NMR and compartmentation in biological tissues

Pmyrrss in NMR Speccrro.~copy, Vol. Il. pp. 241-279. 1985. Printed m Great Britam. All rights reserved.

0079 6565/85/10.00

NMR AND COMPARTMENTATION P. S.

+ so

Copyright 6 1985. Pergamon Press Ltd.

BELTON*

and R. G.

IN BIOLOGICAL

TISSUES

RATCLIFFE~

Food Research Institute, Colney Lane, Norwich, NR4 7UA, U.K. of Agricultural Science, University of Oxford, Park8 Road, Oxford, OX1 3PF, U.K.

*AFRC,

tDepartment

(Received 22 April 1985)

CONTENTS 1.

2. 3. 4. 5.

6.

7.

8. 9.

Introduction Compartmentation in Biological Systems NMR Measurements on Compartmented Systems Exchange Processes Chemical Shift Effects 5.1. Effect of pH gradients 5.1.1. 3’P spectra 5.1.2. ‘H, 13C and isN spectra 5.2. Effect of paramagnetic ions 5.2.1. Shift reagents with a positive charge 5.2.2. Shift reagents with a negative charge 5.3. Miscellaneous effects Intensity measurements 6.1. Kinetic experiments 6.2. Relaxation effects 6.3. Susceptibility effects Relaxation Time Experiments 7.1. The nature of relaxation in compartmented systems 7.2. Applications to unicellular systems 7.3. Applications to muscle and other tissues 7.4. Applications to plant cells and tissues Investigation of Compartmentation by Diffusion Measurements Concluding Remarks Acknowledgements References Note Added in Proof

241 242 244 245 248 248 249 252 252 253 254 256 257 257 258 259 260 260 264 266 270 272 214 214 274 279

1. INTRODUCTION Heterogeneity at a variety of levels is essential to the functioning of living systems” -4) and some non-invasively by NMR. For example, the aspects of this phenomenon can be studied compartmentation of metabolites is a recurring theme in the increasing number of high resolution can be viewed as part of the wider NMR studies of biological systems’5 - lo) and such investigations application of NMR to heterogeneous systems.(‘i -‘v The NMR approach to heterogenous systems is based on the measurement of the usual resonance properties, i.e. intensity, frequency and relaxation behaviour, with due allowance in the subsequent interpretation for the effects of exchange, and we propose to review the general principles of this approach with reference to the compartmental analysis of biological systems and their models. While many different NMR methods have been used to tackle various aspects of the compartmentation problem in selected systems, it often appears that the methods have been considered in isolation. It is an important objective of the present article to bring the methods together so that comparisons can be made and the applicability of the methods can be discussed. Before considering the application of NMR methods to compartmented systems, it is necessary to define the term ‘compartmentation’. Section 2 discusses the various ways in which a system can be compartmented and Section 3 summarises the NMR approach to such systems. Exchange processes can have important consequences for compartmental analysis and these effects are described in 241

P. S. BELTONand R. G.

242

RATCLIFFE

Section 4. The NMR methods for compartmental analysis are classified in Sections 5 to 8 according to the measurable property that is exploited by the method. Section 5 describes chemical shift rnea3~3fim3~rts tiy ceimr763-&7~3. -‘& L S_WkWS.9&TX?k hT&iT>j’ CGl’iL”iT& Wh?h ths? 15PtiS #? $9 @&kTtS an&l_~aram~nb~c’~ons.Se~~~onsb~ are bon%ntimb’-gv rt%ax;i~mn i53133s anb ID some e~~fmf b2E classificaiion &al has ken adopkd is somewhat ar%ary. Section 6 is z-r&r& concerned &h t&e loss 03 s$?&zz%z+ iz?k?z&~, ?&%&2U&X%~ occurs ~~zz..~azz &ti‘err .Jzl&?X~kV2zzJ~~~~~~zz2‘broadens a lineee’nevonh bt%eeilom dd1~eSedimn 3 bescifnes hme born& rtkixaimn measurements >D_~Y~~ZJJX that show no chemical shift dispersion. Section 8 reviews the use of pulsed field gradient methods to detect restricted diffusion. 2. COMPARTMENTATION

IN BIOLOGICAL

SYSTEMS

Some of the features to be expected in a compartmented system can be illustrated by reteerecrceto the simple experiment in Fig. 1, in which it is supposed that a liquid has been placed in a cylindrical vessel and that a similar quantity of the same liquid, containing a very small quantity of a dye, has

FIG. 1.

physical

The time dependence barrier

of the compartmentation of a dye (e) in a model system in which there is no between the two compartments. The initially well defined boundary in (a) becomes blurred in (b) as a result of the diffusion of the dye.

been added to the system. It is assumed: (i) that the dye has no effect on the molecular properties of the liquid; (ii) that the dye can only be detected by a unique spectroscopic method; and (iii) that the second aliquot was added to the first without any mechanical stirring. Two questions need to be considered when analysing the compartmentation of this system. IF’irst~v. to what extent is the svstem in Fi@, 1 comuartmented? Subdivisions within the whole are a necessary condition for compartmentation but they are insufficient without some reference to scale. Although the liquid, which is uniformly distributed throughout the system, contains many discrete molecules it is not compartmented in any usual sense of the term. In contrast, the distribution of the dye ‘IS non-nm’km on a macrosco$c SC&q, wi% a 6>scon%n6@ at ‘rhe ‘oounhary between %e alicpuo& anb soIhe eveis corn~a~rnen~e~wj~~~n~~~ s;vs&m. Sk%&>fi can &‘_?c&7rz?z&e~&~~~~ of &e dye k d&?&&? a&? t&e &U&?&~yJ deikz? Tkere if only one technique that can detect the dye, but the usefulness of this technique for investigating the compartmentation depends on the temporal and spatial resolution of the method. For example, if the detection system has a spatial resolution that is small compared to the dimensions of the system an& 3 ‘rhe measurement \jmescale is Tas1 T&alive to ‘Ihe ti%usive mixing 05 Vne two asiguok3, then ‘rhe position of the boundary will be measured with good precision. If the measurement rate is comparable with the diffusion rate, the position of the boundary is less clear because the discontinuity in the concentration of the dye is replaced by a concentration gradient, and if the measurement rate is slow relative to the diffusion rate then the original compartmentation becomes undetectable. Similarly, the spatial resolution, which depends on the sensitivity of the technique as well as on the design of the detection system, also limits the definition of the boundary. When the spati& resokiron ‘IS _ooor, ?ne kroon&a~ is k%ine& ‘sy a uotiune rakk~ km a plane au& compaicmeutation is unobservable when 1kris u&me kxxornm latp in r3mpatison wiYn the si2e 01 the com_nafimt&s. Thus com_~fimen’ramn is D%~I XTIin%nnrncnV&y k%ne6_&%orn~~n.

NMR and compartmentation

in biological

tissues

243

An important feature of the example in Fig. 1 is the lack of any constraining boundary between the two compartments and the resulting time dependence of the distribution of the dye The investigation of compartmentation in biological systems by microscopy often emphasises the boundaries between the compartments because various methods of staining are available for highlighting the membranes of the subcellular structures. However, defining compartments in terms of their boundaries is too restrictive when spectroscopic techniques such as NMR are used to probe the compartmentation. These techniques detect populations of molecules with particular properties e.g. a given resonance frequency or relaxation time, and evidence for the compartmentation of a molecule is obtained by showing that it exists in two or more populations. This definition of compartmentation is analogous to that used in classical metabolic studies where compartments are identified with quantities of a metabolite that show “uniform and distinguishable kinetics of transformation or transport”.“) In principle an intracellular component A may be partitioned between several populations or intracellular environments: A may exist as a free (hydrated) species in one or more membrane-bound organelles; A may interact with, i.e. be bound to, a variety of other intracellular components including metal ions, small molecules, macromolecules and organelle surfaces; A may be precipitated. In a biological system it is unlikely that the distribution of A will be at equilibrium, with the concomitant requirement for the system to do work to maintain the distribution, and exchange between the different populations may occur over a wide range of timescales. A complete description of the nature, thermodynamics and kinetics of the distribution of A is likely to be essential to a proper understanding of the intracellular role of A. Because NMR is sensitive both to changes in the intracellular environment and to exchange effects, there is thus a considerable incentive to use this technique to probe intracellular compartmentation. Compartmentation also exists at higher levels in biological systems as reflected in the cellular heterogeneity and spatial arrangements of cells in multicellular organisms. Compartmentation on the anatomical or macroscopic scale can be investigated by using NMR techniques in which the signal is a function of the spatial location of the resonating nucleus e.g. using surface coilsJ’@ topical magnetic resonance(‘7) or spin imaging.“s*‘9) This review describes the NMR methods that can be used to distinguish the populations of A in real and model biological systems. The scope of this article is restricted to compartmentation at the intracellular level and although cellular heterogeneity may well be an important factor in the search

a)

b)

0 a

(-tj‘;a 0

._:

0

0

0

FIG. 2. Schematic

preparations,

diagrams of (a) two,(b) three and (c) four compartment systems. The NMR results from vesicie cell suspensions and tissues may be analysed in terms of these models, often with the assumption that any variation in size within the cell population can be ignored.

244

P. S. BELTONand R.G. RATCLIFFE

for this information, e.g. by making it difficult to obtain suitably homogeneous cell preparations, no further mention will be made of the techniques for probing higher levels of compartmentation referred to in the previous paragraph. Specifically, this review considers the methods that have been applied to systems containing up to four populations of A. These systems, which include vesicles, cell suspensions and tissues, may be considered as ensembles or aggregates of the model systems in Fig. 2. For example, suspensions of vesicles or bacteria might be analysed in terms of Fig. 2(a), with a distinction being made between the internal and external populations of A; whereas a leaf tissue might be analysed in terms of Fig. 2(c) with o, a, b and c representing the extracellular space, the cytosol, the vacuole and the chloroplasts respectively. Note that the only physical boundary that needs to exist in these systems is the membrane between the outside and the inside and that the internal partitioning of A does not necessarily correspond to the membrane-bound subcellular compartmentation observable by microscopy. Note also, that while the models used to interpret the spectroscopic data are clearly very simple in comparison with the intracellular complexity that is apparent in any electron micrograph, they provide an accurate discription of the spectroscopic properties of the observed populations of A. Errors are most likely to occur when injudicious comparisons are made between microscopic and spectroscopic data with the aim of specifying the location of the spectroscopically observed population.

3. NMR MEASUREMENTS

ON COMPARTMENTED

SYSTEMS

The assignment of some, or all, of the observed resonances is the immediate aim of any NMR investigation. If the sample is homogeneous, for example a simple solution, it is only necessary to identify the molecular origin of the resonances; but if the sample is heterogeneous, it is necessary to identify the spatial origin as well and it is only when this has been achieved that the spectrum and the spectral changes can be interpreted in terms of the compartmentation of the system. In other words, if a resonance in the spectrum of a heterogeneous system is identified with a molecule A, the assignment is only complete when it has been established which of the possible components A,, A,, At, and A, contribute to the intensity. (In what follows the extracellular compartment will be referred to as o and other compartments as a, b etc.) The problems that arise in assigning the spectra of multicompartment systems can be summarised as follows. Many systems give spectra, e.g. the low resolution ‘H spectra of many cell suspensions and tissues or the 3gK spectra of erythrocytes, that show no chemical shift dispersion and consist of a single resonance that is rarely Lorentzian. The chemical identity of the resonating nucleus presents no problem in such cases, but it is necessary to ask the question: is there any NMR evidence for the existence of more than one component in the resonance, corresponding to a partitioning of the resonating nucleus between different compartments. 7 Similarly in spectra showing several resonances, e.g. the 13C and 31P spectra of living tissues, it is necessary to ask the same question for each resonance and to consider the possibility that a molecule A, e.g. inorganic phosphate (Pi), may contribute more than one set of resonances because it is distributed between compartments with different chemical environments. In principle the various components A,, A,, A,, A, that contribute to the observed resonance(s) can be distinguished by NMR if differences in the chemical or physical environments of the compartments (0, a, b, c) lead to differences in the NMR properties of the resonances from A. All the commonly measured NMR properties have been used for this purpose, including measurements of chemical shift, intensity, lineshape, relaxation times and the diffusion constants determined by pulsed field gradient methods. The application and generality of these different approaches is discussed in the following Sections, but we note here that although NMR may provide evidence for the partitioning of a molecule between two or more environments, the identification of these compartments may be difficult in some cases. This problem can be particularly difficult in the interpretation of water relaxation data (Section 7). A further complication in the interpretation of the data is the dynamic nature of many heterogeneous systems. Exchange between the different compartments will affect the NMR

NMR and compartmentation

in biological tissues

properties of A,, A, etc. in many cases and the possible next Section. 4. EXCHANGE

consequences

of this are considered

245 in the

PROCESSES

A useful way of considering the effects of exchange is to modify the Bloch equations”‘) to take equations. account of exchange. (*I) The modified equations are often called the Bloch-McConnell For simplicity we consider only two sites a and b, the average lifetimes of nuclei in states a and b are 7a and 7b, and the fractions of the nuclei in these states are P, and Pb respectively, the populations are time independent. U and V are the components of nuclear magnetization in phase and out of phase with the rotating radio frequency field and M, is the magnetization along I$,. Following Woessner”‘) the equations can be written,

ua ua

dui,

-(co,-OJW) I+--+-

dt=

2a

dK

x=(wp--w)

v. v,

U.--T--+--cur 2a

dh x=(wb-w)

7a

Ub 7b

vb

7a

7b

7b

7a

M,,

Ub-;-~+~-o, 2b

M,,

dM,a _ 040s-Mm) M,, I Mrb I w v dt

d”zb -=. dt

1

TIa

7a

a

(5)

7b

Mrb I M,, I w v

@‘fob-M,,)

1

Tlb

7b

b

7,

T2 and T1 are the transverse and longitudinal nuclear spin relaxation times; u, and ob are the Larmor frequencies in sites a and b; wi =yB, where y is the magnetogynic ratio of the nuclei and B, is the radio frequency magnetic field strength which rotates at a rate w1 around B,. The solutions to these equations under the appropriate boundary conditions then lead to a series of well known and much used results. Before considering these it is important to realise that a number of assumptions are made which are not necessarily universally valid. These arec2r - 23) as follows. (a) The nuclei make instantaneous transfers from state a to state b. (b) The loss of magnetization by transfer from state a will result in a corresponding gain in state b and vice versa. (c) The relaxation times TaPb are independent of the lifetimes 7+ (d) The relaxation rates are uncoupled except by the exchange process. (e) To calculate the magnetizations following a pulse or train of pulses it is further assumed(22,23) that the radiofrequency field strength B, and the pulse duration t, are such that

In addition it is important to note that the parameter l/7,,, is a first order rate constant(*r) and that if first order kinetics do not apply for the transfer process it is a pseudo first order rate constant. Thus if a reaction of the type A+A%B is involved and k (dm3mol-‘set-‘) is the bimolecular where [A] is the concentration of A in mol dmm3.

rate constant

for the loss of A then 7,’ = k[A]

246

P. S. BELTON and R.G.

RATCLIFFE

Essentially the same assumptions as those given above are implicit in the analysis of exchange processes by stochastic models.‘24,25) Assumption (a) is necessarily an approximation. It implies that the molecule is either in state a or state b, which may be a good approximation for chemical exchange but is not for systems in which exchange is by diffusion. Assumption (b) will be valid if (a) is valid, however as (a) becomes a worse approximation so does the likelihood that there could be loss of magnetization by relaxation during the transfer process. For example this could occur where the rate determining step from state a to state b is translation through a membrane since there will be relaxation during the translation time, r, when these times are of comparable duration. The main problem with these two assumptions is, as Brownstein and Tarr”@ point out in the context of the Zimmerman-Brittin(Z4) equations, that they amount to a discrete rather than a continuous system. In this sense biological systems are continuous, that is exchange processes occur by diffusion between compartments. The diffusion may be between sites where there are no barriers; for example between water hydrating a protein and free water, or, through a barrier of finite dimensions such as a membrane. In either case there is no discontinuous transition from one state to another which can be described by a single time r, rather there will be a continuous distribution of z values(27) corresponding to a diffusion profile. In general such a profile will be strongly dependent on morphology. Assumption (c) may also represent a problem in systems where exchange is by diffusion. The problem in the case of fast exchange has been summarised by WennerstrGm,‘28’ and is essentially that when the exchange lifetime 7ex becomes of the same order as the correlation time governing the relaxation process then it itself will contribute to relaxation. Thus relaxation and exchange become coupled. An example given by WennerstrGm’28) IS when a water molecule exchanges between two environments of different mobility. This situation is clearly pertinent to biological systems. Another is suggested by the inherent anisotropy in many biological systems. If a molecule experiences an anisotropic environment in some region then in this region the static dipolar or quadrupolar interactions will be non-zero. Diffusion between regions of different orientational order will then produce a large contribution to relaxation times, and in particular the characteristic correlation time 7 for this process will strongly affect the value of the transverse relaxation time.(29) This correlation time will depend on the diffusion coefficient of the molecule as it wanders from anisotropic region to anisotropic region, via intermediate spaces with different relaxation characteristics. The exchange time with the intermediate spaces will be related to the correlation time since both are dependent on the lifetime in the anisotropic phase. However since the residual static interactions may be small the correlation time for motional narrowing may be long (in clay water systems 7=0.84 msec(29)), therefore coupling of exchange and relaxation need not imply very rapid exchange rates. A very recent publication by Halle (30) has dealt with this problem in a self consistent way and has pointed out the errors that arise when the continuous nature of diffusive exchange is ignored. Assumption (d) is generally true in compartmental exchange and (e) can be met by adjusting spectrometer conditions suitably. From the foregoing discussion it is clear that the assumptions underlying the Bloch-McConnell equations are not appropriate for compartmented systems. In spite of this their use has been very widespread and very often self consistent results are obtained. This can arise either because the system chosen for study is a good approximation to the ideal one or, perhaps, because the constraints on the model are more elastic than the assumptions imply. However, in general considerable caution is required in the application of these models. Some of the consequences of the limitations of the equations are discussed in Section 7 and in the relevant subject Sections. Even though the applications of the Bloch-McConnell equations should be treated with caution they can be used to illustrate the general effects of exchange and some important exchange limits. One of these is the fast exchange limit. Here 7,: > j(w, -wb)l, G,k. Under these conditions only one resonance line is observed and its frequency w,, is given by (22.23)

%,=Pa %+pb

mbr

(7)

NMR

and compartmentation in biological tissues

247

the line width at half height v is given by, v=P.

v,+Pb

(8)

Vb

and the relaxation times are single exponential and given by

Similar relationships can be derived for a continuous system.(26,31) The opposite limit to fast exchange is slow exchange, and under these conditions

I(w,-wb)l,

T,$ >> T,$.

In this limit of slow exchange two resonance lines are observed whose line widths and resonance frequencies are unaffected by exchange, and the relaxation times associated with each line are also unaffected. Most of the experiments described in Sections 5 and 6 depend upon the assumption that the slow exchange condition obtains. The conditions under which most information on the system is available, and where most complications are likely to occur, are those of intermediate exchange, here t$ -~(o,---ob)J, T&. The simplest parameter to consider under these conditions is ‘& since it is unaffected by the chemical shift. The normalised relaxation curve is given by’2233z’ h(t)= Pi exp( - t/T,‘,)+ Pd exp (- t/T{b).

(10)

These results will also obtain for T2 measurements made from a free induction decay in the absence of chemical shift differences and magnetic field inhomogeneities. In general neither P’ nor T( will represent the actual values of the populations or relaxation times in the sites. Only when T,,mTlb and (rb/Tb)>l are the apparent populations P& approximately equal to the actual populations P,,b.(32)When T,, z=c> Tlb and ~~< Tlb then the relaxation of the b phase is by transfer to the a phase rather than any intrinsic process, thus Pd=O and relaxation is single exponential. The lineshape observed for two chemically shifted sites is given by a series of equations described by Sandstriim. (33)These equations express the well known effects of exchange on lineshape. When t is in the slow exchange limit two separate resonances are seen and the width and position‘ of these are independent oft. As z decreases these lines begin to broaden and move towards each other. They continue to broaden, merge and finally form a single line at the weighted average resonance frequency, and the line then begins to narrow as T further decreases towards the fast exchange limit. The measurement of transverse relaxation by the Carr-Purcell-MeiboomCiill (CPMG) sequence represents a special problem because in this case the pulse spacing becomes an important variable in determining the details of the observed decay. Thus the solutions to the Bloch-McConnell equations for the CPMG sequence may be written as follows’34’ h(t)=K,

exp(t

In&)+K,

exp( if. InA,).

Ki and li are functions of the chemical shift difference, exchange times and relaxation parameters, and tcl, is the 180” pulse spacing in the CPMG sequence. For many cases eqn. (12) must be evaluated numerically,‘34) however, Allerhand and Gutowsky (35)have derived closed solutions for some special conditions. In general three regions of behaviour can be observed depending on the magnitude of tcp.(35) When the pulse spacing is large, i.e. t&’ -4, then relaxation is only weakly dependent on t,, and will be at its most rapid. When top is of the order of T the observed relaxation is very dependent on pulse spacing and declines as tcp declines. When &,I --*cc another limit is approached in which the observed relaxation is slowest and corresponds closely to the intrinsic relaxation rate. The net effect of these phenomena is to generate a dispersion in the observed relaxation as a function of pulse spacing.

P. S. BELTONand R.

248

G.

RATCLIFFE

The Bloch-McConnell equations assume that relaxation at each site is exponential. For nuclei of spin greater than one this assumption is not valid. Hubbard (36) has shown that for spin 312 nuclei, under the conditions of motional narrowing, transverse and spin lattice relaxation in the laboratory frame are biexponential processes. The relative proportions of each component in the process are invariant but the time constants depend upon the spectral density functions. Under conditions of extreme motional narrowing the time constants of both components become equal and single exponential relaxation results. The equations therefore need to be modified to take account of these effects. Bu11t3” has carried out the necessary calculations and shown that in general, for a two site problem, relaxation will be the sum of four exponentials. When one site is in extreme motional narrowing the process becomes biexponential. For systems with spins greater than 312 the behaviour becomes more complex and numerical methods must be used. (38’ Halle has considered the case of diffusive exchange for quadrupolar nuclei in systems with local order.t3’)

5. CHEMICAL

SHIFT

EFFECTS

In general, the most direct and unambiguous NMR studies of compartmented systems are usually made when differences in the chemical environment of the various compartments (0, a, etc.) lead to chemical shift differences in the resonances of a compartmented component (A). The component is usually a solute, although it may also be the solvent or the boundary itself between the compartments. In principle, any change in the chemical environment that affects the chemical shift may be used to distinguish A,, A, etc but in practice the most important changes are those occurring in the presence of pH gradients and those induced by paramagnetic ions. 5.1. Effect of pH Gradients One of the functions of cell membranes is to maintain concentration gradients, more correctly electrochemical potential gradients, between different regions of the cell and between the cell and its surroundings. By this means cells achieve spatial control over many of their intracellular components and maintain intracellular conditions that can be regulated over a wide range of metabolic activity. The regulation of pH is one aspect of this phenomenon and the generation and utilisation of pH gradients, both across the plasma membrane and between intracellular compartments is an important theme in cell biology. The existence of these pH gradients makes it possible to distinguish certain components in certain compartments by NMR. The following criteria must be satisfied if this method is to be applicable: (i) (ii) (iii)

A must have a pH-dependent chemical shift in the pH range encompassed by the different compartments, i.e. d&A)/d(pH) # 0; A must be present in each compartment at detectable levels and it must be in slow exchange between them; the size of the pH gradient (ApH) between the compartments in relation to dG(A)/d(pH) must be such as to cause at least a broadening of the intensity from A and preferably a splitting into more than one resonance.

To what extent can these criteria be satisfied in cellular systems and their models? In a model system, e.g. a phospholipid vesicle suspension, all the criteria can be met by a suitable choice of A and careful manipulation of the internal and external solutions during the preparation of the vesicles. Whereas in a cellular system, there are fewer degrees of freedom: the external compartment (0) can usually be altered in a relatively straightforward way, but the intracellular compartments are often less accessible because the metabolite levels and pH gradients may be tightly controlled making it difficult to perturb a system so that it fulfills the criteria more closely. These general points can be illustrated by reviewing the applications of the pH gradient approach to compartmental analysis. It is convenient to split the description into two parts: the application of “P NMR (Section 51.1) and the application of other nuclei (Section 51.2.).

NMR and compartmentation

in biological

tissues

249

51.1. 3’P Spectra The potential of inorganic phosphate (Pi) as a probe for intracellular pH and thus, via the existence of pH gradients, for compartmental analysis was first demonstrated in 1973.c3’) Inorganic phosphate meets the criteria outlined above in most respects: it is widely distributed in cells in quantities that are well within the detection sensitivity of modern high field spectrometers; the second pK at 6.9 confers a significant pH dependence on the chemical shift of the Pi resonance over a pH range (5.54435) that includes the values (7.0+ 1.0) that are likely to be encountered in cytoplasmic solutions; and Pi exchange across the plasma membrane or across intracellular membranes is expected to be slow in many cases. As a result of this favourable combination of properties, the measurement of intracellular pH, the regulation of the plasma membrane pH gradient and, to a lesser extent, the investigation of intracellular compartmentation are recurring themes in the application of 3’P NMR to living systems.‘6-‘0) H+ transport across model membranes has also by been studied with Pi as the pH-dependence probe, (40+42’ but such studies are far outnumbered the investigation of living systems themselves. Although there may be difficulties in distinguishing Pi, and Pi, in perfused organs such as hearts”) the external signals in the spectra of these systems are usually readily identified by varying the composition of the external medium. In many cases the internal signals are then assumed, or sometimes shown, to arise from a single intracellular compartment, which is usually the cytosol, and the compartmental analysis of the spectrum is complete. However in an increasing number of cases, evidence has been found for intracellular compartmentation in 31P spectra, i.e. for contributions to the Pi intensity from more than one compartment, and we propose to review this phenomenon in the following paragraphs. Note that cellular heterogeneity, i.e. the almost inevitable existence of a range of cell types both in tissues and cell suspensions, could also cause a dispersion in the tissue Pi resonance and this possibility has to be eliminated before an interpretation in terms of intracellular compartmentation can be accepted. For example, the subset of dead cells within the sample has been proposed as the origin of additional Pi resonances in the spectra of a Lolium mult[jlorum suspension culture(43’ and of rat brains in animals dosed with KCN. (44) The effects of sample heterogeneity have also been observed while investigating light dependent pH phenomena in reconstituted bacteriorhodopsin vesicles’4s~46’ and in isolated chromatophores from Rhodopseudomomas sphaeroides.‘47’ One early investigation of intracellular compartmentation focused on the anomalously broad Pi resonance in certain muscle tissues and the possibility that the width of the resonance reflected the overlap of Pi contributions from the sarcoplasm and the sarcoplasmic reticulum.‘48.4g) This explanation, rather than cellular heterogeneity, was eventually shown to be correct, but little further use appears to have been made of the observation. As a result of the pH gradient expected between the mitochondria and the cytosol in functioning cells, separate Pi resonances from these two compartments have been sought in many systems. Isolated liver cells provided an early success: the shoulder observed on the high frequency side of the Pi resonance was assigned to the mitochondrial Pi fraction. (‘O) A resonance has also been attributed to mitochondrial Pi in the spectra of turtle bladder epithelial cells,‘51’ but in general the mitochondrial Pi resonance has proved to be elusive. Bailey et CJ~.‘~‘)discussed the lack of a detectable mitochondrial Pi resonance in heart tissue and emphasised that several factors could be responsible: ApH could be too small to generate a resolved resonance; the quantity of mitochondrial Pi could be too small to be detected; the intensity could be lost from the spectrum because of a broadening mechanism operating on the Pi in the mitochondria. More recently two Pi resonances have been detected in rat hearts and the weak, broad resonance downfield of the dominant cytosolic peak have been assigned to the mitochondria. (53) In a crucial experiment, valinomycin, which caused the mitochondria to swell, was observed to cause an increase in the mitochondrial Pi resonance because the mitochondrial Pi concentration remained constant. Mitochondrial Pi has not been detected in the meristematic tissues of plants(54) and the mixed success that has been reported in the search for this resonance underlines the point that the existence of membrane-bound compartments of different pH does not necessarily lead to separate observable resonances. The mildly acidic vacuole present in many fungi and plant cells has proved to be a more readily detectable organelle than the mitochondrion. Some of the early yeast spectra(s5*56’ showed a third Pi

P. S. BELTON and R. G. RATCLIFFE

250

resonance, in addition to the cytosolic and extracellular Pi and this resonance has been clearly resolved and assigned to the vacuolar Pi fraction in recent work on Candida utilis’57-58’ and Candida albicans.‘5g’ The pH gradient between the vacuole and the cytosol can be much greater than that between the mitochondria and the cytosol and this factor, together with the greater quantity of vacuolar Pi, explains the relative ease with which the vacuolar compartment has been detected in yeasts. The vacuolar compartment often dominates the cell volume in mature plant cells with the result that the vacuolar Pi is often more readily detected than the cytosolic Pi in plant tissues. However the spectra of tissues containing a significant cytoplasmic fraction are characterised by the appearance of two Pi resonances, separated by up to 1.9 ppm in well aerated samples,‘60) and this striking demonstration of the non-invasive detection of intracellular compartmentation by NMR makes it possible to study the H+ and Pi gradients across the tonoplast membrane separating the cytoplasm and the vacuole.(61*62) Table 1 summarises the observations of cytoplasmic and vacuolar Pi that have been made in plant tissues and plant cell suspensions and Fig. 3 shows a representative spectrum. The meristematic tissues in the root tips of maize and pea seedlings have been favoured because of the high cytoplasmic fraction in the tissue, arising from the large population of rapidly dividing, immature cells with small vacuoles. In contrast, the spectra of many cultured plant cells are disappointing because of the fully vacuolated nature of the cells.(s*) In principle, it should be possible to detect three intracellular Pi fractions in photosynthetic plant tissues: the vacuolar Pi (pH m 5.5), the cytoplasmic Pi (pH N 7.4), and the Pi in the chloroplasts. The latter should be dominated by the contribution from the stroma and should be resolved when the sample is illuminated since the stromal pH becomes more alkaline than the cytoplasmic pH in the light.@‘) Unfortunately, the examination of leaf tissues is hindered by the broadening effects of the intracellular air spaces and free paramagnetic ions(75*76**8) and so most 31P NMR studies of the intracellular compartmentation of plant tissues have been restricted to non-photosynthetic tissues

TABLE

1. “P

NMR observation inorganic phosphate

of cytoplasmic in plant tissues

and

References

Plant material Excised root tissues: Zea mays (roots and root tips) Pisum satioum (roots and root tips) Fagus syluatica (mycorrhizal root tips) figna mungo (roots and root tips)

60.63-73 54 61 74

Excised leaf tissues: Triticum aestivum

75

Cell and protoplast preparations: Asparagus ojjicinalis (cells) Spinacia oleracea (protoplasts)

76 76

Cell suspension cultures: Acer psuedoplatanus Catharanthus roseus Elaeis guineensis Glycine max Humulus lupulus Lolium multiforum Nicotiana tahacurn Petroselinum hortense Rosa damscena

77-81 77,78 82 77,78 83 43 84 85 86

Much of the work summarised (61,62).

vacuolar

above has been reviewed

elsewhere

NMR and compartmentation

Cytoplasmic I

I

10

yacuolar

in biological

251

tissues

Pi Pi

I

I

0

-10

I

PPm

FIG. 3. 121.49 MHz 31P NMR spectrum of an oil-palm (Ha& guineenis) cell suspension showing the resolved resonances from the cytoplasmic and vacuolar inorganic phosphate (Pi). Reprinted by permission from Bioscience Reports 3, 1141-l 148, copyright 0 1983, The Biochemical Society, London.

(Table 1). Waterton et Al. using a technique which apparently lacks generality,(“) eliminated the air spaces in wheat leaves by vacuum infiltration, and recorded a spectrum showing two Pi resonances. The larger resonance was assigned to the vacuolar Pi, while the smaller ‘extravacuolar’ resonance was tentatively assigned to the stromal Pi on the basis of its light dependent chemical shift. The absence of a cytosolic Pi resonance, as well as the absence of mitochondrial and intrathylakoidal Pi resonances, was rationalised in terms of the very small fractional volumes occupied by these spaces. Recent experiments on the green alga Chlorella suggest that this organism may be more suitable than leaf tissue for investigating intracellular compartmentation during photosynthesis. Two Pi resonances have been observed in the spectra of Chlorella vulgar-isand assigned to the stromal and cytoplasmic fractions;‘89*90) while the two Pi resonances observed in Chlorellu fusca have been assigned to the chloroplast and the vacuole. (91) The significance of this difference in assignment is not yet apparent, but further work on the Chlorellu system can be expected since light dependent spectral changes are readily observed with both organisms. Light dependent phenomena have also been reported in a number of photosynthetic bacteria, including Synechococcus,“*’ Chromatium vinosum(93s94)and Rhodopseudomonas sphaeroides,‘95’ but while the pH gradient between the cells and their surroundings can be readily monitored, there is no evidence for intracellular compartmentation of the Pi between the cytoplasm and the intrathylakoidal space. Examination of the isolated chromatophores from R. sphaeroides has also been undertaken’47*96) and a marked heterogeneity in the light dependent behaviour of the samples has been observed. Koyama et al.(96) observed two internal Pi resonances in their illuminated samples, but they were unable to distinguish between the possibilty that the sample contained two different types of chromatophore and the possibility that each chromatophore contained two different regions of pH. This type of assignment problem occurs frequently in heterogeneous systems and is one for which there is no general solution.

252

P.S.BELTONand R.G. RATCLIFFE

Although Pi is the most frequently used phosphorus probe for compartmental analysis, several other compounds with pH-dependent chemical shifts have also been used. Monoesters of phosphoric acid such as glucose-6-phosphate, 2-deoxyglucose-6-phosphate and mannose-6phosphate have a pK, of approximately 7 and they can be used to probe pH gradients in exactly the same way as Pi. For example, glucose-6-phosphate has been used to monitor light dependent pH changes inside reconstituted bacteriorhodopsin vesicles(45s46’ and it is often used to confirm estimates of the cytoplasmic pH based on the chemical shift of the cytoplasmic Pi resonance in living systems. Similarly, the chemical shifts of 2-deoxyglucose-6-phosphate(52’ and mannose-6phosphate (67) have been used to identify the intracellular location of these compounds in various tissues. The 31P resonances from ATP and ADP are also pH dependent and internal and external ATP resonances have been observed in the spectra of rat liver mitochondria’97) and chromafhn M ore recently, granules extracted from the adrenal medulla.‘9*-‘00) the cytoplasmic and intragranular ATP resonances from the intact, excised pig adrenal gland have been observed by exploiting the pH gradient between the cytoplasm and the relatively acidic granules.(iol) Although this Section has emphasised the importance of pH and intracellular pH gradients in the application of 31P NMR to compartmental analysis, factors other than pH can be important in determining the chemical shift of a phosphorus compound in uivo. For example, compartmental differences in the Mg*+ concentration contribute to the resolution of the ATP resonances in the work cited above.““’ As is now widely appreciated, the existence of these other factors is particularly important in the determination of intracellular pH and care is needed in setting up an appropriate calibration curve for the pH dependence of a chemical shift.(102) 5.1.2. ‘H, 13C and “N Spectra. In principle, any nucleus with pH dependent chemical shifts can be used to probe pH gradients, but there has been only limited interest in nuclei other than 31P because very few molecules have pH dependent chemical shifts in the physiological pH range. ‘H NMR has been used to distinguish internal and external maleic and fumaric acids in phospholipid vesicles, allowing an investigation of the pH induced transport of the carboxylic acids across the membrane.‘103-105) The pH dependence of the C2 ‘H resonance of the haemoglobin histidine residues has been used to estimate in vivo, the pH inside red blood cells,‘io6) but there appear to have been no ‘H NMR investigations of intracellular compartmentation. In contrast, the i5N resonance of the Nl nitrogen of 15N-labelled histidine in intact mycelia of Neurospora CMSSU(~ “I and the ’ 3C resonances of ’ 3C-labelled malate in the leaves of the CAM plant Kalanchiie t~b~jlorn(‘~~)have been used to probe the vacuoles of these tissues and to obtain values of the vacuolar pH. 31P studies of the acidic vacuole are restricted by the limited pH dependence of the chemical shift of the Pi resonance below pH 6, and these two papers indicate that the 13C and 15N resonances of organic acids and aminoacids could be useful for probing this organelle in plants and fungi. Finally, the pH gradient across the plasma membrane has been used to distinguish the internal and external 13C signals from succinate in E. coli(’ 09) and lactate in Staphylococcus uureus.(11o) 5.2. Effect of Paramagnetic Ions The binding of a paramagnetic ion to a molecule causes the resonances from the nuclei close to the binding site to shift and broaden as a result of the interactions between the unpaired electron density associated with the paramagnetic ion and the nuclear spin. Cl‘I) In many cases, one of the two effects on the resonance predominates and since the geometric dependence of these changes is well understood, shift and broadening probes can be used to investigate the geometry of the binding site. Lanthanide (III) ions have good magnetic properties for this purpose and they have been widely used in conformational analysis.” ’ *) The shifting and broadening properties of paramagnetic ions are readily exploited in compartmented systems by restricting the ion to one compartment, usually the extracellular space. All components with suitable binding sites, whether solvent, solutes or boundaries, will be affected by the paramagnetic ion and the internal and external populations can be distinguished in the the non-uniform distribution of a absence of unfavourable exchange effects. In addition,

NMR and compartmentation

in biological tissues

253

paramagnetic ion in a compartmented system sets up magnetic field gradients as a result of the nonuniformity in the magnetic susceptibility and this can also cause differences in chemical shift and linewidth between components in different compartments. Experiments which exploit the changes in chemical shift are described in this Section; while experiments which depend on paramagnetic ion induced changes in linewidth, intensity and relaxation times are considered in Section 6. The principles of using chemical shifts for compartmental analysis are the same irrespective of whether the origin of the chemical shift changes is Hf binding or paramagnetic ion binding, but the emphasis in the application of shift reagents has been different from that described in the previous Section. In contrast to the work on pH gradients, there is little opportunity to exploit naturally occurring intracellular levels of paramagnetics and little opportunity to study intracellular compartmentation. Much more work has been published on vesicles using shift reagents and the emphasis, both in the work on model systems and in their biological counterparts, has been on the kinetics and mechanism of transmembrane ion transport. It is convenient to divide a description of this work on the basis of the change of the shift reagent. 5.2.1. Shift Reagents with a Positive Charge. One of the earliest reports of this approach was the use of Co(U) ions in the external medium to separate the contributions from the intracellular and extracellular water in the ‘H spectra of frog nerves. (i 13, The success of this experiment depends on the impermeability of the cell membrane to Co(I1) and the relatively slow exchange of the solvent between the two compartments. It is surprising that the possibilities inherent in shift reagents have not been exploited more in the study of water compartmentation. In particular there is a strong possibility of combining these with the relaxation measurements, described in Section 7, to assign extracellular components. So far only one study using shift reagents in combination with relaxation time measurements has been reported(243) (see Section 7.4). Much more interest has been shown in the use of lanthanide cations to distinguish between the inside and outside compartments of vesicle suspensions. In these experiments, vesicles are prepared with a shift reagent, usually Pr3+ or Eu3’, localised in either the internal or external space and the interaction between the paramagnetic ion and the phospholipid headgroups sets up a chemical shift difference between the internal and external headgroup resonances which can be observed in the 1H’1’4,115) 13c”l6’ and 31p(117) spectra. Figure 4 illustrates the effect of external Pr3+ on the ‘H spectrum of phosphatidycholine vesicles.

?uter

choline

headgroups

I Inner

4

choline

3

headgroups

2

I

FIG. 4. 90 MHz ‘H NMR spectrum of egg yolk phosphatidylcholine

0

mm

vesicles showing the resonances from the internal and external choline headgroups. Reprinted by permission from Bioscience Reports 4, 4033413, Copyright Q 1984, The Biochemical Society, London.

P. S. BELTONand R. G. RATCLIFFE

254

Vesicles prepared under these conditions are stable for long periods and the membranes are impermeable to the lanthanide cations. However if a pathway is introduced between the inside and outside compartments, e.g. by adding an ionophore to the compartment containing the paramagnetic ion or by incorporating a channel-forming peptide such as gramicidin A in the membrane, it is possible to model transmembrane cation transport by monitoring the time dependence of the internal and external boundary resonances. Although restricted to vesicle systems (because useful boundary signals are not generally detected in cellular systems) this has proved to be a valuable approach to the investigation of both carrier-mediated and channel-mediated transport. c1‘*-‘23) Springer and coworkers Cl“) have reviewed the literature on this subject (Table 1 in Ref. 120) and have presented a detailed analysis for the timecourse of the inner headgroup resonance for a number of different transport mechanisms. It has been shown that processes that allow the ions to enter the vesicles singly are readily distinguished from processes in which the ions enter in large bursts. Other applications of the same approach include the use of Pr3+ to resolve the internal and external acetic acid resonances in a ‘H NMR study of the transport of acetic acid across vesicle membranes’rz4) and the use of Co(H) to monitor intravesicular precipitation reactions.‘45.125’ In the latter case, precipitation of CoS or Co(OH), was followed by observing the reduction in the chemical shift difference between the internal and external ‘H-N(CH&+ resonances as the shift reagent was removed from solution. 5.2.2. Shift Reagents with a Negative Charge. Although anions such as Fe(CN),3- act as shift reagents and can be used to differentiate the internal and external surfaces of vesicles by virtue of their binding to phosphatidylcholine headgroups, c1*w*‘) the major recent development in the use of anionic shift reagents for compartmental analysis concerns the discovery of a series of complex anions that bind to metal cations in aqueous solution. 02*-134) These anionic shift reagents (Table 2)

TABLE2. Anionic shift reagents for alkali metal cations, Shift reagent

Acid anion

CDYWWJ-

tripolyphosphate,

[DYW’N,I~-

Reference (P,o,,I5-

dipicolinate,

CDY(W,I

6-

-

nitrilotriacetate,

129,130

/

1 CDyW’AL13

128,131,134

2-

coo

‘coo t N(CH,COO), I’-

130

chelidamate,

132

triethylenetetraminehexaacetate,

(OOC.CH,~2N~CH,l,

133 N(CH,&

N iCH,),N(CH,C00)2

I I CH,COO CH,COO

1 6-

Thulium analogues of several of these complexes have also been used. (r3*~t3a)The shifts induced by the thulium complexes are smaller in magnitude and opposite in sign to the dysprosium induced shifts.

NMR and compartmentation are characterised

in biological tissues

by a high charge and as well as shifting

the resonances

255 of the alkali metal cations

(‘Li +, 23Na+, 39K+, *‘Rb+ and rJ3Cs+) they can be used to shift the resonances of 14NH4+ and *‘Mg*+. With an anionic shift reagent in the external compartment, it is possible to resolve the internal and external resonances of the physiologically important cations 23Na and 39K, (Fig. 5) making it possible to study the membrane transport of these cations in vesicles and living cells(135*136)(Table 3).

Internal K+

I ExternalK+

I

I

I

10

0

-10

I

wm

FIG. 5. 16.8 MHz s9K NMR spectrum of human erythrocytes suspended in a medium containing 60mM K+, 6mM Dy3+ and 15mM tripolyphosphate showing the resolution of the intracellular and extracellular potassium. Reprinted by permission from the Biochemical Journal 210,961-963. copyrightOl983, The Biochemical Society, London.

TABLE 3.

NMR studies of compartmented

systems using anionic shift reagents.

System

Shift reagent

Large unilamellar vesicles Human erythrocytes

CDy(NTA),l” CDy(PPP)J[Tm(PPP),]‘CDY(T-THA)I’[Tm(lTHA)]“CDy(PPP),l’ CDy(DPA),l”CDy(NTA)JCDy(PPP)JCDy(PPP),l’CDy(PPP)JCDy(-fTHA)l’-

Yeast cells Millet cells Frog skin Rat hearts

Nucleus

Reference 137 131,136,138,139 139 139 139 140 141 141 142 143 144 145

The application of Z3NaNMR to a number of other systems has been reviewed(‘36)

Three general questions arise cell or vesicle membranes or are any adverse metabolic effects on is represented by the observed

when using this technique. (1) Can the shift reagents penetrate the they confined to the external medium? (2) Do the shift reagents have living systems? (3) What fraction of the total tissue Na or K content internal 23Na and j9K resonances? The first two questions are

256

P. S. BELTON and R. G.

RATCLIFFE

straightforward and there is general agreement in the work summarised in Table 3. There is no evidence for a significant uptake of the complexes by the systems in Table 3 over the timescales required for the experiments and the metabolic and physiological influence of these external ions is slight or negligible. For example, [Dy(PPP),]‘has been shown to be innocuous to red blood cells over a 3 hr timescale” 3*) and to have no adverse effects on the electrophysiological responsiveness of frog skin.044) As regards the quantitative analysis of the spectra, complications can arise because 23Na and 39K are spin-3/2 nuclei and the nuclear quadrupole interaction can have a profound effect on the resonance lineshapes.(146~147) Fo r example, in the case of fast exchange between a very small immobilised pool and a large free pool, 60 ‘A of the intensity will be broadened beyond detection so that the intensity of the observed 23Na or 39K resonance is only expected to correspond to 40 y0 of the total cation content. These effects were poorly understood when in uiuo 23Na NMR first became fashionable(‘46~147) but the significance of the quadrupolar interaction and the need for rigorous quantitative analysis in this type of work is now widely recognised. Detailed quantitative analyses have been carried out in a number of the studies referred to in Table 3. Peak intensities corresponding to the total tissue content, within experimental error, have been reported for 23Na(138,1 39) and 39K(140) in red blood cells, in agreement with the much earlier 23Na data for erythrocytes obtained without the operational advantages of the shift reagents.‘148) In the light of this general agreement, the significance of the 30 % shortfall in 23Na intensity that has been reported for well packed erythrocyte suspensions (13~) is unclear. Quadrupolar broadening in an asymmetric electric field would eliminate 60 y0 of the intensity; but intracellular compartmentation of the Na+, with only one compartment affected by the quadrupolar broadening, or immobilisation of a fraction of the intracellular Na+, with slow exchange between the bound and free forms, could explain the 30 % intensity 10~s.~’36) Springer and coworkers (139) have drawn attention to the lineshape of the intracellular 23Na resonance observed in fresh erythrocytes and have shown by computer simulation that it is the sum of narrow and broad components representing 42 f 10 y0 and 58 f 10 % respectively of the total intensity. A possible explanation is that there are two Na+ pools in slow exchange and each pool gives rise to a motionally narrowed spectrum. The pools would not necessarily be intracellular, since they could arise because of non-uniformity in the cell population. However the preferred explanation assumes the existence of two intracellular pools of very different size in fast exchange, with the small pool experiencing slower electric field gradient fluctuation rates.“39) In yeast cells the broad component is undetectable with the result that the observed 23Na and 39K resonances represent only 40 y0 of the total tissue cation contents. (142) The loss of intensity in the tissue samples can be nicely demonstrated by permeabilising the membranes and observing the increase in the resonance intensity as Na+ or K + is transferred to the external medium.

5.3. Miscellaneous Effects A number of other effects, in addition to those caused by protons and paramagnetic ions, have been exploited in compartmental analysis, particularly in vesicle systems. References to a few of these approaches are mentioned here, but without any attempt to achieve a comprehensive or definitive coverage of the literature. groups in phosphatidylcholine vesicles give rise to The internal and external -N(CH3)3+ resolvable ‘H resonances.” 27~149~1 50) A similar effect is observed in the 31P spectra at 36.43 MHz, with the internal and external phosphorus resonances at different chemical shifts, and this was attributed to a shielding effect associated with the greater density of negatively charged phosphate groups on the inside surface of the vesicles. (12’) The resonances of some solutes show an inherent chemical shift difference across a vesicle membrane, e.g. cadmium ions(r5 ‘) and the tetramethylammonium ion in dimyristoylphosphatidylcholine vesicles,(152) presumably reflecting the interactions of the cations with the two surfaces. Ring current shifts have been used to resolve the internal and external S-hydroxytryptamine ‘H resonances in vesicles loaded with ATP.” ’ 3,

NMR and compartmentation 6. INTENSITY

in biological

tissues

257

MEASUREMENTS

In the absence of appreciable chemical shift effects, the compartmental analysis of NMR spectra hinges on identifying the number of components that contribute to the observed resonances. Relaxation measurements provide the main solution to this problem and our account of this approach starts in this Section with a description of various experiments in which changes or losses of spectral intensity have been interpreted in terms of the compartmentation of a heterogeneous system. 6.1. Kinetic Experiments

Under appropriate conditions, some systems may show time dependent ,resonance intensities reflecting the permeability of the membrane boundaries to particular solutes. For example, the intracellular and extracellular 23Na signals from millet cell suspension cultures have been resolved using [Dy(PPP),]‘as a shift reagent for Na+(see Table 3) and the timecourse of the internal Na+ resonance intensity has been followed under conditions of net efllux or influx.(143) The efRux data could be fitted to a two compartment model with the fast component (t,,,=9.15 min) corresponding to 23 y0 of the initial intensity, apparently reflecting Na+ eftlux from the cell wall and the cytoplasm, and the slow component (t 1,2= 506 min) corresponding to 77 % of the initial intensity, apparently reflecting Na’ efflux from the vacuole. (‘43) The authors suggested that similar techniques could be useful in monitoring potassium fluxes, but preliminary experiments on root tissues at a 39K frequency of 9.33 MHz indicate that the low sensitivity of the 39K nucleus could be a problem in achieving this objective (R. B. Lee and R. G. Ratcliffe, unpublished results). Biphasic sodium efflux kinetics were also observed in a 23Na NMR study of Na+ transport in yeast cells/‘35) but the possible compartmentation of the sodium was apparently not considered as an explanation of the effect. A similar approach to compartmental analysis has been used in a 14N NMR study of plant roots(154) (Fig. 6). In the case of nitrogen it is possible to observe the depletion of the 14N nitrate or ammonium signals as the biological material equilibrates with an external medium containing the

c

.

2’

’ 0

2

4

6

6

Time l hr I

FIG. 6. Time dependence of the peak height of the ammonium resonance in the 21.68 MHz “‘N spectra of excised mature roots from barley seedlings grown with ammonium as the external nitrogen source. The circulating external medium contained an aerated buffer, alone (A), or with added 14NH4’@) or 15NH4+ (0). Biphasic timecourses were observed with A and l . Reproduced from Ref. (154).

258

P. S. BELTONand R.

G.

RATCLIFFE

i5N analogue. This type of experiment is equivalent to the conventional radiotracer technique for the compartmental analysis of plant tissues, in which the tissue is brought to a steady state with respect to an external solution and then the isotopic, but not chemical, composition of the solution is changed. o55, 15~) Timecourse experiments on the nitrate resonance provided no evidence for the existence of more than one large pool in the roots of barley and the results were consistent with a model in which the bulk of the nitrate was vacuolar; whereas timecourses of the ammonium resonance in ammonium grown barley roots were biphasic under certain external conditions, suggesting some form of ammonium ion compartmentation in the tissue.“54’ 6.2. Relaxation Effects As is widely appreciated, it is the freely mobile low molecular weight compounds that are usually detected in the high resolution spectra of biological systems. ‘W The fraction of a metabolite that is immobilised by precipitation or by tight binding to cell walls, membranes or macromolecules is undetectable because the reduction in the motional correlation times invariably leads to efficient spin-spin relaxation and line broadening. It follows that a quantitative comparison between a resonance intensity and an independent assay of the corresponding metabolite could lead to an estimate of the extent to which the metabolite is partitioned between free and bound forms in uiuo. In practice, this apparently straightforward procedure can be frustrated by uncertainties in the analytical methods. In the full quantitative analysis of an NMR spectrum, where the aim is to convert peak intensities into absolute amounts per unit fresh weight of material, it is necessary to calibrate the peak intensities and to determine the quantity of the tissue that is within the detecting region of the probehead. This approach can be both tedious and difficult, with the result that NMR is more usually used to measure relative concentrations in living tissue.‘6,157’ In addition, exchange effects can mix the intensities of the free and bound forms (Section 4) and these effects can be particularly serious for quadrupolar nuclei with I > 1 (Section 52.2.). Conventional analytical procedures rely on freeze clamping, in which the tissue is frozen rapidly and the metabolites are assayed in a suitable extract. This destructive method gives the total quantity of a metabolite in the tissue, i.e. the sum of the free and bound fractions, on the assumptions that enzymic activity is stopped immediately during the freezing and that no errors arise during the subsequent handling of the extract. Good quantitative agreement has been found between the NMR spectra and the conventional chemical analysis for various metabolites in various tissues e.g. for phosphocreatine in muscle’158) and for nitrate in barley roots.‘154) In contrast, 31P NMR detects less than 10% of the expected quantities of inorganic phosphate and adenosine diphosphate in many mammalian tissues.‘6.10’ Shortcomings in the analytical procedures appear to be minimal, suggesting some form of compartmentation between free and bound forms, e.g. in skeletal muscle, ADP may be partitioned between the cytosol and a fraction that is tightly bound to the actin, but to a large extent these discrepancies have not been resolved.” ‘i Natural abundance ’ 3C NMR studies of unicellular algae have shown that only -60 % of the major organic osmoregulatory metabolite (glycerol) is visible in the spectra of Dunaliella salina, whereas all of the corresponding metabolite is detected in Synechococcus sp. (159) A possible explanation for this discrepancy is that the NMR invisible fraction represents the glycerol in the chloroplasts!’ 59) The distinction between free and bound forms depends on an extreme difference in relaxation behaviour that causes a change in the detectable resonance intensity. The intrinsic differences in relaxation between different compartments may be much smaller and these effects are most frequently exploited in the compartmental analysis of systems that show no chemical shift dispersion, using the time domain experiments described in Section 7. Intrinsic differences in relaxation are used only occasionally in high resolution studies of compartmented systems: e.g. indirect evidence for the compartmentation of ATP in heart muscle has been obtained by saturation signals from intracellular and extracellular Pi in liver have transfer experiments;” 60) overlapping with been distinguished using Tl measurements, .(161) the existence of two water compartments different chemical shift properties in leaf tissues has been supported by Tl data.” 62)

NMR and compartmentation

in biological

tissues

259

Changes in relaxation can be induced by the binding of paramagnetic ions (III) and broadening probes have been used in compartmental analysis in much the same way as shift probes (Section 5.2.), with the emphasis on studying transmembrane transport in vesicle systems. The aqueous manganese (II) cation has been used to differentiate between the ‘H signals from internal and external water’113’1 63) (see Section 7), to eliminate the external phospherol pyruvate (PEP 31P between signal in a study of PEP transport across erythrocyte membranes (164) and to distinguish probes include the internal and external surfaces of vesicles. (115*165-167) Other possible broadening which broadens water resonances,‘16g.170’ and Gd3’, Cr(CN)63-,“6*) dextran-magnetite, [Gd(EDTA)]which has been used as a relaxation agent for cations in a study of sodium and lithium transport in vesicles.(“‘) in living systems, e.g. it has Although Mn ‘+ is usually used to label the external compartment been used to show that the polyphosphate resonance observed in the ‘lP spectra of yeast cells includes a contribution from an extracellular fraction,“72’ Mn2+ uptake by maize root tips has also of the vacuolar Pi resonance has been been monitored by 3’P NMR and the selective broadening interpreted in terms of the preferential location of the paramagnetic ion in the vacuolar compartment.‘67’ 6.3. Susceptibility

Effects

The demonstration that spin-echo methods could be used to detect the ‘H resonances of low molecular weight metabolites in red blood cells Cl06) has stimulated considerable interest in the use of ‘H NMR to probe erythrocyte metabolism. (‘73~174) In these experiments, the observed resonances are selected on the basis of their spin-spin relaxation time, by varying the delay between the 90” and 180” pulses, with the result that the sharp resonances of the molecules and residues with long relaxation times are resolved from the broad lines of the slowly tumbling macromolecular components of the cells. During the course of this work, an ingenious method has been developed that exploits the heterogeneity of the cell suspension and makes it possible to identify the compartmental location of the observed metabolites.” ” - ’ “) The differences in magnetic susceptibility between different regions of a heterogeneous sample create magnetic field gradients which can cause linebroadening and a reduction in T;, the spinspin relaxation time measured in a spin-echo experiment using a single refocusing 180” pulse. These undesirable effects can be avoided by susceptibility matching and this technique has been used to reduce the linewidths in powdered solid samples’178) and high resolution seed spectra.(179) Similar field gradients have been observed in suspensions of deoxy red cells and again the undesirable linebroadening and chemical shift effects can be eliminated by susceptibility matching.(‘80) In contrast, the spin-echo method of compartmental analysis depends on increasing the magnetic susceptibility difference between the inside and outside of the erythrocytes. This is achieved by adding to the external medium a complex of a paramagnetic ion, e.g. the diethylenetriamine pentaattic acid complex of dysprosium (Dy DPTA) or the desferrioxamine complex of iron (III).(175) The complexes remain in the external medium and the resulting large difference between the magnetic susceptibilities of the two compartments sets up strong field gradients in the extracellular space. Field gradients also exist inside the cells but these are expected to be smaller so that the internal magnetic field remains relatively uniform. Since T: reflects the local magnetic field gradients, the intensity observed in the spinecho sequence for a molecule in the extracellular space is more rapidly attentuated than the intensity of the same molecule in the intracellular space. Thus the detection sensitivity depends on the compartmental location of the observed molecule and by a suitable choice of the paramagnetic concentration and the delay before the refocusing pulse, it is possible to eliminate the external signal from the spectrum. The transport of a small molecule, such as alanine or lactate, from the external medium to the intracellular space is associated with an increase in the resonance intensity and this provides a direct method for studying the kinetics of membrane transport (i”) (Fig. 7). The importance of the field gradients has been confirmed in a detailed series of T2 measurements focused on the on suspensions of chicken and human erythrocytes. (“w This empirical investigation

Alanine

Lactate

2 min 4min Bmin 14min

35min

1

10

1

1

8

1

I

I

,

6

1

4

1

2

I, , 0

wm

MHz ‘H spin-echo NMR spectra of a red blood cell suspension during an L-alanine influx experiment. The inverted signal at 1.4 ppm is the unresolved doublet of the alanine methyl group and its intensity increases as the amino acid is transported from the extracellular to the intracellular medium. Reprinted by permission from the Biochemical Journal 180, 374, copyright 0 1979. The Biochemical Society. London. FIG. 7. 270

of TTon the diffusion coefficient for the observed molecule, the boundary separation between the erythrocytes and the size and distribution of the field gradients. It was concluded that T: was dominated by diffusion through the field gradients generated at the convex surfaces of the m_em_brane bo1undaries and that the Co~_nartmentnl !ncatin!J Qf a sE_a!! m&cl& CGlJ!d be identified r-- ----------

dependence

on the basis of the observed T: value. “‘w The comparison between the two compartment human erythrocyte suspension (i.e. external medium and cytoplasm) and the three compartment chicken erythrocyte suspension (i.e. external medium, cytoplasm and nucleus) was particularly instructive and the predicted three component spin-echo plot was observed for dimethylsulphoxide (DMSO) in a chicken erythrocyte suspension. DMSO equilibrated throughout the system and in keeping with the cellular field gradient model, the largest T: was observed for the nuclear component.” ‘W The generality of this method in practice is difficult to assess since, with the exception of a study of the glycerol permeability of the cytoplasmic membrane of the alga Dun&e/la salina,(181) almost all of the published results have been obtained with erythrocytes.(175-177*‘81 -184) The spin-echo intensities in erythrocyte spectra have been shown to depend on the osmotic pressure of the external medium(‘s4’ and this result, which arises from the dependence of the cell volume on the osmotic pressure, emphasises the importance of the cell geometry in determining the distribution of the field gradients and hence the T: vaiues. The extent to which the variation in cell volume in cell suspensions that are less homogeneous than erythrocytes complicates the analysis of the T: values has yet to be determined.

7. RELAXATION 7.1. The Nature of Relaxation

TIME EXPERIMENTS

in Compartmented

Time domain experiments in compartmented relaxation time measurements of water and Historically this arose because of the controversy and because of the relative ease with which it can

Systems biological systems have tended to concentrate on ions, with water receiving the most attention. over the nature of water in biological systems” *5) be studied. More lately interest has centred on the

NMR

and compartmentation in biological

tissues

261

role of water proton relaxation times in NMR imaging. (ls6) Measurements on ions show little evidence of compartmentation(‘46*147) and will not be considered in detail here. The nature and significance of water relaxation behaviour in biological systems has been the subject of a number of reviews.“87-194) Th e general features of relaxation in these systems may be characterised as follows.“95’ (i) (ii) (iii)

(iv)

Spin lattice relaxation is slower than transverse relaxation. Transverse relaxation is much faster than in bulk water. Upon reducing the temperature sufficiently the bulk of the water present turns to ice. However there always remaines a residual signal from unfrozen water which often persists down to very low temperatures. Very often relaxation is a multiexponential process.

The.details of the interactions which give rise to these phenomena are still not resolved, but a consensus view seems to be that most of the effects have their origin in the interactions of a fairly small fraction of water interacting with slow moving constituents such as protein and cell walls. Interactions with paramagnetic species may also be important in plants (see Section 7.4). Water relaxation has been studied by observing one of three nuclei--‘H, ‘H or “0. The latter two are usually observed at some degree of enrichment. Protons, naturally, have been the most studied species. Observation of proton relaxation is complicated by signals from components other than water, but replacement of water by deuteriated water usually makes assignment fairly unambiguous. Interpretation of the relaxation characteristics can be complicated by the possibility of chemical exchange with labile protons, particularly from proteins, and cross relaxation effects. Deuterium resonance does not suffer from cross relaxation effects or signals from other components, however the exchange problem remains. “0 relaxation suffers from none of these difficulties but has intrinsically multiexponential relaxation as a result of its nuclear spin quantum number (I = S/2). It is possible to show however’ 196) that T1 will be single exponential, to a very high degree of approximation, where a single nuclear environment exists. Transverse relaxation on the other hand is likely to be multiexponential in circumstances where the spectral density at zero frequency J(0) is significantly greater than that at the Larmor frequency J(oO). This condition obtains in most heterogeneous systems. (19*)It has been argued that in systems where exchange is occurring between sites of extreme motional narrowing and sites where slower fluctuations are occurring that transverse relaxation in spin 5/2 and 7/2 nuclei will be very nearly exponential.(L97) However results for the spin 7/2 nucleus 133Cs in polysaccharide gels(19*3’99)sh ow that this is not the case, which may be a result of rigid binding to the macromolecule, or to anisotropic motion. The point to be taken is that it is dangerous to assume a priori that transverse relaxation will be single exponential in concentrated macromolecular systems such as occur in biological tissue. The most commonly observed features in proton and deuterium relaxation are that (in the absence of relaxation agents) T, processes are single exponential and TIP and T2 processes are multiexponential. “0 relaxation is generally multiexponential in both Tl and T,. Apart from transverse relaxation in ” 0 the most general explanation put forward for these phenomena is that there is a nuclear exchange process between the different compartments present in these systems. For protons and deuterons these processes are fast on the time scale of T1 but slow on the timescale of TIP and T2. “0 relaxation is intrinsically much faster than that for hydrogen nucleides and thus exchange is slow on the time scale of all relaxation processes. This view is not universally accepted however and important objections have been raised to this interpretation. Two types of alternative explanations have been proposed: one based on a diffusion dominated processes and the other on a residual static interaction effect. In common with the compartmentation model these are based on the magnetic inhomogeneity of the system in that they depend on the existence of sites of varying relaxation characteristics, however it is the origin of these sites and the nature of their effects on relaxation which is the subject of varying interpretation. Lillford, Clark and Jones”” have observed that multiexponential relaxation occurs in a large variety of inhomogeneous systems, many of which have no obvious compartmentation. They give examples of soya protein fibres and agarose gels, after a freeze thaw cycle, as well as systems such as

262 mu&

P. S. BELTON and R. G. RATCLIFFE and meat in various forms. Fung ancl fuon 1200)offer support for this observation with their

expelirmenk3on gI_ycerinated mu&e. In this mateita1,. the ceI1 walk aad sarca@smic removed but the myatibrillar strands remain intact. Multiexpanential

Aaxatian

ceticuhm are

is still observed

how~ver.UGorb anh co-workers av!ueYnat agenerA exfiana%on OEVne e%ecl musl’bebaseb on he role of diffusion in exchange processes. They consider a system in which the interfaces between polymer and water offer a potent relaxation sink. Whether or not a water molecule can experience this SjL+r S%J &?&V~ Dz2 i>.v di%~Xe i+D222 il. 15 jZ c2nnD~ &%JSe bDm h pSh>Dn 22 th.2 h?X Of the exciting pulse to the interface in a time comparable with its own intrinsic relaxation time, then it will behave as if no interface exists. On the other hand molecules close to the interface will be profumn&_Y inf’luence~ b_~ h In a ~ys’lem,. tbeTe5ore. where Ihe ti%ance to tie in’lefiace cannol be travexscd wi
2 L exp( -r/7;,“) II= 0 magnetisation at some

(12)

is the appropriate normalised time t for the relaxation time ~_r tX?t <* ls ;t’rc:~m&ciiX~ 1.2&k rP ~?nIqmXtTi Xke YiMxdi~ .a& s@sGbinT ,noikiCahut (T,X.G,,. this solution is that there will be in principle an infinite number of relaxation times but detailed calculations based on models containing only active surfaces show that only the first two or three terms are important, Both Tj and Jn depend on the local geometry, the diffusion coefficient and the strer& of the relaxation sink. When the time scale for diffusion is sufficiently rapid for all the rnolcc&s tifi =v.~vx+E~~~x$+a s&k & r.=B CP5m J&~&lerS m& -V&J V&g& txefla~&GL J&axj 45 observed.(26*3’J In the limit of slow diffusion higher order terms become important and multiexponential decay occurs. Brownstein and Tarr (*“) offer a case study based on the results obtained for transverse relaxation in rat gastrocnemius muscle. They demonstrate that, assuming surf&a95L+Gs&&~ LYasT am&a- L$X&Y ,?$ X+IIT &am; & ~LZXMT? &~v+L*T v~~ti d& predicted multiexponential decay is obtained. It is argued that since the diameter used is in excellent agreement with the expected cell diameter their approach is shown to be correct. The degree to whid, QLG vRI a& &aft 4 ~~X+~7i’z: ~V~EX~ X <+R,id+CJ ^Jf'h +&dP?iCd ~jF~ttTlT~StfC+~%'IIInX&'k problematic. If it is accepted that the main relaxation sinks in muscle are the biopolymers then the model becomes untenable. Although the radii of the cells may be of the right order of size the distz,wes &?NL~~ X~+>S ;V S+ ?DZM&Y G? X&uw a& a&>,~ A& ,%+& &&awes &&~Lw? ~V&+LS are of the order of lo-30nm. In addition the space between the fibrils is filled with sarcoplasmic retic&m and mito&on&ia. Q*2) LiBfo7d et a{. s~ggcst &at rn&&+onentia1 r&xa
&t)

NMR and compartmentation

in biological tissues

263

dimensions. Thus there is probably a large class of heterogeneous systems to which the diffusion approach may be applied properly but to which, as yet, no application has been made. It is important to note that in such systems the application of the Bloch-McConnell types of exchange equation described in Section 4 is incorrect. The recent results of Halle(30’ indicate that even in locally ordered systems a continuous diffusion model is appropriate rather than the Bloch-McConnell model. Thus although the Brownstein-Tarr arguements may not apply it is still by no means certain that exponential relaxation is to be expected or that such non-exponential behaviour can be dealt with on the basis of the discrete exchange models commonly used. Fung and Puon(200) suggest a radically different origin for multiexponential transverse relaxation. They point out that protons and deuterons in water molecules are in exchange with species such as NH2, OH and SH on the protein surfaces, these groups will experience small dipolar or quadrupolar static splittings, which are due to some part of the appropriate static interaction remaining unaveraged by motion. If this splitting is Ao then, provided the fraction of nuclei experiencing this interaction is small, a non-exponential relaxation will be observed if (Aw/lO),<(T;:,+r,‘)
(13)

where t,, is the life time of the group and Tzb is the spin-spin relaxation time of the minor fraction. The authors suggest that reasonable values of Aw for NH, groups would be 104T105sec-’ and for T2,, 10-4sec. They estimate zt, from published results for the protonation of methylamine; 7,, is given by r,=2.7 x lo3 [H+],,

(14)

where [H+], is the hydrogen ion concentration in mol dmm3. It is expected therefore that condition (13) would be satisfied for all pH values less than 10. In support of their contention the authors demonstrate that glycerinated rabbit muscle shows multiexponential relaxation when the pH = 9, whereas at pH 7 and 5 the decay curves are linear until 95 and 98 % of the magnetisation has decayed away. As discussed previously (Section 7.1) compartmentation cannot be responsible for multi-exponential behaviour in this tissue. In considering the model it is important to consider the nature of the splitting Ao. In the case of protons it arises from a static dipolar interaction, and proton exchange will destroy this interaction since its existence is dependent upon the existence of a proton pair. If the modulation rate is of the same order as the splitting Aw, the condition of motional narrowing will apply since the splitting is being switched on and off at a rate comparable to the interaction strength. This will occur when the rate is 1O-4 to 10-5sec-‘, corresponding to a pH in the range 7.4-8.4 which is close to that in muscle. The quadrupolar interaction is not destroyed by exchange and thus while the mechanism is tenable for deuteriated muscle it stretches credibility for protonated muscle. In support of their contention, Fung and Puon argue that the kind of behaviour they see in glycerinated muscle and changes observed in the relaxation characteristics of mouse muscle during the development of rigor (see Section 7.2) cannot be explained by a compartmentation model. Further evidence for this model comes from the observation of residual static interactions in deuteriated frog muscle. tzo4) This was considered as evidence of dynamically oriented water”“) but may be the residium of the static interaction on exchangeable protein groups. Evidence against the model is provided by the work of Oakes(205J who observed single exponential transverse relaxation in bovine serum albumin/water mixtures with up to 70 % protein content. Another piece of evidence comes from the consideration of the non-freezing water in muscle: when muscle is frozen at about - 10°C relaxation which was multiexponential (‘06) from 25 to -5°C becomes single exponential.‘207’ (The previous report of multiexponential behaviour below the freezing point in Ref. 206 is corrected in Ref. 207.) Since this water must be close to the proteins and the exchange time cannot have changed very much, one would expect on the basis of this model to see multiple exponential behaviour below the freezing point. On balance the Fung and Puon hypothesis looks untenable for protons although more tenable for deuterons. The behaviour of glycerinated muscle is consistent with the model of Lillford et ~1.(~‘)

264

P. S. BEL.TON and R. G. RATCLIFFE

especially since truly multicomponent behaviour is seen only at high pH near to where denaturation could occur. Nevertheless, considerations of this particular model apart, it does seem likely that

direct interaction with proteins does have an important role to play. Certainly in spin-lattice relaxation processes in tissues there is good evidence for the importance of cross-relaxation.‘208) There is also direct evidence of proton exchange between water and amide groups causing relaxation at low magnetic fields.‘209) Multiexponential transverse relaxation behaviour may therefore result from intermediate exchange rates between rapidly relaxing sites on protein and water. In the case of deuterons, effects assigned to oriented water(‘92*204)may well arise because of exchange with protein sites with residual static interactions. The importance of exchange is highlighted by Diegel and Pintar. (210)They suggest that some of the multiexponentiality observed in transverse and rotating frame spin-lattice relaxation arises from a ‘slow-diffusion’ process characterised by a time scale of IO-‘set between hydration water and bulk water. It is not clear why this particular mechanism is suggested as exchange between water and protein protons would appear to do just as well. The notion is supported by the observation of a dispersion in T,p at a frequency close to that expected for an exchange process of this order of magnitude. However if the model of Fung and Puon is correct then the breaking of the dipolar interaction on the protein would also give rise to such an effect. In conclusion it may be said that there are a variety of mechanisms by which multiexponential relaxation could arise. All of these may be active jointly or separately in compartmented systems. It is therefore important to interpret results with extreme caution. In particular very little attention has been paid to the Brownstein-Tarr model, although its ideas are relevant to a large number of heterogeneous systems of biological and non-biological origin. 7.2. Applications to Unicellular Systems Suspensions of human erythrocytes can be analysed in terms of two compartments: the intracellular and extracellular spaces. The permeability of the cells to water is high and measurement of water proton relaxation times show simple exponential behaviour, indicating that the two sites are in a fast exchange limit. In order to remove this condition it is necessary to reduce one of the relaxation times to less than the exchange time thus generating a double exponential decay. A direct and simple method of doing this (by doping the extracellular water with manganese to reduce its relaxation time) was proposed by Conlon and Outhred. (*i’) When the relaxation time of the extracellular water was sufficiently short the main relaxation mechanism for the intracellular water was by diffusion out of the cell into the paramagnetically doped medium. In effect the intracellular water is labelled by magnetisation which is lost when it crosses the cell membrane. Under these conditions the apparent lifetime in the cell is given by 1 1 1 (15) r, Tb 7; & is the observed relaxation time for the intracellular water in the manganese doped system, 7i is its intrinsic relaxation time (measured from packed cell preparations). In addition the back flux of the magnetically labelled water into the cell must be considered. A semi-quantitative expression for this effect was found:‘21 ‘)

(16) with r=

exp(d/d) - 1, exp(d/b) + 1

and S = (D TO)*

Q is the exchange time of water in the cell. D is the diffusion coefficient of extracellular water, V and A are cell volume and surface area respectively, and d is the distance between adjacent cells. Of the variables only d and TOcan be manipulated. TO,the relaxation time for the manganese-doped extracellular water, must be small and d should be sufficiently large to make r approach unity. Conlon and Outhred measured transverse relaxation of the water protons in human erythocyte

NMR and

compartmentation in biological

tissues

265

suspensions in saline. They found that provided T, was less than 2 msec and d was such that the packed cell volume was no greater than 40 % of the total suspension volume, then effects due to back flux were not observed They found a value of r, of 8.2 msec at 37°C. Fabry and Eisenstadt(212) have used the same method, measuring T1 rather than T, for the water protons in erythrocytes suspended in plasma in which manganese forms the potently relaxing manganese albumin complex. A difficulty with measuring Tl is that it is necessary to consider the proton signals from haemoglobin, which have a Tl of about 100 msec and must be subtracted from the decay before the relaxation of the remaining components which are due to water can be measured. The authors argue that in spite of this difficulty T, is measured more precisely than T2 since the latter may be affected by chemical shift differences between the manganese doped water and the intracellular water. They show that rather short exchange times (7.X similar to those observed by Conlon and Outhred are found when high concentrations of manganese are used. #en the free manganese is below 5 millimolar, however, the value of r, rises to 22msec. Pirkle et a1.(213)have used both Tl and T2 measurements. They suggest that rather than use the limiting form of the exchange equations the more general form should be used and the data fitted by iterative methods. In addition they carried out a detailed error analysis of the experiment. An important conclusion reached was that the value of z, is strongly dependent on the manganese concentration used and that very low levels are desirable. This conclusion is buttressed by their analysis of high resolution line shapes of doped cell suspensions which showed that for manganese concentrations of 3 millimolar the induced chemical shifts are less than 3Hz Under such conditions the chemical shift exchange contribution to transverse relaxation is negligible. They obtained values close to those of Fabry and Eisenstadt when cells in plasma were used. Conlon and Outhred(2’4) have pointed out that with low manganese concentrations the extracellular relaxation time can be sufficiently long that the difference between T, and 7= in eqn. (16) can be significant. Although this would not be too serious if the erythrocytes in plasma did not aggregate to form rouleaux, they have observed that such aggregation is likely under the conditions used in the low manganese experiments. The manganese in the dead space between adjacent cells in a rouleaux is insufficient to relax water exchanging from inside the cell fast enough to stop a back flux effect. If, however, the manganese concentration is suitably high this problem can be eliminated. The apparently longer exchange times observed at low manganese concentrations probably result from back flux effects.(*14) The authors note that the rouleaux are not formed in saline solutions. More recently Herbst and Goldstein (215.216)have proposed an exchange model which specifically takes account of rouleaux formation. An additional problem with proton T, measurements is that there may be spin-diffusion through the membrane without material transport.(2’4r Fabry and Eisenstadt(*“) have given a detailed analysis of spin-diffusion effects involving protein and have compared results from T1 and T, experiments achieving substantially similar results. An alternative approach to the problem of water transport in erythrocytes is to use “0 which has intrinsically rapid relaxation rates. T, relaxation in an erythrocyte suspension containing i’0 enriched water is double exponential. (*lsi The slow component has a relaxation rate that is faster than that of extracellular water, but the fast component is identical to the single component observed in a packed cell suspension. The slow component is thus extracellular water whose relaxation rate has been increased by exchange with the fast relaxing intracellular water. From the data a value of T, may be calculated and was found to be 9.3 msec at 37°C in Ringer’s solution, in good agreement with Conlon and Outhred.(*il) Notwithstanding the problem of the best conditions for experimentation and the absolute value of r,, the method is of value in comparative studies since large numbers of samples can be examined quickly. The effects of drugs (*i4) lipophilic anaesthetic’2’9) and cryoprotectants(220i have been examined. Significant differences between healthy and pathological subjects have been observed (**I) and the relationship between permeability and cell volume examined.‘***) The effects of osmolarity’217*223’ and addition of dimethyl sulphoxide on permeability have also been reported(224’ An illustration of the complexities to be encountered when experimenting with cells more

266

P. S. BELTON and R. G. RATCLIFFE

structured than erythrocytes is given by Getz et al.(225) They used EPR and NMR to study the effects of manganese on suspensions of erythrocytes and neoplastic mouse cells (Lettree cells). Consistent with other observations, the EPR spectra show only one kind of manganese in the erythrocyte suspensions,. corre.s_oon&ng to Pree Mnl+ . NMR relaxation 01 the water protons shows the usual ‘aiexponential behaviour in the presence of manganese In Eettree cells,. however,. the EPR spectra were interpreted as resulting from three different forms of manganese: free Mn ‘+ in solution, manganese loosely bound to the cell and manganese tightly bound >o rhe ce%.The proton rdaxajjon behaviour is b~u’rh exponen~~d when t’ne manganese concentration is as low as 20~~. (Compare this with the behaviour of erythrocytes where the minimum concentration for the observation of biexponential behaviour is 10Op~.) The relax&on hme 0’ 1-e exXrac&&ar w&er wh’lch ‘1s‘m ccm%icX&X&3 tie c&s ‘IS%as%er&an when ‘11‘IS separated. This implies interaction with the manganese associated with the cells. Finally the protons of the intracellular water relaxed faster than did those of the extracellular water, which is the reverse of the &ma&n wi’lh eryth~om~. T’nix effti pre^yirm’&> arise3 ba>%e 6 inWa&Qn with %i&%y bound intracellular manganese.

An interesting half-way house between the complexities of Lettree cells and the relative simpllicities of erythrocytes is given b_v l_vmphoc_vtes. Spin-lattice relaxation of “0 is biexponential,‘226) in packed rat lymphocytes provided that the cells are viable, whereas in non-viable cells single exponential behaviour is observed. This result, contrasting as it does to packed erythrocytes, is ascribed to intracellular compartmentation. Rat lymphocytes consist of a large nucleoplasm and small cytoplasm whose relative volumes are about 2: 1, this corresponds to the relative populations of the two relaxation components. Thus in this case the NMR result seems to correspond to the microscopically observable compartmentation. The general conclusion to be drawn from the work on even these very simple systems is that accurate quantitative data are difficult to obtain since they are dependent on the details of the experiment and on the model used to interpret it. There are two problems which must be considered when assessing the models. Firstly the effects of such factors as chemical shift or spin diffusion, and secondly whether the system of exchange equations used is valid. In this context it is interesting to note that the diffusion coefficient of water through erythrocyte membranes has been estimated to be of the order of 2 x 10mg cm’sec-’ for a membrane thickness of 100~.(227) Brownstein and Tarr’201’ estimate that for a diffusion coefficient of 2.5 x 10-5cm2sec-1 the size range for the types of effects they describe are of the order 1 to 30 pm. Thus the dimensions and diffusion coefficients for water in cell membranes are both four orders of magnitude smaller than in intracellulir spaces and therefore Brownstein-Tarr considerations may apply. Even if they do not, complex exchange schemes are n ecessa$ “a’ 20 Uke ac~m1 oj tie r&e of the mem&~~ corn&+>& Even given a>> these diffic&ies it dms sell seem
The suggestion that compartmentation was responsible for the multiexponential transverse relaxation of protons in muscle was synchronous with its first observation.‘206’ Three components were characterised which were assigned as extracellular water (0) “water in myofibrils and sarcco&astiic re~~culun5‘ )a> anb”water &s&y assc&ateh*>ti oT Won&> ‘D0unhXn~oi~.n> anGo;r phospholipids” (b). Table 4 gives some typical results. The exact shape of the decay curve was found urd Z&f,%%~?@W to dL+& aa c&f,y ukt =,+&z% W& +. fdf, G~+4i zZWl&W~~&~/*~Z. WXXtfZXX

NMR and compartmentation

in biological

TABLE 4. Transverse relaxation behaviour in frog gastrocnemius are analysed by fitting to three relaxation times, T,, and populations.J(206’

T,(m=)

Temperature

muscle. Results three fractional

f

WI

0

a

b

-5

214 244 350 232 226 243 168

41 37 44 40 42 39 38

13 5 13 11 6 4 10

0 5 10 15 20 25

261

tissues

-0 0.14 0.19 0.13 0.16 0.12 0.14 0.15

a

b

0.60 0.68 0.66 0.64 0.70 0.64 0.67

0.25 0.14 0.2 1 0.19 0.21 0.22 0.18

relaxation rate in the temperature range -5 to 25°C was found to go through a maximum at 20°C (data calculated from Table 1 of Ref. 206). Both of these results indicate that some exchange processes are occurring and that the relative populations of the components cannot necessarily be taken as accurately reflecting the numbers of spins in a particular environment. The assignment of extracellular water to the most slowly relaxing fraction was made on the basis that it is expected to be the most remote from protein and other interfaces and its relative population is found to be close to that of the expected extracellular population. This assignment has also been made by Hazlewood et ~1.~~” but Diegel and Pintar’ ‘lo) disagree. They observed that when the muscle was exchanged with a D,O Ringer solution the proton relaxation still contains a component relaxing at about the same rate. They argued that this must mean that water is not responsible but that the component arises from mobile organic species. Fung and Puon(200) have demonstrated that such a component is more likely to come from an incomplete exchange of protons since the high resolution proton NMR spectrum of D,O exchanged muscle contains a peak at the appropriate chemical shift for water but none of any significant intensity elsewhere. Further support for the assignment of the slowly relaxing fraction to extracellular water comes from the work of Neville and White. (229)They measured the extracellular space in. various frog leg muscles by an ion efflux method, and found a good correlation with the magnitude of the slow component of the proton relaxation process. The single cell muscle of the giant barnacle (Balanus nub&) does not contain extracellular space in the normal sense but does contain clefts on the outer surface which contain about 6 % of the total volume of water. Bumell et u1.(230)were unable to observe any separate signal from this space in blotted muscle. However on adding Ringer’s solution they were able to observe a separate slow relaxing signal which they assigned to solution filled crevices in the folded fibre. Foster et ~1.‘~~‘)did observe a slow relaxing fraction corresponding to about 3 y0 of the total water which they assigned to water in the cleft space. Belton and Packer’ 232)obtained results in close agreement to this (Table 5

TABLE 5. Relaxation of giant barnacle muscle as a function of tonicity.C232’ Three components were observed with relaxation times, TZrand relative populations,f: Tonicity

Isotonic* Isotonic Hypertonic Hypotonic *Result of Foster

TAmW 0

a

400 400 326

35 53 21 123

et ~1.‘~~”

f b 0.34 21 19 49

0

0.03 0.04 0.08 0

a 0.91-0.94 0.46 0.21 0.20

b 0.056 0.5 0.71 0.80

268

P.S.BELTONand R.G. RATCLIFFE

and Fig. 8). In addition they found that when the muscle was first bathed in hypotonic Ringer’s solution (which tends to shrink the muscle and close the cleft space) the extracellular component disappeared. On the other hand hypertonic solutions (which increase the cleft space volume) cause an increase in the amount of the slow relaxing component. Although the relaxing component may be fairly safely assigned to extracellular water, the nature of the faster relaxing components is more problematical. Multiexponential curves are notoriously difficult to fit, and probably the best agreement is usually found in the slowest relaxing component since this can be measured when other components have decayed away and the process is essentially fit obtained for frog single exponential. It has been noted (233) that the three component gastrocnemius muscle can be fitted almost as well by a two component process. The two component fit leaves the slow relaxing component very little changed but the two faster decays are replaced by a single process. In addition (233) the decay characteristics in general are a function of the physiological state of the muscle and the time after excision (see below). It is interesting in this context to compare the results of Foster et ~1.‘~~‘)and Belton and Packer’232) (Table 5). Both sets of authors agree on the long component but differ significantly on the shorter components. This is not an artefact of the fitting since calculation of the curves from the published data generates distinctly different shapes and it probably reflects some physiological change. An additional complication is that some part of the faster relaxing components arise from non-water species. Deuterium exchange experiments(23’*232.234’ have demonstrated a non-exchangeable component with a relaxation time of the order of a few milliseconds. In an attempt to assign the components a frog gastrocnemius muscle was placed in an NMR tube and continuously evacuated. (232) Measurement of the FID as a function of time showed that signal

TIYE/seCx IO-' FIG.8. Transverse relaxation in Balanus nubilis single straight lines represent the three exponential components residual after slowest decaying fraction subtracted, W decaying

cell muscle equilibrated in isotonic solution.(z33) The of the magnetisation decay. A observed decay curve, 0 residual after further subtraction of the intermediate fraction.

NMR and compartmentation in biological tissues

269

loss followed a double exponential process and that the more slowly removed component represented between 15 and 20 o/0of the total muscle water. The changes in the relative intensities of the three components are shown in Fig. 9. The slow and intermediate components, (o and a) disappear first and the fast component (b) begins to go only when both of these have nearly disappeared. At this point it drops from about 12 % of the total signal to 8 % and its T2 drops from 7 msec to 4 msec. It is argued(233i that this fraction represents water closely interacting with the protein. From the deuterium exchange results (231) it is clear that up to 6% of the transverse relaxation curve arises from protein with a T2 of the order of a few milliseconds, this contributes to both the FID and the CPMG echo envelope. The fast fraction therefore probably represents a composite of both protein and water. Possibly the relaxation times of both components are dependent on the water content. Additionally the relative intensity and relaxation time calculated from composite curves will depend on the methods of curve analysis used. The weight of evidence suggests therefore that water in muscle may be assigned to intra- and extracellular regions. The degree and significance of further compartmention is a question of debate.

TIME/MINUTES

FIG. 9. The effects of evacuation on the water content of frog gastrocnemius muscle. The x-axis represents the time of evacuation. The total water content (A), and the relative populations of the A slowest (0). n intermediate (a) and 0 fastest (b) decaying fractions of transverse relaxation are shown. Data from Ref (232).

An interesting issue deriving from the problem of muscle compartmentation is whether or not the changes seen in the water relaxation in muscle post mortem reflect changes in compartmentation. Fung and Puon(200) have argued that the changes in transverse relaxation seen post mortem in mouse muscle’235i could not arise from changes in compartmentation since there was no other evidence of changes in compartmentation post mortem. It was observed (235) that the slow relaxing fraction was not present immediately post mortem but developed in a period of 15 to 60 min. Neville and White(229) have observed similar results in frog muscle: when the muscle was incubated in Ringer’s solution overnight the relative population of the slow fraction was 15 y0 compared to 10 y0 in fresh muscle. This corresponds to an increase in the extracellular space as measured by the ion efIlux method. Chang et al.‘236) measured Tl relaxation of protons in rat muscle and found that, provided sufficient care was taken with the measurement, two components could be observed. The relative proportions of these components change with time post mortem for 6 hr and then remained constant.

270

P. S. BELTON and R.G.

RATCLIFFE

In porcine muscle (237) Ti relaxation is single exponential within experimental error and transverse relaxation is initially single exponential but becomes biexponential after several hours. This double exponential behaviour can be induced by freezing and thawing the muscle. In order to explain the temperature dependence of the relaxation times and the behaviour of Ti and T, processes, two regions of water in slow exchange with each other were postulated. Each slowly exchanging region contains two subsets of water corresponding to bulk water and water interacting with proteins. The amount of interacting water in each region remains constant after death but the population of bulk water in one region declines and that in the other increases. The authors specifically did not attempt to identify the two postulated regions with any microscopically observed regions. In so far as results related to water compartmentation are concerned muscle is the most studied system. The single exponential relaxation observed in squid giant nerve axon has already been relaxation in mylinated frog nerve is triple mentioned in another context. (203) By contrast exponential.(238’ Once again the origin of the two fast components is somewhat problematical but the population of the slow relaxing component (about 20 % of the total) agrees well with the expected extracellular fraction measured by H,O/D,O exchange experiments.(239) A particularly direct observation of the effects of compartmentation has been made by Packer and Bruynooghe.(240) They examined transverse relaxation of water protons in bovine cornea. This tissue consists of the stroma (a stack of lamellae made of collagen) bounded on each side by layers of epithelial and endothelial cells. The relaxation of water protons in the whole tissue is bi-exponetial (Fig. 10). The tissue may readily be dissected into cells and stroma, each of these shows single exponential proton relaxation. When the two curves are combined with appropriate weighting for the relative populations in the whole tissue the original biexponential curve is reconstituted. This result is probably the only completely unambiguous demonstration of relaxation behaviour reflecting compartmentation in complex tissues.

0

12

,;4

TIME / S.C I lO-3

FIG. 10. Transverse relaxation in Cornea. ‘MO)Figure taken from Ref 192.

7.4. Applications to Plant Cells and l&sues NMR studies of the state and transport of water in plants have recently been reviewed.““) The least compartmented plant materials studied so far are thylakoids(242) in the form of broken chloroplasts. Sharp and Yocum (243) have examined water transport in these systems by an ingenious variant of the Conlon and 0uthred(211) method. Instead of using a relaxation agent which would not

NMR and compartmentation

in biological tissues

271

cross the membrane they used an impermeant shift reagent, dysprosium ethylenediamine (Dy (en)3+) in a range of concentrations from 0.5 to 5.0 mM. The exchange rate was then determined by lineshape analysis and the interpulse spacing dependence of the transverse relaxation rate measured by the Carr-Purcell-MeiboomGill sequence (see Section 4). These techniques have the advantage that exchange rates on the millisecond or sub-millisecond time scale can be measured and two independent experiments may be analysed simultaneously. A simple two site exchange model was found to be adequate since the dimensions of the thylakoids are small (500 nm) and the interior is a good approximation to a continuous uncompartmented space. (243) Single exponential relaxation was always observed indicating that the fast exchange limit obtains. The residence time of water within the thylakoid is about 1 msec. This order of time scale would be very difficult to measure using manganese doping because very high levels of paramagnetic material would be required to reduce the relaxation time of the external medium sufficiently. In contrast to the relative simplicity of shift reagent experiments with thylakoids, manganese doping experiments with Chlorella uulgaris are fraught with difficulty.(244) Manganese binds to the cell walls and in addition there are probably already some existing paramagnetic relaxation centres in the cell walls. Since the average cell diameter is 3.2 pm the conditions are similar to those proposed by Brownstein and Tarr (*O’) for the diffusional model of relaxation. Thus great care must be taken in analysing the exchange kinetics in this system. Transverse relaxation of nuclei in the cell suspensions in the absence of added manganese is double exponential and this may be plausibly accounted for by assigning components to intra- and extracellular water. However when cell volumes are packed to high concentration there is evidence (Ref. 244 Fig. 2) of an additional fast component. However, this has not been well characterised, because experimental conditions were chosen which discriminate against the observation of fast relaxing components. Given these complications and the question of which kinetic models should be used means that quantitative interpretation of manganese doping results becomes extremely difficult. Even more complicated systems have been studied, including leaves,(245s246) ivy bark(247) and wheat cell crowns after freeze thaw cyclesJ248), and multiple relaxation is observed in all of these systems. However, the interpretation of the data in plant material needs special care because of the structure and size of plant cells. Typically cells within a single piece of tissue may have a large range of sizes. In Zea mays root tips, for example, this may be from 1 to 300 pm(249) and in Elodea leaves there is a three fold range in sizes, the average cell length being 96 pm and average diameter 27 pm. (245) Plant cells contain vacuoles which may be up to 90 y0 of the cell volume but contain very little in the way of structure of proteinacous matter’249) most of which is contained in the cytoplasm. The diversity of sizes of this order makes plant material fit closely to the conditions described by Lillford et ~1.‘~‘) In cases where external relaxation agents have been added’245*247) the conditions fit almost exactly the assumptions of the Brownstein-Tarr(20’) model, but with the complications of a distribution of cell dimensions. Thus analysis of the relaxation decays into discrete components may be seriously in error and even where discrete components do exist they may well arise from mechanisms other than the proposed simple exchange processes between the compartments. BaEiC and Ratkovii: have used water relaxation measurements to measure the rates of water exchange in Nitella cells(250) and the uptake of manganese in Zea mays roots.(251) The uptake in roots was followed by three methods: total manganese analysis by atomic absorption, measurement of T1 of water protons, and T2 values using a Carr-Purcell spin-echo method. The spin-echo consists of a 90”-r-180” pulse sequence with an echo observed at t=2r, provided that 2r is not greater than 5T2. The conditions were chosen so that 22 was greater than 5T2 for the bathing manganese medium and the repetition rate was fast compared to the root water proton TI in the absence of manganese. Under these conditions an echo was not observed in the absence of manganese, but as manganese diffused into the roots and decreased TI an echo appeared whose intensity was proportional to the manganese concentration. Results from all three methods showed the same three stage pattern of uptake (Fig. 11). The first region, A, is considered to be uptake into the apoplast which is in diffusional continuity with the external solution. Since the roots contain two major regions, the cortex and stele, it is tempting to assign the next two processes to uptake in these. However dissection of the material at different

272

P. S. BELTON and R. G. RATCLIFFE

FIG. 11. Manganese

uptake in Zea mays roots measured Journal 45, 767 (1984) by copyright permission

by T, relaxation. Figure reproduced from Biophysical of the Biophysical Society and the authors.

showed that MnZC ions appear simultaneously in cortex and stele. Region B is assigned to uptake into the cytoplasmic continuum of the root and the final region, C, to uptake into the vacuole. A recent study on plant leaves (162) has indicated that water in some species has orientation dependent spectra and relaxation times. Orientation dependent spectra have been observed before in sciatic nerve/252) dogwood stems(253) and barnacle muscle(230) but have been shown(230*253,254) to be artefacts of geometry. It is thus necessary to exercise great care in the interpretation of such spectra. It was observed that plant leaves from some species give water ‘H spectra consisting of three components. The relative intensities and positions appeared to be a function of the orientation of the leaf to the magnetic field (i6’) Comparative studies with a phantom made of wet blotting paper show a single line only whose position shifts with orientation but which shows no evidence of any structure. Leaves also show orientation dependent EPR spectra and the orientation dependent component of the spectrum was assigned to manganese which is bound to membrane surfaces but is not at the active photosynthetic site since this is usually EPR silent. Measurements of the Ti of the different components in the water resonance shows that different components have different Ti values and that these are angle dependent. It was concluded that the signals must therefore arise from different compartments and that exchange between them is slow on the time scale of Ti. The most likely origin of this orientation dependence is the thylakoid membranes which are known to be highly ordered and contain manganese. Thus the results suggest an interesting new method for studying structure and function in the photosynthetic organs of plants. times

8. INVESTIGATION

OF COMPARTMENTATION

BY DIFFUSION

MEASUREMENTS

The principles of the use of NMR to study translational motion are well established.(25,255) Essentially the problem is to measure the function P(r,t;r,) which is the probability of finding a spin at position I at time r when it was known to be at r. when t=O. (255) This is a one dimensional probability function since the magnetic field gradients used in the experiments are applied in one direction only. In the measurement of diffusion coefficients, therefore, the value obtained is the component of the diffusion tensor in the direction defined by the magnetic field gradient. When diffusion is unrestricted, P(r, t; ro) is given by P(r, t; ro)=(4nDt)-3’2

exp-

(r- 1 ~

[

r012

4Dt

(17)

NMR and compartmentation

where D is the one dimensional diffusion coefficient. Under these conditions intensity observed in the pulsed field gradient experiment is given by(255’

In R= -y2g2D

213

in biological tissues the normalised

signal

a2 (A-:6)

where y is the magnetogyric ratio, g is the magnitude of the linear field gradient, 6 is the width of each field gradient pulse separated by a time A. Equation 18 is valid when the distance, x, between any barriers to diffusion in the system is greater than the root mean squared displacement of the nucleus in the time A, i.e. (2DA)‘/*
214

P. S. BELT~Nand R. G.

RATCLIFFE

is relatively few. This may be in part because of the lack of suitable mathematical models. However, since the phenomenology of diffusion is well understood and powerful computers are readily available it should be relatively straightforward to obtain numerical solutions for the models of interest. This would be particularly useful since it would allow the study of exchange between compartments without recourse to doping or dependence on possibly inappropriate models of exchange.

9. CONCLUDING

REMARKS

There are many NMR methods for probing the compartmentation of heterogeneous systems: some rely on the intrinsic properties of the system, while others depend on the effects induced by external agents; some are empirically based while others are dominated by theory; all are likely to be influenced to some extent by exchange processes. In comparison with the overwhelming structural detail that is apparent in an electron micrograph, the NMR methods for compartmental analysis might appear rather primitive but the diversity of these methods, the fact that it is possible to probe solutes, solvent and surfaces non-invasively in appropriate cases, ensures that NMR has a useful complementary role in characterising the molecular properties of compartments. Acknowledgements-The Mrs. A. P. Christopher

authors thank Prof. K. J. Packer for allowing us access to unpublished material. and for typing the manuscript.

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NOTE ADDED IN PROOF Two papers relevant to the discussion in Section 52.2. have appeared recently. Boulanger et aLCz6” have 23Na signal from erythrocytes is undetectable by NMR, contradicting concluded that z 20 y0 of the intracellular previous results.tr38~‘39r It appears that the long-running problem of estimating the NMR-detectable fraction of the alkali metal cations in biological systems will remain topical. Boulanger et a/.‘*65’ also demonstrated that uncomplexed dysprosium penetrated the erythrocytes under some conditions, again in disagreement with the generally held view that [Dy(PPP),]‘has no effect on the integrity of a cellular system. Finally, Shinar and study of the intracellular sodium in red blood cells, have Navon,r266r in the course of a 13Na relaxation introduced a new technique for estimating the intracellular volume based on the use of an extracellular Co(CN)imarker detected by s9Co NMR. 265. Y. BOULANGER,P. VINAYand M. DESROCHES,Biophys. J. 47,553 (1985). 266. H. SHINAR and G. NAVON, Biophys. Chem. 20.275 (1984).