Monitoring of cellular metabolism by NMR

53

Biochimica et Biophysica A cta, 639 (1981) 53-76 Elsevier/North-Holland Biomedical Press

BBA 86076 M O N I T O R I N G OF CELLULAR METABOLISM BY N M R JUSTIN K.M. ROBERTS a and OLEG JARDETZKY b

a Department of Biological Sciences and b Stanford Magnetic Resonance Laboratory, Stanford University, Stanford, CA 94305/U.S.A.] (Received March 25th, 1981)

Contents I.

Introduction

II.

General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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53 54

III. Experimental measurements and problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Measurement of chemical shifts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Assignment of resonances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Quantitation of metabolite levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The time resolution of NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Maintenance of a controlled physiological state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55 55 56 56 57 58

IV. 31p-NMR in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : .................... A. Quantitation of metabolite levels and metabolite interconversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Intracellular pH measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Study of compartmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Measurement of unidirectional reaction rates in vivo by saturation transfer . . . . . . . . . . . . . . . . . . . . . . .

58 58 60 62 66

V.

68 69 70

13C.NM Rin vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Examin'ati0n of metabolite fluxes and interconversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Intracellular pH measurements by 13C.NM R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VI. 1H-NMR in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Quantitation of metabolite levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Intracellular pH measurements by 1H-NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Membrane transport studied by 1H-NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70 71 72 72

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction Applications o f N M R spectroscopy to the study o f living systems have gained increasing p o p u l a r i t y over the past few years. The earliest r e p o r t e d observations on b l o o d and red b l o o d cells by Odeblad et al. [1] and J a r d e t z k y and Wertz [2] in 1956, as well as m o s t o f the subsequent w o r k prior to 1972, were limited to the study o f w a t e r and electrolytes. A l t h o u g h such studies have revealed some intriguing p h e n o m e n a -

such as differences in relaxation rates for water and alkali m e t a l ions in different tissues - the interpretation o f time-averated N M R parameters o f water and ions, which rapidly exchange b e t w e e n different e n v i r o n m e n t s in c o m p l e x systems, faces formidable theoretical difficulties. Much o f this w o r k has been reviewed elsewhere [ 3 - 6 ] and is included here largely for its historical interest. The focus o f our review is on studies o f small intracellular m e t a b o l i t e s containing 1H, 13C or 31p

0304-4173/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press

54 nuclei. Such studies offer the hope of obtaining information on a wide range of metabolic processes and are in some respects easier to interpret. They became feasible in the early 1970's, when high-frequency Fourier-transform (FT) NMR spectrometers capable of detecting a compound present at or above about 10 -3 M in a matter of minutes became generally available. An impetus for the study of intracellular metabolites came from the report of Eakin and co-workers [7] in 1972, who showed that metabolism of a specifically labelled substrate, [1-13C]glucose, can be observed directly in living cells. In 1973 Moon and Richards [8] showed that the correlation between the chemical shift of the phosphate 31p resonance and pH permitted an estimation of the pH in red blood cells, and in 1974, Hoult et al. [9] reported high-resolution 3~p-NMR spectra of ATP, Pi, phosphocreatine and sugar phosphates in intact muscle. Subsequently, in 1976, Daniels et al. [10] showed that intracellular metabolites in the adrenal gland can be observed using ~H-NMR. More than 100 reports of NMR observations of these nuclei in vivo have appeared in the literature in the last 5 years. For purposes of review at this early stage of the field it is useful to distinguish between feasibility studies, demonstrating that NMR spectra can be obtained from a particular live specimen or dealing with technical developments improving the acquisition and quality of the spectra on one hand, and on the other, studies which address a specific physiological problem. The majority of the published reports still fall into the former category. It is, however, important to identify these problems of physiology and biochemistry to which NMR has made or can make significant novel contributions. This is our objective here. II. General considerations NMR is a form of spectroscopy based on the absorption of radiofrequency radiation by atomic nuclei that possess a magnetic moment (e.g., IH, 2H, 13C, lSN, 19F, 23Na ' 31p and 39K) when they are placed in a stationary magnetic field. Different nuclei have very different resonance frequencies at a given applied magnetic field strength, so a given NMR experiment can only detect resonances from a single isotope; thus, there is no interference problem of one element being confused for another, as can occur

with other analytical tools. NMR plays an important role among methods used to study molecular structure, for the following principal reasons. First, the resonance frequency for a particular nucleus in a given applied magnetic field depends on its chemical environment. The spectrum of a molecule consists of several lines, or~e for each chemical group. The separation between them is termed the chemical shift, 6. Second, the intensity of the signal (i.e., peak area) at a given resonance frequency is linearly proportional to the concentration (not chemical activity) of absorbing nuclei in the sample; Beer's Law * holds over all concentrations above the limit of detection and, furthermore, the extinction coefficient of a nuclear species is independent of the chemical environment (provided saturation is avoided, as discussed below) in contrast to the absorption of visible and ultraviolet light. Third, other parameters which can be measured on NMR spectral lines - line splitting due to coupling of nuclei, the line width, and the related relaxation parameters (T1, T2 and the nuclear Overhauser e n h a n c e m e n t ) - a r e sensitive to interatomic distances, geometric relationships between chemical bonds and the frequencies and amplitudes of molecular motions. Fourth, kinetics of chemical reactions or exchange can be studied by a variety of NMR techniques. NMR spectroscopy of living material can therefore, in principle, be used to identify metabolites present; to provide details of the environment such metabolites are experiencing in vivo (such as pH, occurrence of binding to ions or proteins); to determine their concentrations; and to measure kinetics of metabolic interconversions. No harmful consequences of NMR methods (such as effects of the magnetic field, or the exciting radiofrequency radiation) on living subjects have been observed in conventional one-pulse FT-NMR experiments (e.g., see Refs. 11 and 12), and so it is fair to say that the technique is

* In familiar empirical usage, Beer's law is defined for monochromatic light and holds only as long as line position and width remain constant. However, in its proper theoretical definition which applies here and in all other branches of spectroscopy, it expresses the proportionality between the number of quanta absorbed in a given transition as given by the area under the absorption curve, and the concentration of the absorbing species.

55 basically noninvasive and nondestructive. However, it must be borne in mind that the higher radiofrequency power levels used in certain saturation or decoupling experiments might very well be harmful [13]. The principal disadvantage of NMR is its relatively low sensitivity, which limits in vivo observations largely to low molecular weight metabolites present at relatively high concentrations (about 10-aM or higher). Only proteins present in unusually high concentrations can be studied in intact subjects: 1H-NMR indicated that chromogranin, comprising approx. 50% of the chromaff'm granule matrix protein, occurs as a random-coil protein in vivo [14] (for a review see Ref. 15). In a ~3C-NMR study of [2-~3C]histidinelabelled mouse erythrocytes [16], the spin-lattice relaxation time ( T I ) o f the large histidine peak of enriched hemoglobin was measured and the suggestion made that the rotational correlation time of hemoglobin in vivo is similar to that of aqueous solutions of hemoglobin at a similar concentration. The low sensitivity of NMR has an advantage as well as disadvantages, when the method is applied to complex mixtures such as living cells and tissues, for it results in relatively uncomplicated spectra (particularly in the case of 31p), as only a small number of resonances are apparent. Furthermore, only freely mobile molecular species have sharp resonances. So, only the subspectrum of the more abundant and more mobile molecular species is observed, and this greatly simplifies interpretation. To derive any information from an NMR spectrum it is necessary to assign each spectral line to a chemical group in a specific molecular species; the problem of assignments is tractable only if attention can be focussed on a relatively few readily identifiable molecular species. The theory of NMR methods and relevant experimental techniques are more fully discussed in monographs by Farrar and Becker [17], Dwek ~ [18], Becker [19] and Jardetzky and Roberts [20]. III. Experimental measurements and problems

IliA. Measurement of chemical shifts lane position on NMR spectra are by convention specified relative to a reference standard. The chemical shift, 8, is defined as: 6 = vS - v R X 1 0 6 VR

where us and b'R are the absolute resonance frequencies of the line of interest and of the reference, respectively. If conclusions are to be drawn from chemical shift measurements, the choice of a reference of which the resonance frequency is not affected by the experiment is essential. A reference may be internal, i.e., in the same compartment as the sample, or external, i.e., separated from the sample by a diffusion barrier. Interpretation of chemical shifts with respect to an internal reference in solutions is less problematic [19] but the use of internal references in vivo is rarely practical or free from pitfalls. An external reference compound, contained in a thin capillary placed in the NMR tube, is therefore used in most cases. The problems of interpretation of chemical shifts associated with the use of external standards have been discussed elsewhere [19] and are beyond the scope of this review. Suffice it to say that the separation between the sample and reference lines on a frequency scale may depend on the geometry of the magnet, the position of the capilary relative to the sample and the size, composition and geometry of the sample. Standardization of measurements on a given system is important and comparisons of shifts measured in different laboratories and on different systems are not always possible. Tetramethylsilane has been used as an external reference compound for in vivo laC-NMR (e.g., see Ref. 21), while sodium 4,4dimethyl-4-silapentane (DSS) is useful for IH-NMR studies (e.g., see Ref. 22). I n contrast, a variety of reference compounds have been used for in vivo alp. NMR work. Phosphoric acid (85%), used as a reference compound in most chemical work, is unsatisfactory for in vivo aIP-NMR work - its viscosity results in a broad line, reducing the accuracy of measured shifts. Furthermore, its resonance frequency is close to, or overlaps with, the shifts of certain phosphate metabolites. Phosphoric acid also has a very different magnetic susceptibility from those of water and aqueous solutions [23]. This makes the measured chemical shifts very sensitive to magnet geometry. The extent of the problem depends on the particular reference-sample combination-very similar shifts referenced using 0.5 M methylene diphosphate [24] were obtained for phosphates in plant cells using either a superconducting [25] or an electromagnet [26] and methylene diphosphate is probably the most satisfactory external reference developed so far.

56

The 3~p line of phosphocreatine has often been used as an internal reference (e.g., see Ref. 27). The phosphocreatine signal is strong in fresh tissues, such as muscle and brain, and its shift is not very sensitive to pH in the physiological'range. However, it is not yet clear to what extent the shift can be affected by other changes in the metabolic state and composition of the tissue. The resonance disappears where phosphocreatine stores are depleted and is therefore not useful as a reference in the study of hypoxic states.

IIIB. Assignment of resonances Valid assignment of resonances in a spectrum is an essential, but often problematical, first step in any in vivo NMR study. Few fool-proof methods of assignment exist. Comparison of observed shifts with shifts in solutions of known metabolites under physiological conditions (e.g., of pH, ionic strength and divalent cation concentration) allows certain metabolites to be excluded as contributors to the spectrum. Conventional analysis of extracts of the tissue in question can then be used to check tentative assignments made from measured shifts; for example, if a metabolite is present at a concentration of less than approx. 1 mM (on a tissue wet weight basis), it cannot generate a detectable signal (the precise limit of detection depends on the spectrometer used, and the nucleus under study). Titration of extracts can be used to aid assignment, on the basis of pK a [28]. It is apparent, therefore, that use of in vivo NMR is dependent on detailed information obtained by traditional biochemical methods. This particularly the case for interpretation of broad lines due to overlapping resonances, as occurs, for example, in the sugar phosphate and nucleotide terminal phosphate regions of 31p-NMR spectra. The more complex in vivo 13C(e.g., Ref. 21) and IH-NMR (e.g., Ref. 22) spectra represent an even greater challenge in making assignments. NMR experiments in which two resolvable resonances have been attributed to a single compound that is compartmentalized in distinct environments (e.g., differing in pH) within the sample have been reported (see subsection IVC). Observation that two lines from an in vivo sample become one in a neutralized perchloric acid extract [29] suggests that the two lines originate from a single metabolite but accidental overlap is a possibility. As discussed in sub-

section IVC, assignment of the resonances in distinct intracellular compartments (as opposed to, for example, variation between cells within the sample) can only come from physiological experiments permitting manipulation of the presumed compartmentalized pools (e.g., see Ref. 25).

IIIC. Quantitation of metabolite levels The intensity of a resonance from a particular chemical group is linearly proportional to its concentration in the sample under the usual conditions of data acquisition. This fact allows intracellular metabolite concentrations to be derived from NMR spectra. Two factors must, however, be controlled before this is possible in practice. First, saturation of the resonance by intense radiofrequency radiation must be avoided or quantitatively accounted for. The peak areas of different metabolites depend on the intensity of the radiofrequency and the pulse repetition rate used in the NMR experiment in addition to the relative concentrations of the metabolites (see Fig. 1). Since different metabolites can have different T1 relaxation times, the intensities of different lines can be reduced to a different extent in the same pulse

--4.8--3.2 0 4.6 9.4 pore

18.2

Fig. 1. 31p-NMR spectra from identical perfused mouse livers. Upper spectrum, accumulated in 6 min with a repetition rate o f 30 s-i; lower spectrum, accumulated in 1 h with a repetition rate of 0.2 s-1. Assignments (referenced to phosphoric acid; in ppm): 18.2, t3-ATP; 9.4, a-ATP + a-ADP; 4.6, ~-ATP +/3-ADP; - 3 . 2 , Pi; - 4 . 8 , sugar phosphate monoesters and AMP. The Pi and sugar phosphate resonances have much longer T 1 values than those o f ATP, so that with a short pulse interval the peaks at - 4 . 8 and - 3 . 2 pprn are severely saturated, i.e., in the upper spectrum the relative peak area is not equivalent to the relative concentration. From Ref. 34.

57 sequence. Saturation can be avoided if the interval between pulses is long (approx. 5 • T1). However, in order to maximize the signal-to-noise ratio of spectra it is common practice to repeat pulses much more frequently than 5 • TI. To correct for saturation effects, the relative extent to which different resonances are reduced using different pulse repetition rates can be determined. This enables changes in the relative conconcentrations of metabolites to be accurately followed (e.g., see Ref. 27). To determine absolute tissue concentrations a second factor must be taken into account. A calibration of the spectrometer to determine the sample volume contributing to the NMR signal, together with the proportion of this volume occupied by tissue, is necessary. Using careful calibrations, Dawson et al. [27] demonstrated 'that absolute concentrations of phosphocreatine in muscle determined directly by NMR in vivo are in reasonable agreement with measurements obtained chemical analysis of frozen muscle.

IIID. The time resolution of NMR In the most usual experimental arrangement for in vivo NMR, in which spectra of a sample are obtained before, during and after a particular treatment such as ischaemia, the time resolution for detecting metabolic changes (e.g., in metabolite levels or intracellular pH) is equal to the time required to obtain a spectrum. This may vary from 1 to 30 min. Much better time resolution can be obtained with a cycling experimental system: the timing of the exciting radiofrequency pulses is synchronized to specific periods within the cycle, and scans from each cycle period are stored and accumulated separately. With such an arrangement, Ogawa et al. [30] were able to measure intraceUular pH in Escherichia coli with a rime'resolution of approx. 1 s during an O2-bubbling/nonbubbling cycle; and Dawson et al. [27] followed metabolite levels in muscles during various contractionrecovery cycles (discussed in subsection IVA). By synchronizing the radiofrequency pulses to the measured aortic pressure in beating, perfused rat hearts, Fossel et al. [11] were able to follow Pi, ATP and phosphocreatine levels during the cardiac cycle (of period approx. 200 ms) with a time resolution of approx. 2 ms. The lower limit for time resolution in a cycling

system is determined by the relaxation times of nuclei under study, together with the duration of the exciting radiofrequency pulse. In a conventional onepulse FT-NMR experiment, following a radiofrequency pulse, the signal from nuclei (the free induction decay) decays in intensity exponentially at a rate characterized by the first-order rate constant l/T2. The duration of the free induction decay (a few milliseconds for 31p metabolites in rat hearts [11]) determines the lower limit for time resolution for detecting metabolic changes, and is dependent on the value of/'2 and on the extent to which the nuclei are perturbed from their equilibrium energy distributionthe latter being determined by the duration of the exciting radiofrequency pulse. Free induction decays of shorter duration are observed with nuclei having shorter T2 values, and using shorter radiofrequency pulses. Metabolic changes occurring during a free induction decay will be averaged, and so not detected, although Fossel et al. [11] point out that greater time resolution than that determined by the duration of the free induction decay could, in principle, be achieved by computer analysis of the free induction decay. There is an additional constraint on time resolution in NMR. The acquisition and storage of free induction decay by a computer require a finite amount of time, being equal to N/2W, where N is the number of data points in the spectrum and I¢ the spectral width (in Hz) (see Ref. 31). Successive free induction decays cannot be acquired within intervals shorter than this time, which is usually greater than 0.I s; in other words, successive points in the same cycle cannot be sampled more rapidly than N/2W. Determination of metabolite levels at more frequent time points in the cycle requires comparison of signals from different cycles. The analogy of pulsed NMR with a still camera (alluded to in Ref. 1 I) is useful: pulsed NMR has a short 'shutter speed' (the free induction decay) relative to the interval between 'exposures' (the pulse interval). Even in a noncycling experimental system, time resolution down to seconds is possible simply by adding free induction decays, obtained during a particular period of treatment, from separate experiments (i.e., each experiment is one cycle) until spectra of adequate signal-to-noise ratio are obtained. Dawson et al. [27] were able to improve the signal-

58 to-noise ratio in one study by adding together results from five separate experiments with different muscles, and so to detect small differences in metabolite levels in a contraction-recovery cycle. IIIE. Maintenance of a controlled physiological state Because of the inherent insensitivity of NMR, it is desirable to use as large an amount of material as possible, so that the signal-to-noise ratio in the spectrum is maximized. For fixed sample size, this may mean dense packing. Unless anaerobiosis is the desired condition, maintenance of such a sample in a well oxygenated physiological state in the spectrometer constitutes a major problem. Many early NMR investigations were carried out on aging or inadequately perfused tissues, with the consequence that only limited or tentative physiological conclusions were possible. Stable aerobic conditions have been achieved by bubbling cell suspensions with O2 (e.g., see Ref. 32) (small bubble diameters, 2 mm or smaller, do not significantly increase magnetic field inhomogeneity [28]; by perfusion of organs such as heart [33] and liver [34] in which the adequacy of perfusion was demonstrated by near complete recovery of original metabolite levels in ischaemia-reperfusion sequences; and by performing the NMR experiment on intact animals, without surgery [12,35-37]. Maintenance of a subject in a stable condition for long periods is essential for extensive and thorough NMR investigations to be possible. For example, T1 measurements of resonances, which must be determined for accurate quantitation of metabolite levels [11,27] (see subsection IIIC), and for measurements of unidirectional reaction rates using saturation transfer described in subsection IVD, take much longer than the time required to obtain a conventional spectrum. The same is true for T2 measurements, discussed in section IVC. A somewhat unique feature of in vivo NMR (in particular 31P-NMR) is that, in addition to providing other types of information, the spectra can be used directly to determine the metabolic stability of the subject. For example, McLaughlin et al. [34] were able to maintain initial ATP levels in perfused mouse livers for 2 h. Furthermore, the 3~p-NMR spectra are indicative of the metabolic state of a sample. Thus, it has been commonly observed (e.g., see Refs. 12, 25, 28 and 34) that aerobic cells contain a low cytoplasmic Pi concentration, relative to other phospho-

rylated metabolites; intracellular Pi increases rapidly during anaerobiosis, as these phosphates are hydrolyzed (a similar rise in intracellular Pi occurs during muscle contraction as phosphocreatine is depleted [12,27]. Thus, an increased Pi peak is a very sensitive indicator of tissue hypoxia [ 12]. IV. 3~P-NMR in vivo Detection of naturally occurring 31p resonances in living materials constitutes the majority of in vivo NMR studies. This is partly because experiments do not require feeding an isotopically labelled metabolite as is necessary in laC-NMR (and lSN-NMR)studies; and unlike 1H-NMR studies, the simplest one-pulse FT-NMR experiment can be used to obtain a 31p. NMR spectrum. Furthermore, 31P-NMR spectra are usually simpler than their ~3C and ~H counterparts and hence more readily interpretable (see Fig. 2). 3~p-NMR spectra have been obtained from a great variety of biological materials: blood [8,38], skeletal [9,24,29,37,39-41] and cardiac muscle [33,42], brain [35-37], liver [34,43] kidney [44], adrenal glands [45,46], suspensions of microbiol cells [32, 47-49], including spores [50,51], and mammalian cells [52-53], developing Xenopus embryos [55], a protozoan (Acantamoeba) [56], a marine mollusc (Tapes watlingi) [57], and plants [25,26]. Much of this work is discussed, system by system, in reviews by Burt et al. [58] and Hollis [59]. A table of chemical shifts of 3~p-labelled compounds detected in biological materials is presented by Burr et al. [58]. Most tissue or organ samples have been studied after removal from the organism. However, spectra of brain metabolites have been obtained by placing the animal's head at the center of the radiofrequency coil [35,36]; of heart and kidney metabolites by placing the coil around the organ, by surgery [60-62]; and by placing 'surface' radiofrequency coils next to the tissue of interest [37]. The development of widebore, high-field superconducting magnets of high magnetic field homogeneity has made it possible to record spectra of large organs (human limbs and dog heads) in situ [ 12]. IVA. Quantitation of" metabolite levels and metabolite interconversions In this section we highlight only one of the more

59

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Fig. 2. NMR spectra from red cells: (a) 1H spin-echo spectrum shows hemoglobin (h), glucose (gl), glutathione (g), creatine (c), and unassigned (u) peaks. The magneticfield was 6.3 T, the sample volume, 0.5 ml, and the accumulation time, 4 min. The medium was 2H20/Krebs buffer with 10 mM glucose. (b) 13C.NMR spectrum shows peaks from labelled positions: lactate C-3 (1), glucose C-1 (gl) and 2,3-diphosphoglycerate C-3 (d). The field was 4.3 T, the sample volume, 3.5 ml, and the accumulation time, 10 min. The medium was IH20/Krebs buffer with 10 mM glucose enriched at the C-1 position. (c) 31p-NMR spectrum shows peaks from 2,3diphosphoglycerate (d), Pi (P) and ATP (a). Conditions were the same as for spectrum b. From Ref. 121.

complete alp.NMR studies: that of energy metabolism in amphibian skeletal muscle. Some of this work and related investigations of other systems have also been reviewed elsewhere [23,28,58,59,63-65]. Dawson et al. [27] have studied three particular aspects of muscle metabolism. They first measured

the relative and absolute concentrations of 31p metabolites (ATP, phosphocreatine, Pi and sugar phosphates) during and following contraction of aerobic muscle, using corrections and calibrations as described in subsection IIIC. An apparatus was constructed for perfusing, electrically stimulating, and measuring tension developed in muscles placed in the spectrometer. This, together with the use of amphibian muscle which can function at 4°C, permitted experiments to be performed on a single preparation over several hours, with only a gradual change in metabolite levels. It was found that little or no change in 31p metabolites occurred in frog sartorius muscle during brief contractions (1 s tetani every 2 rain, over a period of about 4 h), whereas during long contractions (25 s every 56 min) about 20% of the phosphocreatine was broken down, with a corresponding increase in Pi; complete recovery occurred about 30 min after contraction. Brief contractions (1 s every 2 rain) in toad muscle gave results similar to those obtained during long contractions in frog m u s c l e - about 10% of the phosphocreatine being broken down following the tetani; this is perhaps due to the large size of the toad muscle, which prevents aerobic conditions being maintained during the 2 h experiment (indicated by the low phosphocreatine : Pi ratio compared to that of frog muscle). The important result was obtained that metabolite levels determined by NMR agreed with those determined by conventional chemical analysis of extracts. Second, these workers examined the relationship between metabolite levels (and derived rates of metabolite utilization) and force development in anaerobic muscle, as muscle fatigue occurred in the spectrometer [66]. They clearly showed that force development is not related in a simple way to the level of substrate available for contraction (phosphocreatine or ATP). In contrast, the decrease in force development during fatique is approximately proportional to the rise in H ÷ concentration (pH being determined from the chemical shift of Pi, as described in the next subsection) and calculated free ADP levels; it was not possible to determine whether this result is indicative of factors controlling force development in muscle, or a mere coincidence. Significance was attached, however, to the linear correlation between force development and the calculated amount of ATP utilized in a contraction. ATP utilization was estima-

60 tion by summing changes in the concentration of ATP, phosphocreatine and lactic acid (the latter being deduced from the decline in pH and the known buffering capacity of frog muscle). Such a correlation is consistent with the idea that hydrolysis of ATP in each myosin cross-bridge attachment and detachment cycle (to and from actin filaments) produces a fixed mechanical impulse. Furthermore, the observation that more force is generated per mole of utilized ATP in repeated contractions of longer duration (5 s vs. 1 s) is consistent with the greater efficiency of longer contractions (e.g., less heat production) observed previously in studies of nonfatigued muscle. Third, Dawson et al. [67,68] studied the relationship between metabolite levels, together with derived parameters such as the free energy (or affinity) for ATP hydrolysis (-dG/d~), and the mechanical relaxation rate following contraction in fatiguing anaerobic muscle. They were able to show that the mechanical relaxation rate was linearly related to measured phosphocreatine and Pi concentrations and -dG/d~, whereas it was nonlinearly related to isometric force developed in the contraction, ATP concentration and the amount of ATP utilized in the contraction. It appeared reasonable to conclude that metabolic factors, rather than independent changes in the activation or deactivation of contraction, determine changes in the mechanical relaxation rate in fatiguing muscle. However, the wealth of information obtained in the study, demonstrating so many relationships between metabolites and relaxation rate, places the interpreter in a quandary: 'Which of these relationships is causal?' [67]. A similar predicament exists in interpretation of data discussed in the previous paragraph. Discernment of critical regulating parameters rests heavily on studies using other techniques. Thus, it was hypothesized [67] that the mechanical relaxation rate may primarily depend on -dG/d~, because this parameter may determine the rate of Ca:÷ uptake (via an ATPase) into the sarcoplasmic reticulum; the in vivo rate of Ca:+ sequestration by the sarcoplasmic reticulum had been previously related to the relaxation rate (see discussion in Ref. 67). These NMR studies allow a direct observation of energy metabolism in working muscle. They reveal the complexity of metabolic regulation, and show that NMR can be used to answer specific questions about a system extensively studied using other techniques.

IVB. Intracellular pH measurements 31p-NMR has been widely used to estimate intracellular pH [8,58,59]. The technique rests on the strong pH dependence of 3~p chemical shifts of phosphates that are present within cells at sufficiently high concentrations for rapid detection by NMR. Pi and, to a lesser extent, sugar phosphates such as glucose 6-phosphate are most commonly used because of their high pK a values (and hence greater pH sensitivity of chemical shift at physiological pH values). Measurement of pH involves relating measured phosphate chemical shifts to corresponding pH values using an appropriate titration curve. The appropriate titration curve is that of the phosphate compound in the intracellular milieu which has to be obtained by careful calibration [57]. Such calibration allows factors other than pH that affect the chemical shifts to be largerly eliminated. This was done in a study of pH inside chromaffin granules [69], which have an unusual composition [70]. From the titration curve of the 7-phosphate of ATP (pK a ~ intragranular pH) present in the isolated granule matrix, the intragranular pH was estimated to be 5.6, with a limit of error of -+0.1. However, because solutions of 'intracellular milieu' of many systems are often impractical or impossible to make, it is necessary to understand the extent to which factors other than pH influence the chemical shift. Analogous problems (together with additional difficulties) are found in calibration of pH microelectrodes, pH-dependent dyes and weak acid or base distribution methods for intracellular pH (see Refs 71 and 72 for reviews). Investigating this problem, we initially observed that the titration curve of Pi in homogenates (undiluted) of plant material [73] is displaced relative to the titration curve of 5 mM potassium phosphate in H20. This result indicates that factors other than pH might be influencing Pi chemical shifts in vivo to a significant extent. The displacements are not extreme, though they indicate that if their contribution to an observed chemical shift cannot be quantitatively accounted for, absolute pH measurements cannot be accurate to within -+0.2 pH units or better. Following this observation, the effects of substances which phosphates could interact with in the cell, viz., proteins, phospholipids and cations- including ionic strength were studied [73]. Acidic proteins (bovine serum albumin, and maize root-tip proteins)

61 and phospholipid dispersions did not detectably influence chemical shifts of Pi and glucose 6.phosphate, perhaps due to their net negative charge at physiological pH. The basis protein protamine did, however, strongly affect chemical shifts of these compounds as do ionic strength (Fig. 3) [22,27] and physiological concentrations of Mg:÷ (Fig. 4) [25]. The Mg2÷ effect could be reversed by addition of ATP or citrate, indicating that metabolites can be ranked with respect to their affinity for Mg2÷. These effects, which have hitherto in general not been appreciated (e.g., see Ref. 58), indicate that the appropriate titration curve of Pi or another phosphate used for intracellular pH determination cannot be chosen without knowledge of intracellular concentrations of substances such as K÷, Mg2÷, ATP, ADP and organic acids. Without such knowledge, uncertainties in pH measurements are of the order of 0.2-0.5 pH units, or even higher (particularly likely in specialized cells which permit a wide range or unusual level of ionic strength of divalent

:7 r I

16 ,

cO

i

I /4!

G6P

:sl

4

5

6

7

8

pH

Fig. 4. Titration curves of Pi and glucose 6-phosphate (G6P) showing the effect of Mg2+ on their respective chemical shifts. '% Pi or glucose 6-phosphate alone; o, Pi or glucose 6-phosphate titrated in the presence of 5 mM MgCI2; -, Pi or glucose 6-phosphate titrated in the presence of 50 mM Mg2÷. Shifts are referenced to methylene diphosphate. Reprinted with permission from Roberts et al. (1981) Biochemistry 20, in the press [73]. Copyright 1981 American Chemical Society.

cation concentration, such as halophilic organisms (see Ref. 57)). Measurements of free intracellular Mg2÷ concentration in various tissues by 31P-NMR [7477] indicate that each tissue must be considered separately. Furthermore, analysis of titration curves in terms of a Henderson-Hasselbalch-type equation, as used by Dawson et al. [27] and Hollis [59], cannot apply in the light of direct effects of Mg2+ on Pi chemical shifts (i.e., effects other than on the pKa of

ck

! /4

G6P

Pi).

/5

/2

4

t7

5

6

7

8

pt-f

Fig. 3. Titration curves of Pi and glucose 6-phosphate (G6P) showing the effect of ionic strength on their respective chemical shifts. ,%Pi or glucose 6-phosphate alone; i, Pi or glucose 6-phosphate titrated in the presence of 0.1 M KC1;i, Pi or glucose 6-phosphate titrated in the presence of 0.5 M K C I . Shifts are referenced to methylene diphosphate. Reprinted with permission from Roberts et al. (1981) Biochemistry 20, in the press [73]. Copyright 1981 American Chemical Society.

These results also indicate that a change in intracellular phosphate chemical shifts can occur without an intracellular pH change occurring. One possible example is the effect of anaerobiosis on tissue: the resultant decrease in ATP levels, with increase in AMP, could result in an increase in free intracellular Mg2÷, that could interact with Pi or sugar phosphates causing their chemical shifts to change. Anaerobic human erythrocytes were shown to contain higher free Mg2+ levels than aerobic ceils [77]. It is apparent that for accurate pH measurements using aIP-NMR, careful evaluation of these complica-

62 tions is needed. Despite these problems, intracellular pH measurements by 3~p-NMR have yielded pH values similar to those obtained using other techniques for E. coli [32] and myocardium [71,78], provided the specimen is maintained in good physiological condition (as discussed in subsection IIIE). In this connection, it has been generally observed (e.g., see Refs. 12, 25, 28 and 34) that tissues under anaerobiosis contain substantially higher levels of Pi, as phosphorylated metabolites such as ATP are depleted; hence, intracellular pH measurements of tissue under 'optimal' conditions may require longer signal averaging compared to tissue stressed, for example, by ischaemia, inadequate perfusion or imposed work. Poole-Wilson [71] noted fundamental differences between the various methods of intracellular pH measurement with respect to their ability to detect and determine pH heterogeneity within cells. Thus, microelectrodes can only give a single pH measurement for each insertion, with little or no indication of variation of pH within a cell (unless large compartments, such as vacuoles in plants, are present). Measurements of pH based on the distribution of weak acids or bases give an intracellular pH value representing an integral of all compartments differing in pH, although comparison of measurements with weak acids and weak bases gives an estimate of the range of pH heterogeneity [71]. The 31p-NMR method will, in contrast, show different lines from phosphate groups in different pH microenvironments if the exchange between the environments is slow. As will be discussed below, in subsection IVC, 31p resonances attributed to separate compartments have been reported. Furthermore, the broad 31p lines seen in living systems are suggestive of microheterogeneity of pH in cells; the problem is complex, however, because other factors (including heterogeneities in the distribution of factors other than H ÷, such as Mga+) can also give rise to line broadening, or multiple, overlapping lines. The importance of these various interactions and factors may differ from one tissue to another and cannot be fully evaluated at present. Shulman and co-workers [32,48] have used 3~p-NMR to measure ApH across the membrane of E. coli under a variety of conditions. For example, addition of glucose to anaerobic cultures resulted in elevated sugar phosphate and nucleotide triphosphate levels, and formation of a large ApH (approx.

1 pH unit); the latter could by inhibited by an ATPase inhibitor (dicyclohexylcarbodiimide, DCCD). Addition of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented formation of nucleotide triphosphates and a ApH. These results are consistent with the chemiosmotic hypothesis - ATP produced by glycolysis being hydrolyzed by a DCCD-sensitive membrane-bound ATPase, thus generating a ApH that can be collapsed by FCCP. In conclusion, it can be stated that 31p-NMR is a useful technique for intracellular pH determination, applicable to many types of active cells and tissues. In addition to being noninvasive, rapid and sensitive (+0.1 pH unit, if appropriate controls are made), the method can in principle give direct information on pH variation within cells.

IVC. Study of compartmentation Compartmentation permits reactions with different, even conflicting, requirements to proceed within a single cell; it also enables reactants in metabolism to be kept apart; other 'functions' of compartmentation can be enumerated. A variety of types of compartment can be identified: the environment within a membrane or enzyme complex, the environment about the charged surfaces of membrane and proteins, and the environment enclosed by a membrane. Cell biologists have had great success in identifying the intracellular sites and properties of enzymatic activities, in determining sites of storage, and in fop lowing the intracellular pathways of large molecules such as ribosomal RNA and secreted proteins from their site of synthesis to final destination. However, knowledge of the distribution of small metabolites, including H +, within cells is more rudimentary, despite extensive information on overall tissue levels [79]. The distribution of metabolites within membrane-bound compartments of cells has been studied by radioactive tracer methods (e.g., Refs 80 and 81), by analysis of uptake or efflux kinetics (e.g., Ref. 82), and by isolation of the organelle followed by chemical analysis (e.g., Refs. 70 and 83). Current issues in the study of compartmentation are discussed in proceedings of two recent symposia [84,85]. Three types of 31p-NMR experiments investigating compartmentation may be distinguished. One kind comprises studies of isolated organelles: NMR has

63 been used as a technique for chemical analysis of organelles, including chromaffin granules from the adrenal medulla [86] and mitochondria from rat liver [87]. alP-NMR has also been used to examine bioenergetic processes in these organelles, since they are known to involve H ÷ movements and so pH changes that can be monitored from measured chemical shifts (see subsection IVB). For example, using the chemical shift of the 7-phosphate of ATP, the internal pH of chromaffin granules (which contain about 0.1 M ATP) was followed during hydrolysis of externally applied MgATP [15,88]. The pH drop that occurred (indicating that the chromaffin granule ATPase is an inwardly directed proton pump) could be followed to within -+0.1 pH unit because the pK a of the T-phosphate is close to the pH values in question, and because careful studies were made to take into account all the possible interactions, other than pH changes, that could influence the "r-phosphate chemical shift [69] (see subsection IVB). A second class of NMR experiments concerns determination of the physical state of metabolites within compartments. Most fully studied has been the binding of 2,3-diphosphoglycerate and ATP to hemoglobin in human erythrocytes. On binding to hemoglobin, 2,3-diphosphoglycerate resonances experience a large downfield shift [8], and by comparing 2,3-diphosphoglycerate chemical shifts observed in vivo with those observed in model solutions (in which binding was directly measured by ultrafiltration) Marshall et al. [89] estimated the amount of 2,3-diphosphoglycerate binding to hemoglobin in human erythrocytes under aerobic and anaerobic conditions. Gupta et al. [90] reported that in model solutions in which ATP or MgATP was known to bind to hemoglobin (as measured by dialysis rates) [91], ATP chemical shifts are not altered. Hence, it is not possible to detect ATP-hemoglobin binding in vivo by comparison of chemical shifts of ATP in vivo with those in model solutions, as attempted by Marshall et al. [89]. A similar lack of a change in ATP chemical shifts on binding to certain enzymes in vitro has been reported [92,93]. However, using dissociation constants for ATP and MgATP-hemoglobin complexes in model solutions [90,91], together with knowledge of ATP levels in vivo and estimates of the level of free frog Mg2÷ in erythrocytes [77], Gupta et al. [90] were able to deduce the proportion of ATP

bound to hemoglobin in aerobic or anaerobic erythrocytes. A similar analysis was applied to the 2,3-diphosphoglycerate-hemoglobin equilibrium [77], and yielded estimates of 2,3-diphosphoglycerate binding in excellent agreement with those of Marshall etal. [89]. 31P-NMR has also been used to show that blood platelet granules contain ATP [94] in a relatively immobilized state. Spectra of intact platelets, in which cytoplasmic ATP had been selectively depleted, did not shown the sharp resonances characteristic of ATP in free solution; such resonances were clearly apparent in perchloric acid extracts of these platelets. (A 19F-NMR study of fluorinated serotonin which had been incorporated into the granules of intact platelets also showed that the motional state of serotonin is restricted [95,96].) The third type of NMR investigations of compartmentation are those that attempt to reveal and study intracellular compartments differing in pH. Because phosphate chemical shifts are sensitive to pH, such compounds, if present in the sample within compartments differing in pH, will show separate resonances for each compartment. However, one must distinguish between studies where spectra showing two clearly resolvable lines are obtained, which can be attributed to a single compound at two different pH values, from studies in which a broad resonance is found in the spectra (often with 'shoulders' apparent) and interpreted as being due to the existence of several overlapping resonances from a compound experiencing a range of pH values. In the former type of study, it is possible to perform physiological experiments in which the two resonances (attributed to separate compartments) can be manipulated independently. For example, 3~p. NMR studies of maize root tips [26] have indicated that Pi resonances from the two major intracellular compartments, viz., cytoplasm and vacuole can be detected (Fig. 5). Depletion of glucose 6-phosphate levels in this tissue during anaerobiosis resulted in an increase in the size of the cytoplasmic Pi resonance, without noticeably affecting the vacuolar Pi resonance; incubation of root tips with 2-deoxyglucose resulted in almost complete elimination of the cytoplasmic Pi resonance (as the resonance attributed to 2-deoxyglucose/6-phosphate increased) while the vacuolar resonance was relatively unchanged in mag-

64

3

3

20 I

'

'

'

0 1

'

'

-20 -qO -60 PPH I

'

'

'

I

•

i

,

I

Fig. 5. An example of the ability of 31P-NMR to distinguish between intracellular compartments. Shown are spectra of maize root tips, with resonances due to glucose 6-phosphate (peak 1) (located in the cytoplasm), cytoplasmic Pi (peak 2) and vacuolar Pi (peak 3). The chemical shifts of the cytoplasmic phosphates indicate a pH of 7.1-7.2, while peak 3 indicates a vacuolar pH of 5.6 -+0.1. B is a spectrum from longer root tips than were used in A, and a larger vacuolar resonance (relative to peaks 1 and 2) is evident; such longer root tips are known to be more vacuolate. Other reasons for these assignments are given by Roberts et al. (1980, unpublished data). Note the overlap of the three peaks, this is discussed in the text. From Roberts et al., unpublished data. nitude [25]. These experiments indicate that the two Pi pools are metabolically separate. Assignment of resonances was made easier by the availability of tissues with differing proportions of the two compartments: resonances attributed to the cytoplasm are relatively smaller in tissue samples containing cells with a smaller proportion of cytoplasm (relative to vacuolar volume), as shown in Fig. 5. Moreover, the fact that the cytoplasmic and vacuolar pH differ so much (pH approx. 7.2 vs. approx. 5.6, respectively) results in maximum separation with respect to the HPO~-~ H2PO?~titration curve (see Figs. 3 and 4). It is commonly observed that intracellular Pi lines are broad, and shoulders may even be apparent, although these are often variable, lessening their apparent significance (e.g., cf. Figs. 1 and 3 in Ref. 50). For muscle, it has been suggested [29,39-41]

that this broad line consists of several overlapping lines due to Pi experiencing different pH values. Support for this hypothesis cannot readily come from physiological experiments. Such experiments as have been attempted seek to perturb the system so that the broad resonance separates into several (usually two) resonances. They suffer from the drawbacks that (a) the perturbing agent results in a large increase in the total intracellular Pi concentration [29,58,97] so that the 'new' resonance(s) seen after perturbation o f the sample may bear no obvious relation to the smaller broader resonance evident before the treatment; (b) the appeareance of some reported spectra before and after the perturbing agent is applied suggests poor reproducibility (cf. Fig. 2 in Ref. 58 and Fig. 1 in Ref. 97). Spectroscopic measurements rather than physiological experiments can in this case provide some information on the origin of the broad Pi lines seen in vivo. This is evident from a consideration of the factors determining line widths in NMR spectra. The width of an observed line in a conventional NMR spectrum is determined by: (a) the spin-spin relaxation time, T2: (b) chemical shift heterogeneity, i.e., 31p nuclei being in different environments (such as pH or magnetic field); and (c) broadening due to exchange of nuclei between two environments (for example, Gadian et al. [23] noted that Mg2+ causes broadening of the 13- and a-phosphate resonances of ATP, due to exchange between MgATP and the small amount of free ATP present, and considered it likely that this process provides an important contribution to ATP line widths in tissue spectra). The respective contributions of these three factors can be estimated by comparison of line widths and T2 measurements of different magnetic field strengths. Thus, if spin-spin relaxation is dominated by chemical shift anisotropy, T2, measured by the CarrPurcell-Meiboom-Gill pulse sequences [17], will be found to decrease in proportion to the square of magnetic field strength; a corresponding increase in line width will be observed, if the line is broadened homogeneously. If 7"2 is found to increase with magnetic field strength, other relaxation mechanisms (such as dipole-dipole relaxation) must be dominant. If chemical shift heterogeneity is responsible for most of the observed line broadening, line width (measured in Hz) will increase linearly with magnetic field strength

65 (after eliminating field-dependent changes in observed line width due to changes in T2, determined as just described, using the relationship AVint = 1/7¢T7~where AVint is the intrinsic line width at half-maximum intensity). This is because resonances from nuclei in different environments are increasingly separated (separation measured in Hz) as field strength is increased. If the observed line width is largely due to chemical-exchange line broadening, observed line widths will decrease with increasing magnetic field strength [19] but the decrease will not match the increase in/'2 predicted from relaxation equations. Pi and phosphocreatine line widths and T2 values have been measured at 120 MHz for rabbit hind leg white muscle [39] and rat vastus lateralis muscle [29], and at 40.3 MHz for frog femoral biceps muscle [41]. Despite the use of different species, we can attempt an interpretation of the data in the light of the considerations above. /'2 values for intracellular Pi and phosphocreatine were similar at both field strengths: 100 ms at 129 MHz, and 80 ms at 40.3 MHz, giving respective intrinsic line widths of 3 and 4 Hz. Intracellular Pi line widths were approx. 40 Hz (at 129 MHz) and !0 Hz (at 40.3 MHz), whereas phosphocreatine linewidths were 15 and 4 Hz, respectively. It may be deduced that the broad Pi line is not a result of rapid relaxation, and that chemical shift anisotropy is not a major relaxation pathway in vivo. The increase in intrinsic line width with increasing field indicates that the line width is not principally a result of chemical-exchange broadening. On the other hand, the almost linear increase in intrinsic line width with magnetic field (approx. 4 vs. 3.2, respectively) is consistent with the view that chemical shift heterogeneity is the cause of the broad resonance observed. If this conclusion is correct, the next problem is to determine what environmental factors are responsible for the heterogeneity. The most obvious candidates include pH and microscopic magnetic field inhomogeneities in the samples. Variation of pH within the sample can occur between cells, between compartments in cells, and within compartments in cells. The first type of variation might largely be due to different physiological states of cells in the sample; careful sample preparation and preservation in the spectrometer should eliminate this contribution. The contribution of pH variation within membrane-bound corn-

partments (e.g., due to nonuniform distribution of H ÷ about the surfaces of membranes and proteins in the compartment, which in general possess a net negative charge) (see Ref. 72 for a discussion) to chemical shift heterogeneity would most readily be determined by study of model systems. Such studies might help account for the observation that 31p-NMR spectra of mitochondrial suspensions [87] show an external Pi line much narrower than that attributed to internal

Pi. The observation that part of the Pi resonance in muscle often has a chemical shift corresponding to unreasonably high pH values (greater than 8) [27] indicates that pH variation in the sample cannot be the sole cause of the observed line widths; magnetic field inhomogeneity is probably also a cause. The line width of phosphocreatine has been used to place a probable upper limit on the contribution of magnetic field inhomogeneity to the observed Pi line width [29]. This is based on the assumption that magnetic field inhomogeneity will produce the same increment in line widths for all resonances, i.e., that the field inhomogeneity occurs on a macroscopic scale of millimeters [29]. However, the existence of large field gradients around particles such as erythrocytes, and within cells containing vesicles or particles, as discussed by Brindle et al. [98], suggests that microscopic field inhomogeneities (on the scale of micrometers) within intracellular compartments might make a significant contribution to chemical shift heterogeneity. This question can be partly resolved by com. parison of T2 measurements using the Carr-PurcellMeiboom-Gill pulse sequence (see Ref. 17) with those obtained using the simple Hahn spin-echo sequence (see Ref. 17), the first sequence gives a value of/'2 independent of line width contributions due to an irthomogeneous field, whereas the Hahn experiment provides an estimate o f / ' 2 that also includes a contribution to the magnetic field gradient experienced by a diffusing molecule (i.e., the gradient within a compartment). If the gradients exist on a microscopic scale within intracellular compartments, T2 estimated by the Hahn experiment will be shorter than that measured by the Carr-PurceU-Meiboom.Gill sequence; whereas, the existence of macroscopic field gradients should not make the two T2 estimates very different, because the restriction of Pi and phosphocreatine within cellular compartments will result in these

66 molecules not experiencing these macroscopic gradients. The Hahn estimate of T2 will not completely determine the contribution of microscopic field heterogeneity to observed line widths, for only gradients across dimensions traversed by the molecules in the pulse interval (approx. 100 ms) will result in a reduction in the measured T2. The question of the causes of broad intracellular lines is important, because unless they are understood and quantified, interpretation in terms of compartments cannot be made without considerable ambiguity. Experiments of the kind suggested may help reduce the uncertainties. Of course, support for an interpretation from in vivo NMR can often be found using other experimental approaches; for example, intracellular pH measurements using weak acids and weak bases (e.g., Ref. 99; see Ref. 71). However, these approaches are also subject to ambiguity.

IVD. Measurement of unidirectional reaction rates in vivo by saturation transfer Measurements of individual reaction rates in vivo are potentially among the most important physiological applications of NMR, since few other noninvasive techniques are suitable for this purpose and most known reaction rates have been derived from invasive experiments. The uncertainties introduced by such procedures as rapid freezing and extraction are considerable and the advantages of rate measurements in an unperturbed tissue or organ fairly obvious. Saturation transfer experiments, introduced by McConnell and Thompson [100] and extended by Forsen and Hoffman [101,102], permit the measurement of unidirectional exchange rates of nuclei between two different magnetic environments. Exchange can occur within the same molecule (e.g., exchange between two conformations) or the exchange can be via a chemical reaction (e.g., the exchange of alp in the reaction ATP + H20 ~ ADP + Pi + H÷)• The range of reaction rates measurable, and the accuracy of the measurements will be discussed following a description of the technique. The two populations of nuclei in exchange must give separate lines in a spectrum for the saturation transfer experiment to be possible. The method has been applied to measurement of rate constants of phosphoryl exchange reactions analyzed as simple equilibria:

kl A - P + B -,~ A + B - P k2

where kl and k 2 are the rate constants for each unidirectional reaction (equal to 1/TA. P and 1/'rB.p, the average lifetimes of A-P and B-P, respectively). In the simplest experiment, the resonance due to A-P is saturated (i.e., the high- and low-energy populations are made equal, so A-P can give no signal) by application of a continuous (except during data acquisition) lowpower radiofrequency field at its resonance frequency. In addition to eliminating the resonance due to A-P, exchange of -P from A to B will result in a reduction in the intensity of B-P line, because the transferred -P retains its spin distribution for a time of the order of the spin-lattice relaxation time T1 hence the term saturation transfer. The decrease in the intensity of the B-P line, on irradiation of the A-P line, can be deduced from the Bloch equations, modified to include chemical exchange [103] (see Refs 28, 104-106 for a more detailed discussion). The equation for the B-P resonance can be written; B B d B M z - M ° + k I M A - k2MBz ~ Mz : _ TB1

(1)

where k I and k2 are rate constants designated above,

T~ is the spin-lattice relaxation of B-P in the absence of exchange, MzB and M A are the instantaneous magnetizations of B-P and A-P, respectively (in this case, when A-P is saturated), and MoB is the magnetization of B-P when it is in thermal equilibrium. In the experiment, saturation of A-P makes M ) = 0, and in the steady state dMBz/dt = 0, hence the above equation reduces to: MB

1

1 + k2 TB provided the magnetizations recover completely between pulses (which occurs after approx. 5. Tp, i.e., a pulse interval of 5 - 1 0 s). More complex treatments cover the case where short pulse intervals (e.g., less than 1 s) are used [105], as is often required in in vivo NMR, in order to obtain spectra with a sufficient signal-to-noise ratio within minutes. Mnz/MBocan be determined experimentally from the ratios of the

67 signals due to B-P in the presence or absence of saturation of A-P (see Fig. 6). Analogous expressions for Kt, when B-P is saturated, can be derived. As noted, T~ must be measured in the absence of exchange, i.e., the in vivo reaction must be inhibited; thus, Brown et al. [107] used to DCCD to inhibit ATPase activity in E. coli cells, so inhibiting observable saturation transfer. An upper limit on the exchange rates that can be measured by the technique can be expressed as R/(VA. P --vB.p)~< 1, VA-p and VB.p being the resonance frequencies of A-P and B-P, respectively, and R being the exchange rate (assuming k~ and k2, i.e., population sizes, are equal) (see Ref. 19). Thus, faster exchange rates can be measured if the chemical shifts of A-P and B-P are large, e.g., on spectrometers of higher frequency. For 3~P-NMR spectra on current instruments the upper limit on values o f k is 50-100 s -1. Below this upper limit, it can be seen by inspection of Eqn. 1 that large values of k2 result in small values of MBz/MB (i.e., large changes in peak height

P~

¢

c

(g-o)

I -5

I 0

I 5

I I0

I 15

I 20

I - 25

|,ppm

Fig. 6. 31P-NMR spectra of aerobic glucose-grownE. coli. The arrows indicate the frequencies of the low-power pulses used in B to saturate t.henucleotide triphosphate (NTP) peak. The peaks labelled pin and pex correspond to intracellular and extracellular Pi, respectively.Peak PP is due to polyphosphate. Peak NTP3, consists of approx. 50% ATP and 50% nonadenine nucleotide triphosphates. NDP# is due to nueleotide diphosphates. The difference spectrum ( A - B) shows transfer of saturation from NTP~.to Pi. From Ref. 107.

with saturation of A-P), hence faster reaction rates can be measured more accurately, all other parameters remaining equal. Furthermore, short T1 values decrease the effect of saturation transfer on peak heights, at a given exchange rate, and so decrease the accuracy of measurements. Eqn. 1 also reveals that as k2 becomes much smaller than T~, the effect of saturation of A-P on MBz/MBo diminishes. This places a lower limit on the values of k2 that can be measured, which is approx. 0.1 s -t if TtB is approx. 1 s. In addition to the signal-to-noise ratio and the absolute values of k2 and T~, error in T~ measurement also influences the accuracy with which k2 can be measured. Usually, at least some assumptions have to be made to derive a value of T B, in the absence of exchange [107,108], so that the values used are not usually accurate to better than 0.1 s. Using saturation transfer, DCCD-sensitive ATPase kinetics in E. coli were followed [107,28] (see Fig. 6). The rate of the reaction ATP ~ Pi + ADP was determined to be 20 -+ 10 s "l, and that of the reaction Pi ~ ATP 0.6 -+ 0.15 s-t; these values were consistent with the steady-st~[te concentration ratio of Pi : ATP of 10 _+5. In studies of human erythrocytes [108], no transfer of saturation between Pi and ATP, or between ATP and 2,3-diphosphoglycerate was observed. Instead, a sizeable transfer of saturation from the {3- to the -r-phosphate of ATP was observed; this was ascribed to the action ofadenylate kinase. Investigation of saturation transfer between ATP and phosphocreatine in resting frog muscle [106] also provided exchange rates consistent with the observed ratio of ATP and phosphocreatine concentrations. In these muscles ATP levels remain constant during long contractions [27], while those of phosphocreatine decline. It is therefore clear that changes in the reaction rates A T P ~ phosphocreatine relative to the steady-state condition are necessary. A preliminary report [105] indicated that reduction of the back reaction rate (ATP--.phosphocreatine) serves to maintain ATP levels during contraction. In contrast to the experiments on frog muscle and E. coli, studies of saturation transfer between phosphocreatine and ATP in beating rat hearts [105,106] indicated that the ratio of the forward to backward rates for phosphocreatine ~ ATP phosphate transfer differed from the observed concentration ratio by more than a factor of three, indicating that the simple

68 two-species exchange model does not apply. A study of rabbit hearts [109] produced a similar result, and it was suggested that intracellular compartmentation of ATP could be the cause of the apparent complexity. However, the observation of large changes in ATP and phosphocreatine levels in beating rat hearts [ 11] (see subsection IIID) indicates that the system is not in a steady state, so that analysis of saturation transfer in the manner described above is not appropriate: concentrations, and forward and backward reaction rates are constantly changing during the cardiac cycle. The observation of steady state in hearts subjected to KC1 arrest [109] supports this explanation. Despite the small number of studies carried out using saturation transfer, it is clear that the technique has the potential for studying metabolic control in vivo in greater detail than was hitherto possible. The major limitations of the technique are that it is applicable only over a narrow range of reaction rates and can only be used to study systems in a steady state. Variants of this technique, such as inversion transfer [110] (see Ref. 111), exist, and may be useful for measurement of exchange rates in particular situations, though none have yet been applied to living systems.

of label in the same molecule from ]aC-laC couplings (resulting in multiplets) (e.g., Ref. 21). Thus, 13CNMR combines some of the analytic capabilities of radioisotope tracer methods and 13C-feeding experiments analyzed by gas chromatography-mass spectrometry, in addition to the advantage of being noninvasive. It has been reported [117] that 13C sensitivity is 2-3-times that of a~p, at the same magnetic field strength, due to narrower line widths, despite the fact that the 13C nuclear moment is about one-fourth that of 31p. The larger range of chemical shifts of 13C metabolites, relative to IH or alp, also permits larger numbers of metabolites to be simultaneously resolved

1[~6

LOC W • =C~

AA

AIO C8 * GIu Ca

GIU (~2 tics I

V. 13C.NMR in vivo

In contrast to ~H and alp, the abundance of 13C is low (1.1% of the total C) rendering in vivo-naturalabundance 1aC.NMR possible only in limited circumstances, such as detection of very concentrated materials present in plant seeds [112], or where accumulation of data can take place over many hours as with slowly metabolizing yeast cells [113]. Thus, most in vivo 13C-NMR investigations require isotopic enrichment by the administration of a 13C-labelled substrate before [7,16,114-116] or during [7,21, 117-119] the NMR experiment. These feeding experiments allow specific metabolic sequences to be investigated, in contrast to what is generally possible using ~H or 31p. And, in contrast to techniques using radioisotopes, 13C-NMR usually provides immediate information on the distribution of label within the various metabolites without elaborate degradative procedures. It can also give details of the distribution

/3-HB

, AfQ I C3 I

8

(el

6 / ~J./tt/12

do

do @,ppm

Fig. 7. 1 3 C - N M R spectra from a pcrfuscd mouse liver at 35°C. (c) 13C natural-abundance background of this liver, accumulated before the substrate was added. The substrate, 8 m M [3-13C]alanine and 20 m M unlabelled ethanol, was then added at 0 rain and again at 120 rain, and a seriesof 13C.NM R spectra were taken. Co) Spectrum measured during the period 150-180 rnin. (a) 13C.NM R spectrum of the perfusatc after the perfusion was terminated, at 240 rain; this spectrum consisted of 5000 scans. The pulse repetition times were 0.5 s for b and c and 2 s for a. Abbreviations: tiC1, ~ I , flC3,5, tiC2, c~C3, ~2,S, aC4, tiC6 and aC6, the carbons of the glucose anomers; Gly C2, glutamate C-2; Gin C2, glutamine C-2; Asp C2, aspartate C-2; AIa2C ,alanine C-2; Lac C 3 , lactate C-3; CB, cell background peak; W, X, Y and Z, unknowns; A A Ca, acetoacctate C H 2 ;and fl-HB Cc~,fl-hydroxybutyrate C H 2 .From Ref. 119.

69 in in vivo ~3C-NMR spectra (see Fig. 7), in contrast to 31P-NMR spectra of cells in which it is often not possible to resolve many resonances because of the smaller chemical shift range and large line widhts; indenti-. fication of metabolites in such cases may be difficult and requires examination of perchloric acid-extract spectra, which have much narrower lines (see, for example, Ref. 52). VA. Examination o f metabolite fluxes and interconversions Here we highlight aspects of the extensive 13CNMR experiments of Shulman and co-workers [ 117, 118], concerning glycolysis in microbial cell suspensions, gluconeogenesis in rat liver cells [21], and alanine metabolism in perfused mouse liver [119]. Some of this work has been reviewed elsewhere [28, 63,120]. When anaerobic cultures of E. coli were fed [ 1-13C] glucose, 13C.NMR spectra of sufficient signalto-noise ratio were obtained for changes in levels of certain metabolites to be determined with a time resolution of 1 min [117]. Spectra revealed the distribution of ~3C label in the fructose 1,6-bisphosphate signal, which was interpreted in terms of the relative rates in the aldolase 'triangle' (see Fig. 8) [63]. It was argued that if the forward flux of carbon through fructose 1,6-bisphosphate and glyceraldehyde 3-phosphate is much faster than the flux in the reverse direction, very little 13C at the C-1 position initially present in glucose (and therefore expected to flow into the C-1 position of fructose 1,6-bisphosphate) will be 'scrambled' into the C-6 position of fructose 1,6-bisphosphate. If, however, the forward and backward fluxes through aldolase are similar, l aC initially present in the C-1 position of fructose 1,6-bisphosphate can be expected to be equi!ibrated between both the C-1 and C-6 positions. In anaerobic E. coli cultures [ 117], little or no scrambling between the C-1 and C-6 positions was observed when [1-1aC]or [6-1aC] glucose was fed, in contrast to similar experiments with anaerobic yeast cultures [118], in which almost complete equilibration between the C-1 and C-6 positions was apparent. These apparent differences between the in vivo activities of aldolase and triosephosphate isomerase relative to glycolytic rates in yeast as compared to E. coli are evident despite comparable glycolytic rates. The reasons for this dis-

kf 6 CH~OPO32-

l

L,~O~H2OPO~-I

Fru-P,

5kH HO ~ 2 H ~ O H

OH

k=

H

ka

Dihydroxyacetone phosphate

1 iH2OPO32-

Glyceraldehyde 3-phosphate

k3

2 C-~-O

I

3 CH2OH

Aldolase

4 HC=OI =

k.

5 HCOH 6

I

CH2OPO3=-

TPI

Fig. 8. Reactions catalyzed by aldolaseand triosephosphate isomerase (TPI); Fru-P2, fructose 1,6-bisphosphate. From Ref. 118.

crepancy, however, are not clear. Among the findings in a study of laC.labelled alanine and ethanol metabolism in perfused livers from starved mice [119] was the elegant demonstration of the point of entry of the alanine label into the Krebs cycle in the presence or absence of ethanol. As illustrated in Fig. 9, the position of laC label in glutamate derived from [3-1aC]alanine is determined by whether pyruvate (derived from alanine) enters the Krebs cycle as oxaioacetate or acetyl-CoA; the former gives rise to glutamate labelled in the C-2 and C-3 positions, while the latter generates [4-1aC]glutamate. When [2 -13C]alanine is supplied to liver, glutamate is labelled in the C-2, C-3 and C-4 positions, indicating appreciable flux through both pathways. Addition of unlabelled ethanol ([2-13C]alanine still present) resulted in an absence of [4-1aC]glutamate, indicating that aianine was entering the Krebs cycle only via pyruvate carboxylase. If [2-13C]ethanol was substituted for the unlabelled ethanol [4-13C]gluta-

70

H 13CH3-i-OH

3C-~-COOH -------~13CH3-C-COOH NH2 Pyruvate

Alanine

~ CO2

~Co

Ethanol A-SH

¢

¢ HOOC-13CH2-~-COOH

Oxaloacetate f

Aeetyl CoA

~,I~CoA-Slt bH

HOOC-13CH2-q:-CH2-COOH COOH Citrate [2-13C]-Glutamate (some[3-13C]-Glutamate

13CH3-~-S-Co A

I

AcetylCoA

~

Oxaloacetate

~ " ' - ~ CoA-SH HI HOOC-CH2-!- 3CH2-COOH

COOHCitrate [4-13Cl-Glutamate

is formed due to scrambling, attributed to maIate dehydrogenase and fumarase activity).

Fig. 9. The flow of carbon from L-[3AaC]alanine and [213C]ethano1 to glutamate via the Krebs cycle.

mine did appear, indicating that ethanol replaces alanine as the dominant source of acetyl-CoA. This effect of ethanol was attributed to the previously known conversion of pyruvate dehydrogenase (pyruvate ~ acetyl-CoA) to an inactive form in the presence of NADH and acetyl-CoA, both being produced by ethanol oxidation. The activation of pyruvate carboxylase by acetyl-CoA was also considered to be a possible factor controlling changes in alanine metabolism on addition of ethanol. These experiments show that 13C-NMR can provide detailed information on particular aspects of cell metabolism. These aspects differ somewhat from those that can be studied by 3~p. and ~H-NMR, and in some instances complementary studies using two isotopes may be possible (e.g., Refs. 28 and 121). Variants of experiments of the type just described can be used to study different metabolic pathways.

VB. Intracellular pH measurements by ~3C-NMR To date, no intracellular pH estimates from measured chemical shifts of intracellular ~3C resonances have been reported. It was suggested [64] that the chemical shift of H l 3CO~/13CO]- would be a feasible monitor for intracellular pH, these species being in rapid exchange so that at any given pH only one line is seen (as with 31P-NMR of the H2POJHPO ]equilibrium). However, the high pK a of HCO~ results in little sensitivity of HCOS/CO]- shifts over the pH range 7 - 8 (less than 0.2 ppm). Given the great sensitivity of the I aC nucleus to its chemical environment, compounds with suitable pK a values (such as certain amino acids) could be sensitive indicators of intracellular pH, but have not yet been used for this purpose. Carbonic anhydrase, in vitro, catalyzes rapid exchange between CO2 and HCO~, causing the ~3C resonances of these species to collapse to a single chemical-exchange-averaged line [122]. If carbonic anhydrase present in cells were to catalyze such rapid exchange, the resulting resonance would be a sensitive indicator of intracellular pH. However, on addition of 13CO2 to whole blood suspensions [116] and maize root tips (Roberts et al., unpublished observations), sharp resonances assigned to HCO~ and CO2 were identified, with no intermediate resonance apparent. Thus, it would appear that even in cells containing large amounts of carbonic anhydrase, such as erythrocytes, the exchange rate between CO2 and HCOa is slower than in aqueous, homogeneous solutions of this enzyme.

VI. IH-NMR in vivo In vivo ~H-NMR studies appeared after 31p. and 13C-NMR work because IH resonances from metabolites of which the concentration is low compared to 55 M H20 cannot be readily detected in the conventional one-pulse FT-NMR experiment. The H20 signal-to-noise ratio contained in a single free induction decay is several thousand at 100 MHz (being even greater at higher fields strengths), whereas for solute protons this ratio is usually less than one. Present day signal detection and data processing systems cannot separate the latter in a reasonable amount of time. This is the so-called 'dynamic range problem'. There are four ways to avoid this problem (for reviews see

71 Refs. 123 and 124), and three have already been applied to living systems. The simplest approach is to exchange most of the H20 present in the material with 2H20 [10,22,125, 126] which reduces the H20 signal by a factor of several hundred. Alternatively, the large H20 signal can be selectively eliminated by destroying most of the solvent magnetization at the time the conventional 90 ° observation pulse is applied [22,121]; this is the socalled 'WEFT' (water-elimination FT) [ 127]. A third approach is correlation NMR spectroscopy ('rapid scan NMR') [128,129], which allows solute proton resonances not near the H20 resonance to be detected without observing the H20 peak. Using this technique, in vivo concentrations of organic acids (which have chemical shifts approx. 2 ppm or more from H20) above 0.1 mM have been quantified with a 5 min acquisition time [130]. Thus, sensitivity is a full order of magnitude greater than for in vivo studies of 31p resonances (e.g., Ref. 28). Finally, the Redfield technique [ 1 3 1 - 1 3 3 ] - a low-power, long-pulse sequence-enables 1H resonances as near as 100 Hz from the H20 resonance to be detected [132]. In the Redfield experiment, the amplitude of the radiofrequenct pulse (centered around the region of interest) is adjusted so that the magnetization vector associated with the H20 resonance returns to its original direction at the end of the pulse [134], and the H20 signal is absent from the free induction decay. The technique, widely used for studies of exchangeable protons in solutions of macromolecules, has not yet been used in vivo. The above discussion indicates that a variety of more complex NMR experiments can be used to obtain 1H-NMR spectra of living specimens, compared to most laC- and 31P-NMR studies which invariably use a simple one-pulse FT-NMR technique. Although these various techniques apparently generate comparable results- enabling quantitation of levels of small, mobile metabolites - t h e y do differ considerably with respect to important practical aspects such as sensitivity, how close to the H20 resonance 1H signals can be observed, flatness and stability of the baseline, and observation of the entire or only part of the 1H-NMR spectrum. Consequently, the choice of technique largerly depends on the problem and sample to be studied; many spectrometers are now capable of performing all of the above experiments.

VIA. Quantitation of metabolite levels Using the method of correlation NMR, Ogino and co-workers [130,135] have followed concentrations of ethanol, lactate, pyruvate and other organic acids in anaerobic cultures of E. coli, from spectra as in Fig. 10. By feeding these cultures with [1-1aC]glucose, they were able to follow glucose catabolism via glycolysis separately from glucose catabolism via the pentose monophosphate shunt [130]. This was possible because organic acids derived from the latter ~C

TIME(hour) 7:15

E 8

5:45

33

0

,I

~

2:15 ~ , . ~ . , ~ . p ~ 1:00

ppm

Fig. 10. The 100 MHz correlation IH-NMR spectra of a suspension of E. coli cells grown in an M9 medium with glucose as sole carbon source. E. coli, 0.5 • 109/ml; glucose, 0.1 M; pH 7.2, 3 0 ~ . Incubation times (under anaerobic conditions) after inoculation are given in hours. Each spectrum was accumulated for 5 min in the correlation mode. Chemical shifts are given in ppm from external DSS. Assignments: A, succinate; B, pyruvate; C, acetate; D, lactate; E, ethanol (see text). Reprinted with permission from Ogino et al. (1978) Biochemistry 17, 4 7 4 2 - 4 7 4 5 [135]. Copyright 1978 American Chemical Society.

72 pathway only contain ~2C, since the 1 3 C label is lost as 13CO2 in the phosphogluconate dehydrogenase step. On the other hand, in the glycolytic pathway the label is transferred to organic acids and ethanol, resulting in some IH lines being split, due to 1H-~3C coupling. In ~H-NMR studies of cells and tissues such as adrenal glands [10], octopus salivary glands [125] and erythrocytes [22], conventional one-pulse FTNMR spectra were dominated by broad resonances (principally from proteins) that obscured the sharp lines from mobile, small metabolites. Daniels et al. [10,125] and Brown et al. [22] used two-pulse 'spinecho' experiments, originally devised by Hahn (see Ref. 17) to eliminate selectively the broad resonances. This technique makes use of the fact that line widths in an NMR spectrum are determined in part by the magnitude of T2 (the spin-spin relaxation time), according to the relationship A/;in t = 1/nT2 where Avin t is the intrinsic line width at half-maximum intensity. The signal (or 'echo') following a 90°-r 180°-T pulse sequence, r being the interval between pulses (see Ref. 17), contains a greater contribution from nuclei with long T2 values, relative to nuclei with shorter T2 values, as ~- is lengthened. Therefore, at appropriate values of r, resonances giving rise to narrow lines can be observed alone (see Fig. 11). This spin-echo method permits metabolites present at approximately millimolar concentrations to be quantitated in about 5 min [22], i.e., a sensitivity about 10-times lower than that obtained using correlation NMR [130]. T2 selection (i.e., elimination of broad lines from spectra) can also be performed by suitable processing of a conventional one-pulse FT-NMR experiment [136]. Spin-echo spectra of erythrocytes contained resonances assigned to lactate, pyruvate, glutathione and certain hemoglobin residues [22], and these have been followed in cells subjected to various treatments [22,121,126]. For example, the regeneration of reduced glutathione was observed in cells that had been treated with the oxidizing agent t-butyl hydroperoxide [22]; the rate of regeneration provides an estimation of the rate of metabolite flux through the pentose phosphate pathway in these cells [ 121 ].

VIB. Intracellular pH measurements by IH-NMR In their spin-echo studies of erythrocytes, Brown

fl

w

Fig. 11. Elimination of broad lines using a Hahn spin-echo sequence. 270 MHz 1H-NMR spectra of glucose-depletedred cells at 37°C. (a) Normal FT spectrum. (b) Spectrum obtained using a 90°-'r-180°-r spin-echo sequence, with r = 20 ms. (c) Spin-echo spectrum with r = 60 ms. For assignments of lines in spectrum c, see Fig. 2a. From Ref. 22. et al. [22] detected 1H resonances assigned to [2~3C]histidine residues of hemoglobin. Previously published [ 137] titration curves of the histidines of hemoglobin solutions indicated that these histidine resonances detected in vivo correspond to an intracellular pH of 7.4. It is not clear if factors present in erythrocytes other than pH could influence the observed chemical shifts and so the accuracy of this measurement is not known. The pH sensitivity of chemical shifts of other meabolites, such as organic acids [135,138], might also be useful indicators of intracellular pH in some systems.

VIC. Membrane transport studied by 1H-NMR Two pulse spin-echo NMR experiments have been widely used to measure movement of molecules across membranes. Most studies consist of adding a

73 paramagnetic ion, such as Mn2÷ (Brindle et al. [98] reported that Dy3÷-diethylenetriaminepentaacetic acid is effective at significantly lower concentrations), to the extracellular medium so that the apparent T2 values (i.e., /'2 values not corrected for diffusion effects) of extracellular molecules are made much shorter than the corresponding intracellular molecules [139]. A Hahn spin-echo sequence applied to such a system can then be used to detect principally the intracellular resonances, because it can selectively eliminate the signal due to nuclei with short apparent T2 values (as described in subsection VIA). Water exchange between erythrocytes and plasma [140,141], a rapid process (half-time for exchange less than 10 ms), has been studied extensively using spin-echo techniques. Here, the intensity of the H20 signal is plotted as a function of the interval between pulses, r, in the 90°-T-180°-r echo experiment (see Ref. 17). At first, as r increases the signal (echo) intensity declines sharply, as the signal from extracellular water is eliminated. With further increase in r the decline in echo intensity becomes essentially linear; the slope of this part of the curve is determined by the water exchange t i m e - for the echo decays much more slowly (more than 10-fold) as r increases in the absence of extracellular Mn 2÷. The technique yields water-exchange times in good agreement with those of most alternative techniques [142]. Applications to other cells have been few; Stout et al. [143-145] have attempted to use the technique to estimate water permeability in a variety of plants, but have encountered uncertainties because of apparent cell wall-Mn 2÷ interactions (perhaps generating extracellular magnetic field gradients very different from those around erythrocytes) and problems due to the use of multicellular material. A study of plant cell protoplasts, isolated cells (including the cell wall) and pieces of tissue , from a single source, might make it possible to understand such complications. Evaluation of the data, as Stout et al. [145] discuss, is further made more difficult because of an absence of reliable determinations of water-exchange times using other techniques. Such an experience is common in in vivo NMR studies: NMR findings obtained can almost always be more easily interpreted on systems which have been extensively studied using other techniques (data collection by NMR, however, being generally much faster, and technically easier). In fact, so far,

very few in rive applications of NMR have been reported in areas or on systems where other techniques have failed. Molecules that are transported more slowly (time scale of equilibration in minutes) across erythrocyte membranes have been followed by plotting the FT of a spin-echo free induction decay, at r values where the echo amplitude from an intracellular molecule exceeds that for an extracellular molecule, as demonstrated by Brindle et al. [98]. These workers measured lactate and alanine influx into these cells as a function of time, and obtained influx rates in agreement with those determined previously using other techniques. Advantages of this spin-echo method are its relative technical ease together with the speed with which measurements are possible: a complete uptake experiment can be performed on a single sample of cells that require no manipulation after the experi. ment is initiated. The principal disadvantage is the inherent insensitivity of NMR, so that higher concentrations of compounds (1 mM or greater) are required than may sometimes be desirable. Other two-pulse techniques have been used to study membrane transport, including estimation of water.exchange rates by T1 relaxation measurements [142,146] and by pulse-gradient NMR [147,148]. Adistinct advantage of the pulse-gradient NMR technique is that it does not require the use of paramagnetic ions. These two-pulse experiments could be applied to follow transport of any molecule containing a nucleus that can be detected by NMR. Acknowledgements This research work was supported by National Science Foundation Grants PCM7809230, PCM7807930 and GP23633, and National Institutes of Health Grant RR00711. References

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