13 C NMR Studies of Intact Cells and Tissue

Raymond S. NortonRoche Research Institute of Marine Pharmacology

Dee Why, N.S.W., Australia

I. INTRODUCTION 29II. THEORETICAL CONSIDERATIONS 30III. BIOLOGICAL MEMBRANES 34IV. NATURAL ABUNDANCE 1JC NMR OF INTACT TISSUE 36

A. Terrestrial Plants 36B. Marine and Halophilic Organisms 38C. Other Systems 41

V. STUDIES EMPLOYING "C LABELLING 41VI. CONCLUDING REMARKS 44References > 46

I. INTRODUCTION

Nuclear magnetic resonance (NMR) has becomefirmly established as a powerful biochemical toolduring the past decade due to its ability to providedetailed information about biologically importantmolecules in solution. Recently, its influence hasspread to other areas of biology following recogni-tion of the fact that many of these molecules can alsobe studied directly in living cells and tissue by NMRspectroscopy.

This review focuses attention on studies of thistype which have employed 13C NMR spectroscopy,either alone or in conjunction with NMR studies ofother nuclei. Its principal aim is to illustrate the widerange of problems which can be examined by thistechnique. The topic will be interpreted somewhatliberally to include studies on organelles and otherintact subcellular preparations where the principlesemployed have been similar to those applied instudies of whole cells or tissue.

The term "intact" implies that the sample retainsits biological integrity throughout the experiment.This has not always been demonstrated in thestudies to be described herein, although it must beacknowledged that in general the rigour of the

criteria applied in establishing biological com-petence will depend to some extent on the type of in-formation being sought. It is to be hoped that infuture studies greater attention will be given to thestate of the sample, and hence to the biologicalsignificance of the NMR results.

Because of the low natural abundance of the 13Cnucleus (1.1%) and its low sensitivity in an NMR ex-periment (0.016 relative to the same number of 'Hnuclei, 0.24 relative to 31P), "C NMR studies atnatural abundance require significantly longer ac-cumulation times than 'H or 31P spectra. For thisreason, natural-abundance "C NMR studies of intacttissue have so far been limited to systems which arerelatively stable as a function of time. In order tostudy more rapid biochemical or physiological pro-cesses it has been necessary to resort to 13C enrich-ment. Even then, spectrometers of high magneticfield strengh are required to follow events on thetime scale of minutes.

The incentives to overcome the limitations impos-ed by low sensitivity are, however, quite significant.13C NMR spectra are very well resolved because achemical shift range of approximately 200 ppm isobserved for most biologically interestingmolecules (compared with about 10 ppm for 'H and35 ppm for 31P) and all resonances appear as singletsin the presence of complete proton decoupling dueto the lack of homonuclear coupling. "C has a spin of1/2, so that quadrupolar effects are not observed. Fur-thermore, I3C spin-relaxation parameters are readilyinterpreted in terms of molecular motion. Finally,although the low natural abundance of 13C createscertain problems, there is the possibility of using 13Cenrichment to study biosynthesis and metabolism ofspecific compounds without background in-terference.

Vol.3, No. 1Duplication of Bulletin of Magnetic Resonance, in whole or in part by any means for any purposes is illegal.

29

Indeed, the first studies of living systems by UCNMR were carried out with the aid of 13C enrichment.Matwiyoff and Needham (1) found that 13C NMR spec-tra of whole blood samples treated with 13CO, 13CO2,and 13CN~ ion contained resonances from thesespecies in the intact blood cells, thus demonstratingthe feasibility of observing intracellular constituentsin situ by 13C NMR spectroscopy. Subsequently,Matwiyoff and co-workers (2) examined whole cellsof the yeast Candida utilis grown on acetic acid ran-domly enriched to 20% with C3C. The 13C NMR spectraof these cells contained many resolved resonancesfrom amino acids, carbohydrates and lipids. Themetabolism of [1-'3C] glucose by intact C. utilis cellswas also examined. The label from C-1 of glucose ap-peared in C-2 of ethanol, as expected, and subse-quently in another unidentified compound, possiblyglucose-6-phosphate. From this work it was clearthat many intracellular metabolites were sufficientlymobile to yield sharp, resolved resonances in vivo,at least for C. utilis cells. The corollary (2,3) that itshould be possible to observe the metabolism oflabelled substrates in whole cells was also shown tohold true.

In spite of the considerable promise demonstratedby this initial work, relatively few studies of intacttissue were carried out during the next few years.This probably reflects the fact that adequate in-strumentation was not available in many centres.Furthermore, UC NMR spectroscopists at the timewere still taking the first steps toward characteriza-tion of biopolymers and their building blocks (4), sothat sufficient experimental and theoretical data tointerpret experiments on intact tissue had in manycases not yet been acquired. Nevertheless, London,Matwiyoff, and co-workers (5,6) continued to ex-amine intact cells labelled with 13C during growth,and Schaefer et al (7) analysed the metabolism oflipids and saccharides in soybeans photosyn-thetically enriched with 13CO2. During this periodthere was a dramatic increase in the use of 13C labell-ing to elucidate biosynthetic pathways to natural pro-ducts (8,9), but these studies were not carried out onintact tissue. It is only in the past few years that in-terest in the use of 13C NMR to follow metabolic andbiosynthetic reactions in vivo has regeneratedsignificantly. This renewed interest probably owes agood deal to the outstanding success of recent 31PNMR studies of intact cells and tissue (10-12), cou-pled with the realization that metabolites not contain-ing phosphorus must also be studied in order to gaina full understanding of cellular biochemistry andphysiology. In addition, NMR instrumentation hasimproved significantly during the past few years,

with the availability of spectrometers of highmagnetic field strength equipped with quadraturedetection and probes designed to accept largediameter sample tubes. The improved sensitivity andversatility of these instruments has opened the wayfor a number of NMR experiments on biologicalsamples which could not have been carried out evenfive years ago.

In the following sections we will consider some ofthe factors which affect I3C NMR spectra of tissueconstituents, then examine a number of applicationsof 13C NMR both at the natural abundance level andfollowing 13C enrichment.

II. THEORETICAL CONSIDERATIONS13C NMR spectra of intact tissue are usually.obtain-

ed using techniques employed in conventional NMRstudies of molecules in solution. Under these condi-tions only the relatively mobile tissue constituentsare observed. In order to understand why this is so,it is necessary to consider in brief the phenomenonof I3C spin relaxation. This leads to a discussion ofhow 13C spin relaxation parameters may be inter-preted in terms of molecular motions. The subjectwill, however, be covered very briefly; a full descrip-tion of the phenomenon of nuclear spin relaxationand the derivation of the relevant equations aregiven by Abragam (13).

Many interactions can contribute to I3C spin relax-ation (13-15). However, in diamagnetic molecules,most protonated carbons relax by 13C-'H dipole-dipole interactions with their directly bondedhydrogens (13-17). Therefore, it is appropriate toconsider the equations which describe these in-teractions. The simplest case is that of a '3C nucleusin an isotropic rigid rotor, for which the spin-latticerelaxation time T, is given by

r, To2 V 2,. 2 (D

where yc and yH are the gyromagnetic ratios of I3Cand 'H, respectively, r is the distance between the13C nucleus and'H, and x is given by

( C O W - O V ) 2 T R2

6TR (2)

Here wc and coH are the resonance frequencies in ra-dians sec1 of 13C and 'H, respectively, and TR is thecorrelation time for overall rotational reorientation.

30 Bulletin of Magnetic Resonance

3 _

Figure 1. Plots of 7", (in sec), W(in Hz), andNOE VSTR (in sec) for a "C spin relaxing by adipolar interaction with a single proton 1.10 A NOEaway in a molecule which behaves as anisotropic rigid rotor. Magnetic field strengthis 47 kG (corresponding to 'H and 13Cresonance frequencies of 200 and 50.3 MHz,respectively). Plots for T, and Ware log-log,and for NOE semi-log. The dashed lines in-dicate experimental TR values for (from left toright) glycine (18), AMP (19) and humanhemoglobin (17, 20).

2 .

1 .

10

The corresponding expression for the spin-spinrelaxation time (7"2) is

T2 20X+4T« +

6TR (3)

The natural linewidth W of a I3C resonance is equalto {nT2)-'. Finally, the nuclear Overhauser enhance-ment (NOE), which is the ratio of the intensity of a 13Cresonance under conditions of complete protondecoupling to its intensity in the absence of decou-pling, is given by

NOE = 1 + —YX

—YcX

6TR

(CO H +CO C ) 2 T R2 (4)

For a protonated carbon, relaxation is dominatedby interactions with the directly-bonded hydrogens.The dependence of 7",, W, and NOE of a methinecarbon (rCH = 1.10 A) on rR at a magnetic fieldstrength of 47 kG (corresponding to a lH resonancefrequency of 200 MHz) is illustrated in Figure 1.Typical TR values are shown for glycine (18), AMP(19), and human hemoglobin (17,20). Figure 1 showsthat 7", is long for small molecules in non-viscousmedia and for solids, but passes through a minimumwhen molecular motions have the same frequencyas the observation frequency. The linewidth in-creases monotonically with rotational correlationtime, while the NOE decreases from 2.988 in mobilemolecu les to 1.153 in s low ly movingmacromolecules. Therefore, while resonances from

protonated carbons in small, mobile tissue consti-tuents (e.g., free amino acids) are sharp and havethe full NOE, resonances from immobilisedmolecules (e.g., structural proteins) are broad andhave minimal NOE. Thus, the former class ofmolecules tends to dominate conventional NMRspectra of most tissues.

The relaxation of quaternary carbons is usuallydominated by 13C-'H dipolar interactions with non-bonded protons at low magnetic field strengths (15-17). Because C-H distances are greater for quater-nary carbons this relaxation mechanism is much lessefficient for these carbons. Thus, althoughresonances from quaternary carbon atoms aresharper than those from protonated carbons (for thesame TK and Ho), Tx values are much longer, so theformer resonances are more prone to saturation dur-ing repetitive pulse excitation.

A further consequence of the weaker 13C-'Hdipolar interactions experienced by quaternary car-bons is that other relaxation mechanisms may com-pete more effectively with this mechanism, therebyreducing the NOE of these resonances (the max-imum NOE at any value of TR is observed only whenspin-lattice relaxation occurs exclusively via 13C-'Hdipolar interactions). This situation is likely to obtainat high magnetic field strengths, where thechemical-shift-anisotropy (CSA) relaxationmechanism (13,14) becomes s igni f icant .Measurements at a magnetic field strength of 63 kG(equivalent to a 'H resonance frequency of 270 MHz)

Vol.3, No. 1 31

have shown that CSA dominates the relaxation ofquaternary aromatic, olefinic, and carbonyl carbons(21,22). Because the contribution of the CSAmechanism to spin relaxation times increases in pro-portion to the square of the magnetic field strength(13,14), this mechanism may be expected todominate the relaxation of quaternary carbons forwhich the anisotropy of the shielding tensor isgreater than 100 ppm in studies carried out at highmagnetic fields. The only compensation for the lossof NOE and increase in linewidth accompanying theCSA contribution is a reduction in the 7, value(21,22). The relaxation of olefinic and aromaticprotonated carbons will be affected slightly by CSAinteractions at magnetic field strengths in the range70-100 kG. Aliphatic carbons will be less seriously af-fected because their shielding tensors are muchsmaller (less than 25 ppm). As above, quaternaryaliphatic carbons will begin to be affected at lowermagnetic field strengths than will correspondingprotonated carbons. To summarise, while quater-nary carbon resonances will in some cases be morereadily observed than protonated carbonresonances, greater caution must be exercised in in-terpreting their relaxation behaviour.

For those carbons fully relaxed by 13C-'H dipolarinteractions, 7,, W, and NOE measurements canprovide information on molecular motions. As shownin Figure 1, W increases monotonically with TK,whereas 7, exhibits a biphasic dependence on T«.Therefore W is, in principle, the more usefulparameter for determination of TR. In practice,however, accurate measurement of W can be dif-ficult. For short TK values (less than 0.1 nsec) the con-tribution of "C-'H dipolar interactions to the ex-perimental linewidth is less than contributions fromfactors such as magnet inhomogeneity, exponentialbroadening, and limited digital resolution, while forlong TR (greater than 20 nsec) peak overlap preventsaccurate linewidth measurements (unless the spec-trum is very simple or specific "C enrichment isemployed). Peak broadening due to compartmenta-tion in intact tissue (23) or to exchange between twoor more sites in the tissue (24) can further complicateinterpretation of linewidth measurements.Therefore, 7, measurements are usually preferable,although they are more time-consuming. Each 7,value yields two values for TR from Equation (1) (seeFigure 1), However, it is usually straightforward todecide which TR value is the correct one byestimating Wor NOE.

Ideally, 7,, W, and NOE values should bemeasured for all carbons in the molecule of interestin order to establish that the '3C-'H dipolar

mechanism dominates 13C relaxation, and that themolecule behaves as an isotropic rigid rotor.Measurement of the frequency dependence of theseparameters provides an even more rigorous test ofthe applicability of this model. Once this is estab-lished, the equations given above can be used todetermine the t> value. This in turn is useful forcharacterising the viscosity of the intracellular spaceoccupied by the molecule, and for detecting inter-molecular interactions. In this respect 13C NMR has aconsiderable advantage over many other spin Vznuclei, such as 'H, 3'P and 15N, employed in studiesof intact tissue. Frequently, these nuclei are relaxedby a number of competing mechanisms, so that theirspin relaxation behaviour cannot be analysed readilyto yield TR values.

There are many instances where a molecule doesnot behave as an isotropic rigid rotor due toanisotropic reorientation, internal rotation, orsegmental motion (17,20,25,26). Under these cir-cumstances, it has been common to derive an "ef-fective" rotational correlation time T.,, from Equa-tions (1)-(4) in place of TR (25). This parameter hasbeen useful in many cases, but is of limited quan-titative significance. While efforts are being made todescribe internal motions quantitatively (e.g., 27-30),it is beyond the scope of this review to considerthese in detail.

For the sake of completeness, it should be men-tioned that although protonated carbons will berelaxed by '3C-'H dipolar interactions in most cases,other mechanisms can operate. For very rapidlyreorienting carbons, such as those in freely rotatingmethyl groups, spin-rotation (13,14) can contribute torelaxation (31). Alternatively, high local concentra-tions of paramagnetic metal ions can also affectrelaxation (24). These interactions reduce themeasured NOE. As a reduction in NOE can also oc-cur for carbons fully relaxed by '3C-'H dipolar in-teractions but undergoing slow molecular tumbling(Figure 1), it is important to be able to recognise thispossibility. One criterion could be exact agreementof the measured 7,, W, and NOE values with thosecalculated from Equations (1)-(4) with the appropriateTR. Measurement of the field dependence of 7, andNOE (32) may also be useful in proving that relaxa-tion occurs by 13C-'H dipolar interactions in the caseof large molecules. However, it should be empha-sized that these criteria apply to molecules whichbehave as isotropic rigid rotors.

A knowledge of the 7,, W, and NOE values forobservable resonances in a 13C NMR spectrum of in-tact tissue is useful not only for characterisingmolecular motions in vivo, but also for quantitation

32 Bulletin of Magnetic Resonance

of metabolites. In order to relate the measured inten-sity of a "C resonance to its absolute, or even itsrelative, concentration in the tissue, it is necessaryto establish first that the resonance is fully relaxed.This will normally be achieved with the use of pulserecycle times longer than 3.5-4 times the relevant 7",value. The NOE values for the various 13Cresonances must then be determined if, as willusually be the case, the spectrum was obtained us-ing complete proton decoupling. It is possible,however, to avoid the need to determine NOE valuesby recording a spectrum under conditions of gatedproton decoupling (33), in which complete protondecoupling is gated on only during data acquisition,and off for a period 9-10 times the relevant 7", (34).This technique retains the advantage of dealing withfully decoupled UC resonances, but avoids problemsassociated with variations in NOE values. However,it requires longer than normal spectral acquisitiontimes because of the absence of NOE and thenecessity of waiting 9-10 x T, between successivepulses.

When dealing with small mobile molecules in livingcells, protonated carbon resonances will be moreuseful than quaternary carbon resonances for pur-poses of quantitation because they have shorter 7",values, narrow linewidths, and, in most cases, thefull NOE. For molecules the size of small proteins,protonated carbon resonances will be much broader(17) and may not be usable. However, in these casesquaternary carbon resonances have more useful 7",values and can be used for quantitation, at least forlow magnetic field strengths (17). A further require-ment for accurate quantitation is adequate digitalresolution (25).

As pointed out at the beginning of this section,most 13C NMR studies of intact tissue have been car-ried out using the conventional NMR techniques ap-plied to relatively small molecules in non-viscoussolutions. Figure 1 indicates that carbons inmolecules with long TR values(e.g., macromoleculesor small molecules immobilisd by interactions withinthe cell) give very broad resonances, with long 7",values and minimal NOE. Thus, resonances fromthese intracellular constituents will not normally beresolved from the baseline noise. In order to observesuch molecules, it is necessary to utilize specialtechniques developed for high resolution NMR ofsolids. These include high power (dipolar) protondecoupling, to reduce broadening caused by 13C-'Hdipolar interactions (35), cross-polarisation, whichenhances the 13C signal-to-noise ratio and allowsfaster pulse recycle times to be used (36), and"magic-angle" spinning (37), which reduces

HbS ERYTHROCYTES

200

Figure 2. Natural-abundance 13C NMR spectra (at 15.1 MHz)of a suspension of intact sickle erythrocytes at 37°C.(a) Oxygenated sample, recorded with scalar decoupling.(b) Deoxygenated sample, recorded with scalar 'H decou-pling, (c) As (b), but with dipolar 'H decoupling, (d) Dif-ference spectrum c-b. (e) Proton-enhanced, cross-polarisation spectrum of deoxygenated sample. Note thatchemical shift scale is relative to external CS2. Re-produced with permission from Biochemistry(38).

broadening due to residual chemical shift anisotropywhich for mobile groups in solution is normallyaveraged out by rapid molecular motion.

An example of the distinction between mobile in-tracellular molecules detected by conventional NMRtechniques and immobilized constituents detectedby "solid-state" techniques is given in a recentpaper on hemoglobin S(HbS) gelation in sickleerythrocytes (38). Spectra of sickle erythrocytes areshown in Figure 2a (oxygenated) and Figures 2b-e

Vol.3, No. 1 33

(deoxygenated). Figures 2a and b were recordedwith routine 'H decoupling power (0.7-G fieldstrength, referred to as "scalar" decoupling). Thereduction in intensity in Figure 2b is due largely togelation of deoxyhemoglobin S to form an insolublepolymer, which does not give observableresonances under these conditions. Figure 2c wasrecorded with the same sample as in 2b but with theuse of high power "dipolar" decoupling (14-G fieldstrength). In this spectrum, the polymerized HbS isobserved in addition to the isotropically tumblingHbS molecules observed in Figure 2b. The dif-ference spectrum c-b (Figure 2d) thus correspondsto the polymerized material alone. This fraction canalso be observed directly in a proton-enhancedcross-polarization spectrum, where only the semi-solid HbS is detected. The intensity in Figure 2e isgreater than that in Figure 2d, as expected (seeabove). By comparing Figures 2b and 2c it is possibleto estimate the fraction of HbS polymerized upondeoxygenation of the sample, the value obtained be-ing 0.75 at 37°C (38). It was also found that the cor-relation times of the motionally restricted form ofHbS were on the 10" sec time scale, which indicateda reduction in the rate of molecular motion of at least4 orders of magnitude relative to the isotropicallyreorientating state. The proton-enhanced cross-correlation spectrum in Figure 2e is very broad duein part to residual chemical shift anisotropy (38). Itwould be interesting to observe the improvement inresolution afforded by magic-angle spinning.

The techniques described above have been ap-plied to a number of biological systems and will beused increasingly in the near future now that spec-trometers equipped with the necessary facilities arecommercially available. Schaefer and co-workershave obtained well resolved 13C NMR spectra forsolid samples such as wood (37), ivory (37,39),various collagen-containing tissues (39), and intactseeds (see below). Dipolar decoupling and cross-polarization have also been employed by Torchiaand co-workers to study molecular motions incollagen-containing tissues at the natural-abundance '3C level and in reconstituted fibrils ofchick calvaria collagen enriched with [1-'3C]- and[2-I3C]glycine (40,41). Applications to biological mem-branes will be discussed in the following section andother examples appear in a recent review (42).

III. BIOLOGICAL MEMBRANES

Our understanding of the details of molecularorganisation and dynamics in both model and

biological membranes has increased significantlyduring the past decade. NMR spectroscopy has con-tributed substantially to this development, withstudies being carried out on 'H, 13C, 19F, and, morerecently, 2H and 31P. One goal of these studies hasbeen to observe individual sites in the membraneand then to characterise the interactions and mobili-ty of those sites. 13C NMR spectroscopy is wellsuited to such applications because of its goodresolving power and the possibility of utilising 13Cenrichment.

As a consequence of the low natural abundance ofI3C and its low NMR sensitivity, good quality '3C NMRspectra of intact cells or tissues at naturalabundance cannot usually be obtained in reasonabletime periods (less than 1 hr), unless one or morecellular constituents is present in relatively high con-centration. In the case of membranes, this impliesthat membrane-bound constituents in tissues, cells,or organelles will be observed only if the system isenriched in membranes, either naturally (e.g.,chloroplasts) or artificially (e.g., by osmotic shock toremove cytoplasmic components). An alternativeprocedure is to make use of 13C enrichment. We shallnow consider some examples of each of these situa-tions. The discussion will be restricted to biologicalmembranes since 13C NMR studies of artificial mem-branes have been adequately covered elsewhere inthe literature (26,43).

Metcalfe and co-workers (44) examined thenatural-abundance I3C NMR spectrum of sealedvesicles isolated from sarcoplasmic reticulum.Resonances from methylene carbons and the ter-minal methyl of lipids were identified, along witholefinic carbons and +N(CH3)3 groups. The intactcanine sciatic nerve was investigated by Williams etal (45), who concluded that the readily observedresonances arose from triglycerides present in thenerve interstices (probably associated with adiposetissue), rather than from lipids of the nerve myelinsheath. The latter were observed only in lipid ex-tracts of the nerve. This 13C NMR study cast doubt onan earlier 'H NMR investigation of rabbit sciaticnerve (46) in which the relatively sharp protonresonances were ascribed to myelin lipids in a fluid-like state.

Other intact biological membranes studied by con-ventional natural-abundance '3C NMR includechloroplast (47) and mitochondrial (47,48) mem-branes and erythrocyte ghosts (47,49). More recent-ly, Nicolau et al (50) investigated the effect of SV 40virus-induced transformation on membranes ofhamster embryo fibroblasts using 13C NMR. Theirresults indicated an increase in molecular motion in

34 Bulletin of Magnetic Resonance

173 ppm 130 100

Figure 3. "C NMR spectrum (at 25.2 MHz) of osmotically shocked Candida utilisceWs grown on acetic acid randomly enrich-ed to 20 atom % I3C. Reproduced with permission from Biochemistry (53).

the transformed membranes. In general, however,these studies were characterised by limited signal-to-noise ratios which restricted the amount of in-formation obtainable.

13C enrichment of membrane components hasbeen used extensively as a means of overcoming thelow sensitivity inherent in natural-abundancestudies. A logical application of such I3C labelling isto the study of molecular motions in biological mem-branes, taking advantage of the increased sensitivityas well as the capability of enriching individual sitesselectively, and using the same approaches as formodel membrane systems (26,43). Thus, Metcalfe etal (51) studied the mobility of 13C-enrichedphospholipids in membranes from Acholeplasmalaidlawii cells grown on a medium containing13C-labelled palmitic acid. They concluded from the13C NMR studies and concurrent electron-spin-resonance studies employing spin labelling that theorganisation of the bulk lipids in these membraneswas indistinguishable from the simple bilayer struc-ture. An interesting study of lipid organisation in theenvelope of the vesicular stomatitis virion was car-

ried out by Stoffel and Bister (52). Host cells (babyhamster kidney cells) were first labelled with either80-90% 13C-enriched [11-13C] oleic acid or [Me-13C]choline and then infected with virion. After thevirions were enveloped with 13C-labelled host celllipids, they were purified and examined directly by13C NMR. Spin-lattice relaxation measurements ofthe [11-'3C] oleic acid-containing virions showed thatthe mobility of this hydrocarbon chain was morerestricted in the virion envelope than in chloroformsolution or in liposomes, at least in the region of the13C label. Similar measurements for the [Me-'3C]label also indicated motional restrictions, whichwere ascribed partly to the high content ofcholesterol in the virion envelope (52).

The challenge of characterising molecular motionsin intact membranes has also been taken up by Lon-don, Matwiyoff and co-workers. Following their earlywork on C. utilis cells labelled by growth on 20%l3C-enriched acetic acid (2), they investigated themembranes of these same cells following osmoticshock (53). The 13C NMR spectrum of such cells,shown in Figure 3, is dominated by resonances from

Vol.3, No. 1 35

fatty acid residues of the lipids, as well as by glucoseand mannose residues of the cell wall pep-tidoglycan. The good resolution observed in thisspectrum is partly due to the increased membranefluidity engendered by a high content of polyun-saturated fatty acids (53). 7", measurements for fattyacid carbons in the yeast cell membranes revealedthe presence of a mobility gradient along the fattyacid chain similar to that observed in sonicatedlecithin vesicles, although some quantitative dif-ferences were noted.

The same workers examined Chinese hamsterovary cells grown in a medium supplemented with[Me-liC] choline (6). 1JC NMR spectra of cellularsuspensions showed that about 42% of the label wasassociated with cellular lipids, the remaining 58%being found in the water-soluble fraction of the cell,primarily as phosphorylcholine. The latter had a 7i of0.55 sec at 6°C in intact cells, compared with 0.66 secfor phosphorylcholine in solution. This suggestedthat the intracellular viscosity was about 20% higherthan that of water. The lipid-bound choline could bestudied in detail only in ceils fixed with form-aldehyde and washed to remove free choline andphosphorylcholine (6,54).

Smith and co-workers (55,56) studied the yeast-likefungus Aureobasidium pullulans grown on [1-13C]acetate and [2-"C] acetate (see Figure 4). Singly-labelled acetate precursors were used to avoid thecomplications introduced by "C-'3C spin-spin coupl-ing. 7", measurements revealed once again dynamicbehaviour similar but not identical to that of a modelmembrane. However, it should be noted that in-tracellular neutral lipid may have contributed to theobserved spectra. The membranes of a number ofother microorganisms have also been labelled bygrowth on 13C-enriched acetate (56,57), although notall of them yield spectra as well resolved as thoseshown in Figure 4. Membrane-associated car-bohydrates have been observed in spectra ofMicrococcus freudenreichii cells (56). Thetransverse distribution and movement of "C-labelledphosphatidylcholine (58) and lysophosphatidyl-choline (59) incorporated in isolated sarcoplasmicreticulum membranes have been studied.

The above studies on biological membranes havebeen carried out by conventional high-resolutionmethods. Accordingly, information has been obtain-ed only about the mobile constituents. There aremany membrane-bound molecules which arerelatively immobile, and many cells in which eventhe bulk lipids do not yield well resolved spectra(53,56). These cases lend themselves to study by"solid-state" NMR techniques.

Cross-polarisation spectroscopy and magic-anglespinning have been employed to obtain spectra ofunsonicated membrane dispersions (60-62). Thecombination of these two techniques afforded wellresolved spectra of unsonicated dispersions ofdimyristoyl- and dipalmitoyl-phosphatidylcholine(62). Rapid sample spinning (2.6 kHz) did notsignificantly affect the structure of the liposomes(62). Cross-polarisation spectroscopy has also beenapplied to observe lipids in intact biological mem-branes of red blood cell ghosts, chloroplasts, andmyelin (63). These spectra were obtained withoutmagic-angle spinning so that the residual chemicalshift anisotropy could be used to characterisemolecular motions in the intact membrane.

The advent of dipolar decoupling and cross-polarisation spectroscopy holds considerable prom-ise for the study of membranes as these techniquesgreatly extend the range of membranes and mem-brane components which can be visualised by "CNMR. The general utility of magic-angle spinning canbe evaluated only by further studies directed towardestablishing how stable biological membranes areunder these conditions. Application of these tech-niques, and of 13C labelling, is likely to yield a greatdeal of information on membrane structure anddynamics in the near future.

IV. NATURAL-ABUNDANCE 13C NMROF INTACT TISSUE

As pointed out earlier, natural-abundance "C NMRstudies of constituents of intact tissue can be carriedout only if these constituents are present in highconcentration or if the tissue is stable under the ex-perimental conditions, which require long spectralaccumulation times. In this section we shall considerexamples where one or both of these criteria issatisfied.

A. Terrestrial Plants

Among the first tissues to be studied successfullyby natural-abundance 13C NMR were intact plantseeds. Shoolery (64) examined soybean and cornseeds, and Schaefer and Stejskal (65) showed thatsoybean and radish seeds gave spectra in which fat-ty acid resonances were sufficiently well resolved topermit analysis of the composition of the 3 major un-saturated fatty acids in the intact seeds. The highsensitivity achieved in these spectra is illustrated in

36 Bulletin of Magnetic Resonance

o

**-C-4>6

I 2 (3-7J g 9 10

^2_C-I8

I/3& I I I r r i

\ c

(3-7J 8 9 l 0

C-5,7-

^2—c-9

I I I

<Z-C-I7

1 1 1 1 1 I

Figure 4. 13C NMR spectra (at 20 MHz) of packed whole cells of Aureobasidiumpullulans (in D2O at 30°C) grown on (a) [2-13C] acetate, and <b) [1-'3c] acetate.Resonances are assigned to oleic acid as indicated. Reproduced with permissionfrom Biomolecular Structure and Function (55).

Vol.3, No. 1 37

Figure 5. Natural-abundance "C NMR spectrum (at 22.6 MHz) of an intact soybean (obtained in 20 min with a 10-mm probeand quadrature detection). The spectrum covers a chemical shift range from 0 to 200 pm from Me4Si. Reproduced with per-mission from J. Am. Oil Chem. Soc. (65).

Figure 5. A number of intact seeds from other com-mercially important plants were examined (66-68).The spectra were dominated by fatty acidresonances in most cases, although a cyanolipidwas observed in seeds of Stocksia brachuica (68). Incontrast, intact seeds from the fruit-bearing plantAucuba japonica (aoki) afforded a natural-abundance I3C NMR spectrum composed ofresonances from sucrose and the iridoid glucosideaucubin (69). The skin and flesh of the aoki fruit didnot contain these compounds, but showed promi-nent resonances from glucose and fructose. Thesame authors also examined pericarps and seedsfrom star anise fruit (70). The spectrum of the seedscould be accounted for by triglyceride fatty acids,while that of the pericarps was dominated by theessential oil anethole. The spectrum of wholenutmeg seed consisted of resonances from essen-tial oils and triglycerides (70). Starch also gave rise toobservable resonances in plant tissue which hadbeen boiled (71). Maltose was observed in the excis-ed endosperm of wild oats following digestion ofstorage carbohydrate during germination (68).

Plant tissues such as those just described arefavourable systems for NMR analysis because theyare easily handled, relatively stable as a function oftime, and contain significant pools of mobile consti-tuents which give rise to sharp resonances. Indeed,it is only the relatively mobile constituents which are

readily observed using conventional NMR tech-niques, as we have already noted. However,resonances from the mobile constituents can besuppressed and those from motionally restrictedcomponents enhanced by use of the cross-polarisation technique. This is illustrated in Figure 6which shows the natural-abundance 13C NMR spec-trum of a single soybean seed obtained with dipolardecoupling and transfer of polarisation from 'H, aswell as magic-angle spinning (72). The prominentresonances in this spectrum come from starch andprotein, in contrast to conventional 13C NMR spectraof soybean seeds (64,65) recorded without dipolardecoupling or cross-polarisation, which exhibitresonances from lipids only (see Figure 5). This ex-ample provides a further demonstration of how thesevarious techniques may be manipulated to obtain in-formation on different classes of tissue constituentswithout the need for disruption of the tissue.Schaefer and Stejskal had previously reported spec-tra of various plant tissues obtained with dipolardecoupling (73) and cross-polarisation (66), but theresolution in these spectra was inferior to that ob-tained with magic-angle spinning.

B. Marine and Halophilic Organisms

Relatively few high-resolution NMR studies ofcells and tissue from marine organisms have been

38 Bulletin of Magnetic Resonance

A. Adductor muscle

B. Foot muscle

C. Hearts

Figure 6. Natural-abundance 13C NMR spectrum (at 22.6MHz) of an intact soybean recorded with cross-polarisation(from protons) and magic-angle sample spinning. Spectralaccumulation time was 16 hr. The spectrum covers achemical shift range of about 350 ppm, which en-compasses the 200 ppm region shown in Figure 5. The ma-jor low-field peak is due to carbonyl carbons (primarilyfrom proteins), while the small peak furthest downfield is aspinning side-band. Reproduced with permission fromBiochem. Biophys. Res. Commun. (72).

carried out. Williams and co-workers examined thesalivary gland of the octopus by 'H NMR (74), while"P NMR spectra of abalone muscle (75) and crayfishnerve (76) have been reported. This technique hasalso been used to study energy metabolism in tissuefrom the marine bivalve mollusc Tapes watlingi (77).Recently, natural-abundance 13C NMR spectra ofwhole tissue and tissue homogenates from T. wat-lingi were described (78). Prominent resonancesfrom free taurine, betaine, and glycine were observ-ed (see Figure 7), as well as small resonances fromother free amino acids and sugars. The good signal-to-noise ratio of resonances from the three majormetabolites, taken in conjunction with their sharp-ness, indicated that the metabolites were present inhigh concentration and were quite mobile within thetissue. These observations indicated a role for thesemetabolites in osmoregulation in tissue from T. wat-lingi. This was subsequently confirmed by exposing

240 220 200 180 160 140 120 100 80 60 40 20 0

ppm from Me4 Si

Figure 7. Natural-abundance UC NMR spectra (at 15.04MHz) of intact tissue from the marine bivalve molluscTapes watlingi. A. Adductor muscle (1 gm wet weight; totalaccumulation time 2.9 hr). B. Foot muscle (1.1 gm; 2.9 hr). CCompounds released from four hearts (aproximately 0.3gm wet weight). Reproduced from (78).

specimens of this mollusc to media of varying salini-ty for two-day and two-week periods, then assayingthe levels of these metabolites in tissuehomogenates by 13C NMR (79). The concentrations oftaurine, betaine, and glycine all showed a strong cor-relation with environmental salinity after a period oftwo weeks (79). "C NMR studies on tissue from T.watlingi were facilitated by its good stability, asdemonstrated by 31P NMR (77). Tissue from a numberof other marine molluscs was also screened by I3CNMR, which revealed that taurine, betaine, andsome polyols were present in all species, andglycine was present in some (78). As metabolitessuch as these have been found to act asosmoregulatory solutes in many species of molluscs(80), it appears that natural-abundance I3C NMR willbe useful in the study of osmoregulation in marine

Vol.3, No.1 39

organisms. Spectra of live tissue can be obtainedquickly without T3C enrichment because of the highconcentrations of these solutes in the cytoplasm,and the responses of all classes of compounds(e.g., free amino acids, sugars, and polyols) can bestudied simultaneously.

A range of marine microorganisms has been ex-amined to explore the general utility of this ap-proach. The spectrum of the unicellular blue-greenalga Synechococcus sp. is dominated byresonances from the glycoside 2-O-a-D-glucopyranosylglycerol (81). I3C NMR analyses ofcells grown in media containing NaCI concentrationsranging from 0 to 10% (WIV) show that thissubstance is the principal organic osmoregulatorysolute in this organism (81). Intact cells of Dunaliellasalina, a unicellular halophilic microalga, yield anatural-abundance "C NMR spectrum dominated byresonances from free glycerol, as shown in Figure 8(82). The good signal-to-noise ratio obtained with areasonably short accumulation time indicates thepossibility of using 1JC NMR spectra of D. salinacells to follow the time course of changes in the levelof glycerol in response to alterations in external

salinity or other environmental stress, and studies ofthis type are underway (82). Glycerol is known to bethe major osmoregulatory solute in a number ofDunaliella species (83). Good quality spectra havealso been obtained from other unicellular marinealgae (82).

Because L3C NMR spectra of intact cells or tissuefrom marine organisms can be obtained in relativelyshort times, they should also be useful forcharacterising the spin-lattice relaxation behaviourof the major osmoregulatory solutes in intact tissue.These data act, in turn, as a probe of interactionswithin the cell, as well as of intracellular viscosity.

While the high concentrations of osmoregulatorysolutes in tissue from marine organisms willfacilitate the study of these substances by natural-abundance l3C NMR, they will hamper attempts tostudy other metabolites not involved in osmoregula-tion unless specific UC enrichment of the latter isemployed. In the case of photosynthetic marinemicroorganisms, specific enrichment may be dif-ficult because CO2 or bicarbonate is the only organiccompound taken up by many of them. However,s,ome of these microorganisms will grow in media

CH9OHI z

CHOHICH2OH

- C H 2 -

- C H -

140 120 100 80 60

ppm from Me4 Si

20

Figure 8. Natural-abundance I3C NMR spectrum (at 15.04 MHz) of a suspension (0.15 gm wet weight in 1.5 ml) of intact cellsof Dunaliella salina grown on a medium containing 12.5% w/v NaCI. Cells were harvested by centrifugation and resus-pended in growth medium prepared with D2O. Spectral accumulation conditions (500 scans, 5.0 sec pulse repetition time,total time 42 min) were chosen to ensure complete I3C relaxation between 90° pulses, but a partially saturated spectrumwith comparable single-to-noise ratio can be recorded in less than 10 min. A spectrum of the extracellular medium removedfrom the sample after nearly 4 hr in the probe showed that less than 15% of the intracellular glycerol had leaked out of thecells during this time (82).

40 Bulletin of Magnetic Resonance

containing l i t t le or no NaCI {e.g., theSynechococcus mentioned above), and under theseconditions the intracellular concentrations of theosmoregulatory solutes should be low enough topermit observation of other metabolites.

C. Other Systems

Sharp and Richards (84) analysed the 13C and 'HNMR of bovine chromaffin granules in an attempt tocharacterise the internal aqueous phase of theseorganelles. Good quality spectra were obtainedbecause of the high concentrations of the majorcomponents epinephrine, ATP and the random coilprotein chromogranin A. Based on 13C and 'Hchemical shifts, intensities, and linewidths, it wasconcluded that these three major components werepresent in a fairly fluid aqueous phase in which mo-tion was essentially isotropic (84). This eliminatedcertain other models which had been proposedearlier. Subsequently, molecular mobilities withinthe granules were probed in greater detail byanalysis of spin-lattice relaxation times (85). The datawere consistent with the concept of a storage com-plex based on electrostatic interactions betweenchromogranin and ATP and epinephrine, in whichATP crosslinks cationic sidechains of the protein.

The process of fermentation by baker's yeast cellswas investigated by Kainosho et al (86). The timecourse of the reactions initiated by addition ofglucose and adenosine to anaerobic, acetone-treated yeast cells was followed directly in the NMRtube. The accumulation of ethanol, glycerol, andATP was observed, with fructose-biphosphate beingdetected as an intermediate. The latter two com-pounds were also observed by 31P NMR. The 13CNMR spectra showed that trehalose was formed andthat this was the unidentified glucose metaboliteobserved by Eakin et al (2) in their earlier study ofthe metabolism of [1-'3C] glucose by C. utilis (seeabove).

An interesting study has been carried out on theinteraction of human plasma lipoproteins with aortictissue. Using a high field spectrometer (63.4 kG),Hamilton et al (87) examined low-density lipopro-teins (LDL) from normal and hypercholesterolemicindividuals, aortic tissue from normal subjects, andaortic tissue containing fibrous atheroscleroticplaques. Spectra of LDL from normal and hyper-cholesterolemic plasma were essentially identical.Spectra from non-atherosclerotic arterial tissuewere dominated by triglyceride resonances, eventhough the tissue contained phospholipid,cholesterol, and triglycerides in approximately equal

concentrations. By contrast, spectra of tissue con-taining fibrotic lesions exhibited cholesteryl esterring resonances as well as numerous narrowresonances from fatty acids, but no peaks at-tributable to unesterified choiesterol and only abroad choline peak from the phospholipids. The lat-ter spectra were similar to those of heat-denaturedLDL, which differed from those of native LDL in ex-hibiting marked broadening of resonances frompolar lipids. Thus, these spectra provided informa-tion on the mobility of the various classes of lipids inthese tissues.

The 'H and 13C NMR spectra of rat skin have alsobeen examined (88). While the 'H NMR spectrum wasdominated by a water resonance, a well resolved 13CNMR spectrum was obtained, which showed thattrioleoylglyceride was the dominant free lipid in thistissue.

V. STUDIES EMPLOYING 13C LABELLING

Most of the biological samples discussed in theprevious section were atypical in one way or anotherso that natural-abundance I3C NMR spectra could bereadily obtained. The majority of biological systems,and in particular samples of mammalian tissue, canonly be studied with the aid of '3C enrichment, atleast at the present level of development of NMR in-strumentation. While this creates some problems, italso paves the way for in vivo studies of metabolismand biosynthesis using 13C-labelled samples withoutsignificant background interference from theunlabelled components of the tissue. Thisrepresents an advantage of '3C NMR over 'H and 3IPNMR, particularly in view of the small perturbationcaused by the I3C isotope (3). Indeed, studies of thistype constitute the most rapidly growing branch ofthe general area of research covered by this review.

As indicated in Section I, the first application of 13CNMR spectroscopy to intact tissue employed '3Clabelling (1,2). The same workers subsequentlyenriched mouse hemoglobin by feeding L-[2-13C]histidine and compared the spin-lattice relaxationbehaviour of the histidine residues of hemoglobin inintact and lysed erythrocytes (5). The rotational cor-relation time for hemoglobin in the intact cells wasonly 25% higher than in solution, indicating that theintracellular viscosity was not unusally high. Asimilar attempt to observe the methyl carbons of thee-N-trimethyllysine and methionine residues ofcytochrome c in Neurospora crassa cells grown on[S-Me-'3C] methionine was less successful becauseof overlap with resonances from a number of lowmolecular weight labelled compounds (89). The

Vol.3, No.1 41

protein-bound methyl groups were readily observedin purified cytochrome c.

The experiments described above on l3C-labelledhemoglobin in erythrocytes (5), [Me-'3C]phosphorylcholine in Chinese hamster ovary cells(6), and various constituents of bovine chromaffingranules (85), indicated that 13C spin-lattice relaxa-tion parameters were, as expected, a useful probe ofthe intracellular environment, particularly its viscosi-ty. Neville and Wyssbrod (90) employed a similar ap-proach to characterise 13C-labelled glycine ac-cumulated in frog muscle. The '3C 7", and NOEvalues for glycine, measured at 1°C to minimisemetabolism and transport, indicated that the in-tracellular space occupied by the glycine had ahigher viscosity than free solution. However, thedata provided no evidence for special "organisa-tion" of the intracellular water or for binding ofglycine.

Studies have been carried out on plant tissuelabelled with I3C by photosynthetic incorporation of13CO2. Thus, Schaefer era/ (7) examined metabolismin soybeans which had been exposed to I3CO21 to 3days earlier. The initial spectra contained prominentresonances from sugars and lipids. Three days afterthe '3CO2 treatment, the intensity of the sugarresonances had decreased and that of the lipidresonances increased. Consideration of 13C-I3Ccouplings led to the conclusion that some of thesugar was oxidised via the pentose phosphate cycle,thereby demonstrating how 13C labelling could be us-ed to obtain information about metabolism in intacttissue.

Cells of the blue-green alga Agmenellumquadruplicatum grown with 20 mol % 13CO2 have alsobeen examined (91), this level of 13C enrichment be-ing chosen to enhance sensitivity while minimisingI3C-'3C couplings. The spectra contained prominentresonances from sugars, lipids, and protein car-bons, and were similar to spectra of another blue-green alga Anacystis nidulans reported previouslyin connection with a biomembrane study (53). Thesugar resonances arose from polysaccharides aswell as from low molecular weight carbohydrates in-cluding glucosylglycerol. The latter was alsodetected by natural-abundance 13C NMR in a marineblue-green alga Synechococcus sp. (81), as describ-ed above. In cells of A. quadruplicatum grown forlonger periods, the intensity of the proteinresonances decreased due to nitrogen depletion,and there was a corresponding increase in intensityof the polysaccharide resonances. Signals from freeglutamate were also observed during the exponen-tial growth phase of the cells, but subsequently

decreased in intensity following nitrogen depletion(91).

Information about the organisation ofmacromolecular components of intact viruses hasalso been obtained by 13C NMR. Tobacco mosaicvirus (TMV) enriched with 13C to a level of 12% wasfound to yield an observable spectrum using conven-tional NMR techniques (92). Calculations predict thatcarbons held rigidly in intact TMV would not bedetectable under the conditions used, so the observ-ed spectrum was interpreted to indicate that asignificant fraction of the TMV protein experiencedinternal motion within the virus. A number ofunicellular organisms have been cultured on13C-enriched media in order to facilitate '3C NMRstudies of their membrane components (see SectionIII).

The remaining studies in this section are concern-ed with the metabolism of exogenous 13C-labelledprecursors or substrates by intact cells. Shulman etal (12,93) investigated the metabolism of [1-'3C]glucose by suspensions of Escherichia coli cells by13C NMR at 90.5 MHz. Because of the excellent sen-sitivity achieved by use of 13C labelling and the highmagnetic field strength, spectra were obtained withas little as 1 min accumulation time, thus allowing thestudy of fairly rapid processes in living cells. Spectraobserved immediately after addition of [1-"C]glucose to an anaerobic suspension of cells showedresonances from the a and p anomers. Subsequentspectra indicated that the cells preferentiallycatabolise the a anomer to produce lactate, as wellas smaller amounts of succinate, acetate, ethanol,alanine, and valine. Fructose-1,6-biphosphate wasobserved as an intermediate and was found to belabelled at C-6 as well as at C-1. Labelling at C-6 canoccur as a result of scrambling by the action oftriosephosphate isomerase coupled to resynthesisof fructose-1,6-biphosphate by aldolase, although itwas not demonstrated unequivocally that thispathway was responsible for the observed scram-bling (12). The two anomers of fructose-1,6-biphosphate were found to be in equilibrium duringglycolysis, a result which could only be establishedby a noninvasive technique such as NMR becausethe time for anomerisation is of the order ofseconds.

The effect of oxygenation on glucose metabolismby E. coli was also examined. Under these condi-tions glutamate was formed. Consideration of theobserved 13C-labelling patterns in this amino acid in-dicated that the label enters the tricarboxylic acid cy-cle as acetyl-CoA rather than oxaloacetate. At latertimes, the glutamate was converted to glutamine.

42 Bulletin of Magnetic Resonance

C6-Fru-P2

C1-£Fru-P2

ETHANOL

100 80 60 40 20 0

8,ppmFigure 9. "C NMR spectrum (at 90.5 MHz) of a suspension of yeast (Saccharomyces cerevisiae)cells recorded 10 min afteraddition of 75 mM [1-'3C] glucose. Spectral accumulation time 68 sec. Signals observed are from C-1 of a- and /3-glucose, C-1and C-6 of the intermediate /3-fructose-1,6-biphosphate, and the end products glycerol (C-1) and ethanol (C-2). Peak A isassigned to C-1 of a-fructose-1,6-biphosphate. Reproduced with permission from Proc. Natl. Acad. Sci. U.S.A. (94).

These results demonstrated the power of the com-bined use of 13C labelling and a high magnetic fieldstrength spectrometer to study metabolism in vivo.The excellent sensitivity achieved by this combina-tion permits reasonably fast processes to becharacterised, while the position of the labels in themolecules provides useful information on metabolicpathways and relative fluxes.

Similar experiments have been carried out on theyeast Saccharomyces cerevisiae (94). A spectrum ofan anaerobic suspension of yeast cells recorded 10min after addition of [1-"C] glucose is shown inFigure 9. As with E. coli, the two anomers of glucoseare observed, but in Figure 9 the resonance from thea anomer is significantly reduced due to its morerapid uptake. The principal end products ofglycolysis, [1-'3C] or [3-'3C] glycerol and [2-'3C]ethanol, are readily observed, as well as smalleramounts of fructose-1,6-biphosphate labelled at C-1and C-6. By comparing the amounts of label in-corporated at these two positions in the presence of

[1-'3C] glucose and [6-13C] glucose, quantitative in-f o r m a t i o n w a s d e r i v e d a b o u t t h ealdolase/triosephosphate isomerase triangle (seeabove) which indicated that forward and reversefluxes through aldolase were almost equal.

Mammalian cells have also been examined bythese methods. For example, gluconeogenesis from[2-13C] glycerol and [1,3-'3C] glycerol in suspensionsof rat hepatocytes has been studied by the samegroup (95,96). Longer accumulation times (17-30 min)were required for these spectra, but 70-90% of thecells were still viable at the end of the experiments.The labelling patterns observed were consistentwith expectations. Quantitative analysis of thedistribution of the 13C labels in glucose indicated thatapproximately 10% of the hexoses had passedthrough the pentose pathway, and that the trans-aldolase reaction is essentially irreversible, whereasthe transketolase reactions are reversible. Further-more, gluconeogenesis in normal rat liver cells wascompared with that in rats made hyperthyroid by

Vol.3, No.1 43

treatment with the thyroid hormone triiodothyronine.In these cells, the rates of glycerol consumption andglucose formation were twice the normal value,while the L-glycerol-3-phosphate level was reducedto 40%. These results were interpreted to indicate anincreased activity of mitochondrial glycerolphosphate dehydrogenase in the hyperthyroid ratcells (96,97).

Gluconeogenesis from [3-'3C] alanine in rat livercells (97) and perfused mouse liver (96,98) was in-vestigated. A series of 13C NMR spectra of mouseliver is shown in Figure 10. The numberresonances in Figure 10c were assigned totriglycerides of palmitic, oleic, and palmitoleic acids(98). These compounds are present in liver in neutralfat droplets, which accounts for the reasonably nar-row linewidths. By contrast, the natural-abundance"C NMR spectrum of a suspension of rat liver cellsshowed no prominent resonances after a com-parable spectral accumulation time (97).

Figure 10b was accumulated 150-180 min after addi-tion of 10 mM [3-13C] alanine and 20 mM unlabelledethanoi to the perfusate (98). Peaks from glucoseand several other compounds were observed. Allcarbons in the glucose were labelled to some extent,whereas only C-1 and C-6 would have been labelled ifthe alanine had proceeded directly to glucose. Thescrambling of the label was interpreted in terms ofthe known pathways of gluconeogenesis fromalanine (98). Figure 10a was obtained from the liverperfusate, and indicates that glucose, lactate, andminor amounts of a few other metabolites were pre-sent. Those peaks present in Figure 10b but not in10a came from intracellular metabolites.

Gluconeogenesis from "C-labelled alanine andethanoi by perfused mouse liver was also examined(98). In these experiments, labelled C4 of glutamateand glutamine and C2 of acetate were prominent, in-dicating that ethanoi was the dominant source ofacetyl-CoA. The time courses were followed for anumber of labelling experiments, and detailed inter-pretations have been made (98).

13C NMR has been employed in conjunction with 'Hand 3'P NMR to study metabolism in humanerythrocytes (99,100). Label from [1-13C] glucose wasfound to be incorporated into 2,3-diphospho-glycerate and then lactate, thus providing informa-tion about the so-called 2,3-diphosphoglyceratebypass. Phosphorus-containing metabolites wereobserved in the same sample with a spectrometerdesigned to observe 13C and 3IP NMR spectrasimultaneously (99), while 'H NMR spectra wereobserved independently. The simultaneous observa-tion of different nuclei within the same sample per-

mits a more complete interpretation of metabolicprocesses, free from the potential uncertaintiesassociated with variations among different samples.The metabolism of [1-13C] and [6-'3C] glucose by rab-bit erythrocytes under anaerobic conditions hasbeen examined by Scott and co-workers (101).

I3C labelling has been used extensively to studythe biosynthesis of secondary metabolites (8,9), butonly recently have attempts been made to carry outthese studies on intact cells (101,102). Informationabout the vitamin B,2 biosynthetic pathway wasderived by incubating intact Rhodopseudomonasspheroides cells in the presence of [5-13C]aminolevulinic acid (102). The spectra illustrated theformation of labelled coproporphyrinogens I and III.Similarly, administration of [11-'3C] porphobilinogento a suspension of Propionibacterium shermanii ledto formation of coproporphyrinogen III labelled at dif-ferent positions. As natural-abundance I3C glucosewas present in the cellular suspension at high con-centration, its metabolism to propionate and acetatewas also monitored as a function of time (102).

Polyketide biosynthesis was also studied in themould Penicillium urticae (101) by following the timecourse of metabolism of [2-'3C] sodium acetate. After12 hr, the acetate had been metabolised to citrate;after 24 hr, resonances from l3C-labelled gen-tisalcohol were observed; and subsequently, 13Clabel appeared in patulin. Saturated fatty acidslabelled at the "even" carbons accumulated (101).

VI. CONCLUDING REMARKS

Reviews of the applications of 13C NMR spec-troscopy to areas of organic chemistry andbiochemistry quite justifiably emphasize the "ex-plosive" growth which has occurred during the pastdecade or so. It would be inappropriate to describethe field of interest covered by this article in theseterms, although there is every indication that thisstage may be reached in the near future.

The range of applications covered in this reviewtestifies to the general utility of 13C NMR. As NMRspectrometers operating at higher magnetic fieldstrengths become available, it is likely that manynatural-abundance 13C NMR studies will becomefeasible in addition to those described above.However, the largest growth in the application of I3CNMR to intact cells and tissue is likely to come from13C-labelling studies. A number of studies of primarymetabolism in vivo have already been carried out,and increasingly sophisticated experiments may beexpected in the near future. Interesting applicationsof this technique to the study of secondary

44 Bulletin of Magnetic Resonance

35*C

xl/4

100 90 20

Figure 10. 13C NMR spectra (at 90.5 MHz) of a perfused mouse liver at 35°C. Spectrum (c), which shows the 13C natural-abundance background of this liver, was accumulated before the substrate was added. The substrate, 8 mM [3-13 C] alanineand 20 mM unlabelled ethanol, was then added at 0 min and again at 120 min and a series of 1JC NMR spectra were taken.Spectrum (b) was measured during the period 150-180 min. Spectrum (a) is the "C NMR spectrum of the perfusate after theperfusion was terminated at 240 min. This spectrum consisted of 5000 scans. The pulse repetition times were 0.5 sec forspectra (b) and (c), and 2 sec for spectrum (a). The abbreviations used include: fiC,, a d , /3C3,,, |3C2, aC3, aC2,s, aC, /JC6, aC,carbons of the glucose anomers; Glu C2, glutamate C2; Gin C2, glutamine C2; Asp C2, aspartate C2; Ala C2, alanine C2; Lac C,,lactate C3; CB, cell background peak; W,X,Y and Z, unknown; AA Ca, acetoacetate CH2; and p-HB Ca, /3-hydroxybutyrateCH2. Reproduced with permission from Proc. Natl. Acad. Sci. U.S.A. (98).

Vol.3, No.1 45

metabolism, the metabolism of drugs and otherforeign substances, the interaction of smallmolecules with receptors on cells, and so on, shouldalso be forthcoming. Furthermore, information aboutthe kinetics of processes in vivo can be obtainedfrom saturation transfer experiments, as has beendemonstrated with 31P NMR (11,12).

Perhaps the most informative studies will be thosewhich employ 'H, 13C, and 31P NMR concurrently(e.g., reference 100). Each of these nuclei has ad-vantages not shared by the others, so that the mostcomplete description of a biological process will bebest obtained by the combination of techniques.

In recent years there has been considerable in-terest in the application of NMR spectroscopy to thestudy of intact animals, with the ultimate aim of usingthe technique for clinical diagnostic purposes inman. Thus, studies have been carried out by 'H NMRon malignant tumors (103), the human abdomen(104,105), and other systems (106), and by 31P NMR onthe leg muscle and brain of a living rat (107) and on in-tact human limbs (108). So far, 13C NMR has not beenemployed in this way. A potential barrier to this sortof study by 13C NMR is the need for proton decoupl-ing, which may cause unacceptable heating of thespecimen under investigation. However, this prob-lem should not be insurmountable, and studies ofthis type may be carried out in the near future.

It is worth noting that a feature of many of thestudies described herein is their ease of interpreta-tion. Once the observable resonances have beenassigned to specific molecules in the tissue, a taskwhich is relatively straightforward, the NMR spectracan be readily interpreted in terms of biological pro-cesses with a minimum of spectroscopic knowledge.This is likely to lead to a more rapid acceptance bybiologists of this application of NMR spectroscopythan of other areas which require a more intimateunderstanding of the technique (e.g., the use ofparamagnetic agents in structural studies) and whichare sometimes characterised by conflicting inter-pretations that leave the nonexpert confused and ex-asperated. The greatest problem in the applicationof NMR spectroscopy to intact tissue will often bybiological, that is, how to maintain a viable sample,rather than spectroscopic.

ACKNOWLEDGEMENTS

I would like to express my gratitude to a number ofcolleagues at the Roche Research Institute for theirconstructive comments on the manuscript, and tothose who were kind enough to send me preprints oftheir work prior to publication.

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