 35, 82). This approach, especially the original agarose threads variant (35), accounts for


NMR OF CELLS

779

most of the recent publications on NMR spectroscopy of perfused cells. Its wide use is due to its simplicity and the great cell density attainable. The drawback is that in this system cells are placed in a non-physiological environment and consequently do not grow within the embedding matrix. This is especially true for anchorage-dependent mammalian cells that require a substrate attachment (7). In order to overcome this limitation, Daly and colleagues developed a variant of the agarose gel procedure in which cells are embedded in a basement membrane matrix (25). Results with this system appear to be superior to those obtained with the agarose threads. Neverthe less, some doubt remains about the ability of this system to support cell growth at a rate comparable to that seen under normal tissue culture con ditions. For instance, the 31p NMR spectra of cells perfused in this system over 39 hr indicated very little growth (25). It is not clear from the published data why the cells do not grow, although one possibility may be a problem of diffusion of nutrients and metabolic products to and from the embedded cells, especially at high cell density. In addition to the agarose and matrigel thread systems, two other variants deserve mention. One is a technically elegant system in which cells are mixed with agarose, as in Cohen's procedure, and then gelled into beads by stirring in paraffin oil. This method, described in detail by Bental (10), is simpler than the agarose thread method and shares the same advantages and disadvantages. The second system involves embedding cells in calcium alginate gel beads (82), and this technique has been used successfully by at least two groups (56, 82). As is the case with the preceding variants, this method is simple and has the same advantages and disadvantages. However, a concern with this gel entrapment method is the high concentration of calcium in the embedding matrix, which may have a modulating effect on the physiological and/or biochemical parameters being measured. In summary, the gel entrapment methods are simple and likely to remain useful in future studies, especially where the physiological milieu of the cell is not a crucial parameter. CELLS ON MICROCARRIER BEADS This method was the first system used for maintaining perfused immobilized cells in the NMR spectrometer. In this system, which is applicable only to anchorage-dependent cells, cells are grown on the surface of small polymer beads. The cell-covered beads are then placed in a specially designed NMR sample chamber where they are retained by filters placed at either end of the chamber, while medium is perfused over this column (1l5). In the original version of Ugurbil et al (1l5), the microcarriers were permeable polydextran Cytodex® beads. The use of these beads presented two problems. The first was a possible ambiguity in differen tiating between signals originating from the intracellular volume vs


780

SZWERGOLD

those coming from the internal bead volume. The second concern was the relatively high sample conductivity, also due to the presence of medium within the beads. A solution to these problems was found by substituting the permeable Cytodex beads with the impermeable polystyrene beads (Biosilon®) (114). The advantages of the microcarrier approach are twofold. First, it is compatible with standard, well-established, tissue culture equipment and techniques. Second, cells are cultured in an environment indistinguishable from that of a standard tissue culture dish. Consequently, as has been demon strated in a number of studies, perfused cells on microcarriers grow at the same rates as do cells in tissue culture dishes (84, 110). The clear dis advantage of this method is the inherently low cell density attainable due to the fact that about two thirds of the sample volume is occupied by the beads. The cell density attainable is also limited by the surface area of the beads, which is an inverse function of bead size. In practice, even with optimized bead size (70-100 }Lm diameter), at most 10% of the sample volume is cellular. An additional limitation of this method is that not all microcarrier beads can be used. For instance, glass beads, which are cheap and on which many cells grow quite readily, are incompatible with NMR because of magnetic susceptibility problems, while collagen-coated beads are not suit able for 31p NMR because of the collagen-bound phosphate, which produces a large hump in the 31 P NMR spectrum. Despite these limitations, the microcarrier method has been adapted, with modifications, by a number of groups and has become the second most widely used method for NMR studies of perfused cells (84, 110).

(DIALYSIS ) FIBER SYSTEMS This method, proposed by a number of groups (41, 43, 50, 72, 76), has been developed most extensively by Gillies and colleagues (41). In this approach, cells are incubated and/or grown in a special chamber between hollow (dialysis) fibers. Cells are nourished and oxygenated by medium perfused through the lumen, which then diffuses out to cells through the porous walls of the hollow fibers. The principal positive attribute of this system is that high cell density is obtained, which results, in the best cases, of up to 70% of the sample volume occupied by cells (41). This results in a markedly improved signal-to noise ratio and/or greatly improved temporal resolution. While initially de signed for anchorage-independent suspension cells, this system can also accommodate anchorage-dependent cells by including a basement membrane gel in the interstitial space between the fibers. However, this system is relatively complicated and expensive and requires special equipment and methods to grow and perfuse the cells and to assess their number and viability. Complexity and cost have been considerable

HOLLOW

�

NMR OF CELLS

781


impediments in the adaptation of this otherwise superior method, and con sequently, it has been used only in a small number of studies. MESH SUPPORT SYSTEMS Another method, used to a very limited extent, is the mesh support system. Cells are attached and/or grown on a woven support such as a nylon mesh (B. Szwergold, unpublished results) or a polyester filter (84). This system is comparable to the hollow fiber approach by offering a high cell density with considerable complexity and cost. One possible advan tage of this system over the encapsulation and the hollow fiber approach is that the perfusing medium is in direct contact with the cells, thus circumvent ing the possible diffusion problems that can arise with these other methods. On the negative side, unlike the hollow fiber method, this system is not suitable for perfusing anchorage-independent cells. At present there are three mature methods of immobilizing cells for perfu sion: encapsulation, microcarriers, and hollow fiber systems. An additional related approach is the spheroid technique, where cells are grown in relatively large, spherical multicellular aggregates that can be perfused without the need for immobilization (37). Which of the above methods is chosen depends on the cell system being studied, but regardless of the approach taken, the problem of cell immobilization need not be a limiting factor in NMR spectroscopic study of cells.

Perfusion Systems To maintain viable cells within the NMR spectrometer they need to be maintained at an appropriate temperature (usually 37°C) and perfused with appropriately conditioned medium to provide oxygen, nutrients, and to re move waste products. A perfusion system designed to perform these functions needs to include a pump and some means to maintain the proper pH, tempera ture, and oxygen tension. For a one-pass minimal system all that is needed is a peristaltic pump. The temperature can maintained by a temperature-regulating system of the spectrometer, and the other parameters are controlled by the composition of the medium (25). Such minimal systems, however, are in sufficient for long term experiments in which the system needs to be main tained in a sterile state, with recirculating medium. A number of groups using sterile perfusion systems, such as the one noted in Reference 41, have maintained a continuous perfusion of cells for many days (41, 43, 84, 110).

Indicators (Reporter) Compounds While most NMR spectroscopic studies of biological systems to date were performed using signals from normal constituents of cells, there are many cellular parameters that cannot be measured using endogenous metabolites. In order to extend this limited scope of NMR spectroscopy, considerable efforts

SZWERGOLD

782

have been devoted to the development of exogenous NMR indicators of physiological parameters such as pH, Ca2+, Mg2+, Na+, redox state, O2 tension, cell volume, and membrane potential. Following is a brief survey of this work. For a recent detailed discussion on the design of indicators see

Reference

70.

pH INDICATORS

These exogenous indicators are important for cells and


tissues in which thr usual endogenous NMR pH indicator, inorganic phos

phate (Pi)' cannot be used. The requirements for such an indicator include pKa

close to physiological pH

(7.0--7.4), large effect of pH on chemical shift

(Ll8ILlpH), ability to be taken up and retained by cells, and low toxicity. Even

though no such single indicator has been found, a number of phosphorylated

and fluorinated compounds have been developed and used as indicators of pHi' While there is no recent general review on this subject, the use of 19F NMR for pH measurements has been discussed in detail by Deutsch & Taylor (28, 29). Table 1 gives a comparable list of the 31p pH indicators.

CALCIUM

19p NMR indicators for Ca2+ were developed by two groups.

The first compound was synthesized by Smith et al (104), who modified the fluorescent indicator

Table 1

BAPTA to an 19p NMR observable Ca2+ chelator

3lp NMR pH indicators'

Compound

pK.

M/apH at pK.

Pi

6.71

1.3

2-deoxyglucose

6.20

1.0

Uptake and retention

very good

Toxicity

Reference

none

40

relatively

83

high Methylphosphonate

7.53

-2.1

very poor except in

none

27, 65, 103

low

R.

RBCs and E. coli 3-aminopropyl-

6.9

-2.1

phosphonate (3APP)

slow uptake, good retention

J. Gillies

personal communication

2-amino-4-phosphono-

6.90

-1.7

excellent

high

110

7.35

-1.9

very good

low

110

7.60

-2.0

good2

none

110

butyric acid (APBA) 2-amino-5-phosphonovalerie acid (APVA) 2-amino-6-phosphonohexanoic acid (APHA) 'For 19F indicator compounds see References 28, 29. 2In amino acid free medium.


NMR OF CELLS

783

5-fluoro BAPTA (5FBABTA). Subsequently, because of a mismatch between the Kd of this indicator (708 nM) and the typical intracellular concentrations ofCa2+ (50-500 nM), other chelators, e.g. quinM (67) and DIME-5FBAPTA (60), were developed with about a tenfold lower dissociation constants. While these compounds have proven useful (4,59,67,79), their utility is limited by the inherent conflict between the limits of NMR sensitivity and the very low concentrations of free Ca2+ in cells, since in order to measure Ca2+ concen tration, the Kd of the indicator needs to be near the level of free Ca2+ (70), while in order to obtain an NMR signal, about - 100 JiM intracellular concentrations of the compound are needed. Given these constraints, it is easy to see that the indicator will buffer relatively large amounts of Ca2+, thus affecting the parameter it is measuring, especially in cases where rapid changes in Ca2+ concentrations occur (59, 60). MAGNESIUM Intracellular Mg2-t- concentrations can, in most cases, be as sessed from the chemical shift of the f3 peak of ATP (47). This measurement is somewhat ambiguous, however, since the chemical shift of f3 ATP, in addition to being affected by Mg2+, is also modulated by intracellular pH and temperature. Moreover, the accuracy of the technique is further limited by the fact that in most cells ATP is nearly saturated with Mg2+ because of the low Kd of ATP for Mg2+ (- 100 JL M). In order to address these problems, Smith et al developed a fluorinated citrate chelator (+) Fcit (59). Recently Levy et al have synthesized two additional19F NMR Mg2+ indicators, MF-APTRA and 5F-APTRA (68). Since typical intracellular free Mg2+ concentrations are on the order of 500 JiM, these indicators do not perturb the cations' homeostasis to the same extent as do calcium indicators and may therefore prove to be more useful than the Ca2+ NMR probes. In the limited number of studies in which these indicators have been used, they have provided measures of intracellular Mg2+ consistent with other methods (59, 68, 97). For additional discussion see References 70, 78.

Sodium 23 is a spin 3/2 NMR visible nucleus with good sensitiv SODIUM ity. Unfortunately, intra- and extracellular Na+ normally resonate at the same frequency. To address this problem, Springer & Gupta (46, 47, 107) de veloped membrane-impermeable shift reagents that selectively change the chemical shift of extracellular Na+ without affecting the resonance of the intracellular ion. While these compounds have found extensive use (13, 46, 1 07), they do have serious limitations, most notably toxicity ( 109). Therefore it would be useful to have another method of measuring intracellular Na+ levels, preferably by 19F NMR, since 19F is not normally present in cells, has high sensitivity, and a large chemical shift dispersion range. One such indicator has been synthesized by Smith et al (lOS), but thus far has not been used in studies on cells or tissues.


784

SZWERGOLD

REDOX POTENTIAL Ability to measure redox potential noninvasively would be useful, especially in the opaque interior of tissues that are not accessible to the currently used methods such as measurement of NAD(P)H and flavin fluorescence (48). Attempts have been made to measure this parameter by NMR by measuring the lactate/pyruvate and ,B-hydroxybutyrate/acetoacetate ratios (55), but quantitation is difficult. The most direct approach was made by Unkefer et al using l3C NMR and a specifically l3C labeled nicotinamide (11 6). Cells grown on this labeled vitamin incorporate it into NAD and NADP where the l3e label can then serve as a direct reporter of the NAD(P)/ NAD(P)H ratio. Results of preliminary studies using this approach in E. coli and Saccharomyces cerevisiae appear promising. Recently, another approach using l3e NMR was reported, but with a different indicator, a phosphorothio ate compound WR3689 (69).

Oxygen concentration in tissues is another parameter that would be useful to access by NMR. Several studies on possible O indicators 2 utilizing the effect O2 on the T) of pertluorinated compounds (112, 1 1 3) have been reported, and preliminary results utilizing this technique with perfused cells appear promising (74). Another approach, used in preliminary studies on the rat brain (54) and human forearm (11 7), is to measure the intensity of a proton resonance of a histidine proximal to the heme of myoglobin at 80 ppm. OXYGEN

MEMBRANE POTENTIAL Thus far there have been two reports on membrane potential measurements by NMR. In one study a hypophosphite ion was used in conjunction with 31p NMR (58), while the second study used the trifluoro acetate ion and 19F NMR (71). Data appear to be quite convincing, although both studies used erythrocytes as a model system. These cells are not a generally useful model system because they contain large amounts of hemog lobin and differ substantially in their transport properties from other cells. A n earlier report by Ogawa (87) on the difficulties in the use of 31NMR to measure membrane potential in isolated mitochondria using tetramethylphos phonium suggests that these methods may indeed not be generally applicable.

Attempts to measure this parameter by NMR have used 31p NMR dimethyl-methyl-phosphonate (95) and 19F NMR of trifluoroacetamide (71). Since both of these studies used erythrocytes as model cells, the same caveat applies here as above. Other methods of measuring cell volume include the use of deuterated sorbitol/endogenous D20 as the extracellular/total volume markers (R. Gillies, personal communication) and the use of 23 Na+NMR (41). Overall, the most promising of these techniques appears to be the 23 Na+ method described by Mancuso et al, which compares total CELL VOLUME

NMR OF CELLS

785

sample Na+ to that in an external standard. As cell density increases, the total Na+ in the sample decreases (72).


TEMPERATURE There is no record of a serious effort to develop an NMR thermometer. The only brief discussion of the subject is by Gorenstein (44), who has used the temperature-dependent chemical shift difference between trimethyl-phosphate and inorganic phosphate to measure temperature in vivo. OTHER PROBES In addition to the specific examples listed above, other exogenous compounds have been used in conjunction with NMR to obtain biochemical and/or physiological information. These include various sub strates labeled with NMR detectable nuclei such as 13C and 19F. Two recent studies have used 133CS+ as an NMR probe for ionic fluxes in cells without the need for shift reagents (26, 92). Another example, described below, uses 31p NMR and a phosphonium analogue of choline (102) to follow choline metabolism in intact cells. A recent development that has the potential of substantially broadening the scope of possible NMR metabolic studies on cells is the use of 3H NMR (86) through the National Tritium Labeling Facility.

AREAS OF STUDY NMR spectroscopy has been used in several areas of research that are briefly outlined below with a few selected examples cited for each area.

Bioenergetics Since the 3 1p nucleus is 100% abundant and since high energy phosphates and Pi are some of the most prominent features of the 3 1p NMR spectrum, bioenergetic was one of the first and most obvious applications of NMR spectroscopy to cellular systems (5, 6). While results of such studies from intact tissues have yielded important results (6, 38, 62), experiments with isolated cells have been less informative. This is because, unlike muscle, where there is a relatively straightforward method of measuring tissue func tion as force, correlations between energetic status of other cells and their functions are extremely difficult to assess. An attempt to make such a correlation, albeit in isolated mitochondria rather than cells, was made in a series of studies by Ogawa et al (87). In terms of most experiments with perfused cells, the bioenergetic profile obtained by 31p NMR is useful mainly as an indicator of cell density and/or viability. While there have been some reports on differences in bioenergetic profiles between drug-resistant and drug-sensitive tumor cells (22, 61), these findings have not been generally confirmed and it now appears that the

786

SZWERGOLD

bioenergetic profile of a cell provided by 31p NMR is not indicative of a pathological state (37). On the other hand, there are some indications that cellular bioenergetic status may be an early predictor of the response of tumor cells to chemotherapy (99, 108).


Intracellular pH In parallel with bioenergetic studies, aided by the relatively easy and noninva sive measurement of ATP and phosphocreatine, the presence in cells of inorganic phosphate (PJ, which can serve as an endogenous 3 1p NMR pH indicator (40), has prompted numerous NMR studies on the regulation of intracellular pH. In addition to the use of Pi, many of these studies utilize a variety of other NMR pH indicators (29; Table 1). Overall, these results show that in most cells intracellular pH is an important parameter that is closely and actively regulated (29, 40, 64, 110). A more detailed description of one such study (110) is given below. At present, pH measurements by NMR have been validated repeatedly by comparison with other methods, and NMR has be come the method of choice for measurement of intracellular pH in intact tissues.

Sodium and Other Ions Studies of intracellular ions have focused largely on 23 Na+ (13). Most notable among these have been experiments on renal proximal tubules (22). As mentioned above, a number of studies have been carried out on Ca2+ (59, 79) and Mg2+ (59, 68, 97), and this area is likely to remain of interest, especially as the improved Ca2+ and Mg2+ probes become more widely available. NMR spectroscopy of other ions such as 23K and 39CI has been limited to a relatively few studies because of lower sensitivity and NMR visibility prob lems that are especially acute with 35 CI (14).

Metabolic Pathways As is the case with bioenergetic studies, most of these investigations were carried out using 31p NMR. Some of the more interesting findings from this work have been the observation of a number of novel metabolites, the most recent being the discovery of sorbitol-3-phosphate and fructose-3-phosphate in the mammalian lens and erythrocytes (91, Il l ). An important methodolog ical aspect in these studies has been the combined use of in vivo NMR with analysis of tissue extracts by NMR and other analytical techniques (17, 32). Of the other nuclei, 13C has been the second most often used, mostly in studies on glycolysis and the citric acid cycle. While it is impossible to summarize all these studies, a notable and concerted effort has been made by Jans & Leibfritz (52) in studies on renal cells. In addition to 31p and Be, increasing numbers of metabolic studies have been utilizing 19F NMR, includ-

NMR OF CELLS

787

ing a study on metabolism of 3-deoxy-3-fluoro-D-glucose (11), 2-deoxy-2fluoro-D-galactose (45), and a study of fluorinated polyamines (51). Studies utilizing the 2H and 15N labels have been far fewer, and none of these was carried out on mammalian cells. One notable trend in metabolic studies has been an increasing focus on


phospholipid metabolism (23, 24, 56, 64) largely prompted by the apparent metabolic pertubations of these reactions associated with tumorgenic transformation (64, 73, 85, 89, 99, 108).

Pharmacokinetics Closely associated with metabolic studies are experiments of the biotransfor

mation of exogenous drugs (9, 57). More prominent among such studies are experiments on the metabolism of fluoropyrimidines (75), difluoromethylor nithine (53), and cyclophosphamide (15, 106). Given the number of drugs with NMR visible nuclei, it is likely that this field wi ll ex pand. SPECIFIC STUDIES Two examples of work from our laboratory involving NMR experiments with perfused mammalian cells are given below.

Intracellular pH in Mitogenically-Stimulated Cells As a result of an intense study of intracellular pH in the early 1980s, it appeared that mitogenic stimulation of quiescent mammalian cells was ac companied by an elevation in intracellular pH (77). Even though the magni tude of this alkaline shift was small, -0.15 units, these observations led to a hypothesis linking this alkalinization to the process of mitogenic stimulation (66, 77, 98). To test this hy pot hesis in a noninvasive and nondestructive manner, we conducted an NMR study on NIH-3T3 cells using a system in which intracellular pH and cell growth could be measured on the same cohort of cells. Cells were grown and perfused on Biosilon microcarrier beads (Figure 2) and pHi was measured using 2-amino-6-phosphono-hexanoic acid (APHA) as a 31p NMR pHi indicator (Table 1). Results of this study do not support the notion that intracellular alkaliniza

tion is linked to mitogenic stimulus. In fact we showed that in a physiological, bicarbonate-buffered medium, the mitogen-associated alkaline shift in pHi does not occur and is not required for those cells to grow. The previously reported, mitogen-associated alkaline shift of 0.15 pH units is observed only in cells perfused with a non-physiological, bicarbonate-free medium (Figure 3) (110). Since almost all of the previous studies on this phenomenon were conducted using such non-physiological media (77), these results suggest that the mitogen-associated alkalinization of pHi was probably artifactual. These

788

SZWERGOLD

120

A

yNTP

aNTP

E

OJ

'Qj


.t: � «l m

c. a.. fZ ?o-

B

C

100

.... .. .. M, ...... A

80

A

•

,,�·M

A

A A A

60

·ft·· A

40

• .

..4A11>•..

'

20 0 iii IS

0

10

20

30

40

Time of perfusion (hours)

50

Figure 2 Growth of NIH 3T3 cells in the perfusion system: (A) 31p NMR spectrum of NIH 3T3 cells at the beginning of pertusion at a cell density of 4 x 106 cells/ml beads; (B) 31p NMR spectrum of the same cell sample after 42 hr of perfusion in the NMR spectrometer at a cell density of 12 x 106 cells/ml beads; (C) time course of increase in the peak height of the yNTP during the course of the experiment. Data were acquired at 162 MHz in 3600 scans using 30° pulses and 0.4 sec recycle time. Chemical shifts were referenced to aNTP set at -10.06 ppm. Peaks identities are PME·phosphate monoesters, Pi-inorganic phosphate, PDE-phosphodiesters, NTP-nucleoside triphosphates. The Pi signal observed is caused by extracellular phosphate in the perfusion medium.

findings, while initially controversial, were subsequently confirmed by stud ies from other groups (12, 39). Thus it appears that while pHi is an important intracellular housekeeping parameter, it is not a part of the cascade of second messengers linking the external mitogenic stimulus to cell growth and mitosis. We believe that in this case the resolution of this issue was aided sub stantially by the nondestructive and noninvasive character of NMR spectros copy, which permitted measurements of pHi and growth on a single sample of cells.

Phospholipid Metabolism in Normal and Transformed Cells This second example is from an ongoing NMR study on the metabolism of choline in normal and tranformed cells. This project was prompted by the findings from in vivo 31p NMR that in many tumors and transformed cells the concentration of phosphory1choline, a key intermediate in phospholipid syn thesis (90), is consistently higher than in normal tissues (64, 73, 85, 89, 99, 108). This elevation appears to be qualitatively different from that observed in

NMR OF

CELLS

789

A aNTP


POE

•

i I Ii I. f

31

B

--- +FBS

---FBS

'

25

iii I' i' i , ••iii' i i j iii ii" IS

20

III

5 PPM

II

'YNTP

i' ii' i • 'I' i Ii"

-5

c HIGH C02

--- +FBS

II

--- FBS

-

I ll

-IS

iii I'

-28

i I

LOW C02

II

is Figure 3 (A) A typic al 31 p NMR spectrum of NIH 3T3 cells perfused with serum free DMEM at pH 7.0 and 37°C. For acquisition parameters, see Figure 2. Peaks identities APHA-intracellular

pH indicator 2-amino-6-phosphono-hexanoic acid, PPA-extracellular pH indicator phcnyl phosphonic acid. (B) Expanded region of the above spectrum showing APHA in serum-starved cells before (90 min, solid line) and after (50 min, dashed line) addition of 15% FBS to the bicarbonate buffered perfusion medium. (e) Spectrum of APHA in serum-starved cells in a low bicarbonate medium (pC02 0.8 Torr, pH e 6.95) before (60 min, solid line) and after (40 min, dashed line) the addition of 15% FEBS. =

=

nonnal growing tissues such as the brain (88), regenerating liver (20), or ,developing fetus (17), where the predominant phospholipid precursor is most often phosphorylethanolamine. In addition, a number studies have shown that concentration of phosphorylcholine in tumors often declines following ther apy (33, 93, 99).


790

SZWERGOLD

In order to better understand the biochemical basis for these phenomena, we have undertaken a 3 1p NMR study of choline metabolism in a series of related, well-characterized normal and transformed cells: primary rat embryo fibroblasts, a rat embryo fibroblast cell line, RAT-2, and the ras and src transformants of RAT-2. In conducting this work, we used an 31p NMR visible analogue of choline, phosphonium-choline (P+Cho), in which the trimethyl-ammonium group has been replaced by a trimethyl-phosphonium (Figure 4B). This compound, first described in 1975 (101), has proven to be an excellent surrogate that can substitute for choline in mammalian systems virtually without any detectable deleterious effect on cell growth or function (30, 102). In the 31p NMR spectrum, the phosphonium nucleus of P+Cho resonates at 25.7 ppm, far downfield from other phosphorus-containing compounds such as phosphomonoesters and phosphodiesters. In addition, this compound has an another, extremely useful attribute; unlike the phosphoryl resonance of phospholipids, which is very broad, the phosphonium peak of the analogue is relatively narrow and thus easily observed, even following its incorporation into membrane as phosphatidyl-P+Cho (Figure 5) (100).

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GL YCERO· PHOSPHORYL· PHOSPHONIUM· CHOLINE (GroPP+Cho)

Figure 4 Structural fonnulae of choline. its phosphonium analogue and their phosphorylated derivatives. (A) Cho·choline. (B) P+Cho.phosphonium.choline, (C) PCho·phosphoryl·choline, (D) PP+Cho.phosphoryl-phosphonium-choline, (E) GroPCho-glycero-phosphoryl·choline, (F) GroPP+Cho·glycero·phosphoryl·phosphonium.choline


NMR OF CELLS

H

791

---

SOp.p.m. Figure 5

31p NMR spectrum of unsonicated vesicles made with a phosphonium analogue of

phosphatidylcholine. The peak on the left results from phosphonium (P+) resonance whereas the broad peak on the right is from the phosphodiester of the phospholipid. From 101.

When primary rat embryo fibroblasts are perfused with P+Cho in a physi ological medium containing fetal bovine serum (FBS), the analogue is taken up and metabolized, as can be seen in Figure 6A. At the beginning of perfusion with 20 IJ-M P+Cho, there is no detectable P+Cho signal (upper panel), after 10 hr of perfusion there is a large peak at 26.2 ppm, which is the expected chemical shift position of the phosphonium peak of phosphorylated P+Cho. Concomitant with the appearance of this peak, there is also a sub stantial increase in a peak at 3.6 ppm, the chemical shift position of the phosphoryl group of phosphoryl-P+Cho. These results indicate that in the presence of serum P+Chol is metabolized primarily to phosphoryl-P+Cho in these cells. In contrast to the situation described above, when these Same primary cells are rendered quiescent by withdrawal of serum, the pattern of P+Cho metabolism changes dramatically (Figure 6B). The compound is taken up and phosphorylated as evidenced by the increase in the phosphonium peak at 26.2 ppm. However, there is little parallel increase in the phosphoryl peak at 3.6 ppm, which would be expected if P+Cho was metabolized primarily to phosphoryl-P+Cho. The most plausible explanation for this observation is that the phosphonium peak at 26.2 ppm is due mostly to signal from phospha tidyl-P+Cho which, as shown in Figure 5, is much narrower than the corre sponding phosphoryl resonance. If correct, these results imply that in quies cent primary cells most of the analogue accumulates as the phospholipid. Similar experiments performed with an established cell line, RAT-2 (de rived from rat embryo fibroblasts) and its RAS and SRC transformants show

SZWERGOLD

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31p NMR spectra of cells at the beginning (top spectra) and after 10 hr (bottom

spectra) of perfusion with 20 /LM P-Choline. (A) Primary rat embryo fibroblasts perfused with serum-containing medium. (B) Primary rat embryo fibroblasts perfused with serum-free medium. (C) Rat embryo fibroblast cell line RAT-2 perfused with serum free medium. Peak identification: I-phosphonium peak, of phosphorylated-P+Cho (including phosphoryl-P+Cho, phosphatidyl P+Cho and glycero-phosphoryl-P+Cho); 2-phosphoryl peak of phosphoryl-choline and phos phoryl-P+Cho; 3-phosphoryl peak of glycero-phosphoryl-choline and glycero-phosphoryl P+Cho, a, {3, yNTP-a, {3, y phosphates of nucleoside triphosphates.

that in these cells most of P+Cho accumulates as phosphoryl-P+Cho (Figure 6C). However, in contrast to the primary cells, in these immortalized cells the accumulation appears to be serum-independent. These results suggest that immortalization of primary fibroblasts, which constitutes a partial transforma tion event (7), alters the pattern of P+Cho metabolism, which leads under appropriate conditions to the accumulation of phosphoryl-P+Cho. While the above observations are clearly preliminary, they illustrate how NMR spectroscopy of cultured cells, combined with the use of an appropriate indicator, can provide a unique approach to the study of biochemical process in intact cells. SUMMARY/FUTURE DIRECTIONS The survey presented above suggests some directions in which NMR spec troscopy of cells most likely will move. Refinement and improvement in the methodologies, both in terms of the NMR hardware and software, and in terms of the cell immobilization and perfusion, will undoubtedly continue. The continued evolution of NMR indicators of physiological and biochemical parameters is probable. Beyond the expected improvement in the sensitivity of NMR with an increase in the spectrometer field strength, another likely source of increased sensitivity will come from a wider use of indirect detec-


NMR OF CELLS

793

tion methods (8, 34). Such enhanced sensitivity is likely to stimulate new experiments, including ones with hitherto relatively neglected nuclei such as l3e and 15N. Much remains to be done in terms of choosing appropriate model systems that will maximize the advantages that NMR methodology offers for metabol ic and physiological studies. It should be emphasized that in addition to its noninvasive and nondestructive character, NMR spectroscopy has another positive attribute often neglected in discussions of the subject. This property is its non-specificity in detection and quantification of cellular metabolites, i.e. its capacity to observe and measure concentrations of metabolites in complex mixtures without a need for a specific assay. This is well illustrated by the recent discovery of sorbitol-3-phosphate and fructose-3-phosphate for which NMR spectroscopy remains the only assay (91, 111). Given that fact, one way in which NMR spectroscopy can contribute to increased understand ing of cellular biochemistry and physiology is by focusing on studies in which this particular strength is more fully exploited. Literature Cited 1. Ammann, H., Boulanger, Y., Legault, P., Vinay, P. 1989. An incubation sys tem for the NMR study of kidney tubules. Magn. Reson. Med. 12:339--47 2. Avison, M. J., Gullans, S. R., Ogino, T., Giebisch, G. 1988. Na+ and K+ fluxes stimulated by Na+ -coupled glu cose transport: Evidence for a Ba2+-in sensitive K + efflux pathway in rabbit proximal tubules. 1. Membr. Bioi. 105:197-205

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Ultrahigh-resolution NMR spectroscopy.

1H NMR spectroscopy of DNA.

Fully resolved NMR correlation spectroscopy.

Chiral discrimination in NMR spectroscopy.

Investigation of glycofullerene dynamics by NMR spectroscopy.

C-NMR spectroscopy of tropane alkaloids.

Proton NMR spectroscopy of Canavan's disease.

Exploring RNA polymerase regulation by NMR spectroscopy.

NMR spectroscopy for metabolomics and metabolic profiling.

Fast multidimensional NMR spectroscopy for sparse spectra.

31P NMR spectroscopy in chronic adriamycin cardiotoxicity.

Visualizing transient dark states by NMR spectroscopy.

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells.

[Non-invasive analysis of proteins in living cells using NMR spectroscopy].

G-quadruplex DNA and ligand interaction in living cells using NMR spectroscopy.

[NMR-Spectroscopy in hetercyclic compounds. 4. 13C-NMR spectroscopic coordination of 1,4- and 1,5-substituted imidazoles].

Self-healing capacity of nuclear glass observed by NMR spectroscopy.

Rapid characterization of molecular diffusion by NMR spectroscopy.

Examination of amber and related materials by NMR spectroscopy.

Kinetics of enzyme-coenzyme interactions by NMR spectroscopy.

Observation of diastereotopic signals in (15) N NMR spectroscopy.

Investigation of native and mutant plant peroxidases by NMR spectroscopy.

Solution structure of endothelin-3 determined using NMR spectroscopy.

Structure determination of helical filaments by solid-state NMR spectroscopy.