NMR active nuclei and solid state NMR Biomedical

REVIEW ARTICLE

Simon G. Patching

School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

Table of Contents

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a principal well-established technique for analysis of chemical, biological, food and environmental samples. This article provides an overview of the properties and applications of NMR active nuclei (39 nuclei of 33 different elements) used in NMR measurements (solution- and solid-state NMR, magnetic resonance spectroscopy, magnetic resonance imaging) with biological and biomedical systems and samples. The samples include biofluids, cells, tissues, organs or whole body from different organisms (humans, animals, bacteria, fungi, plants) for detecting and quantifying metabolites or environmental samples (water, soils, sediments). Isolated biomolecules (peptides, proteins, nucleic acids) can be analysed for elucidation of atomic-resolution structure, conformation and dynamics and for characterisation of ligand and drug binding, and of protein-ligand, protein-protein and protein-nucleic acid interactions. NMR can be used for drug screening and pharmacokinetics and to provide information in the design and discovery of new drugs. NMR can also measure translocation of ions and small molecules across lipid bilayers and membranes, characterise structure, phase behaviour and dynamics of membranes and elucidate atomic-resolution structure, orientation and dynamics of membrane-embedded peptides and proteins.

Keywords: biological and biomedical applications; drug screening; dynamics; magnetic resonance imaging; membrane proteins; metabolomics; MRI; NMR active nuclei; nuclear magnetic resonance; protein structure

1. Introduction

Nuclear magnetic resonance (NMR) spectroscopy is one of the principal techniques used for analysis of biological and biomedical systems and samples. This can include the identification, quantification and monitoring of ions, small molecules and biomolecules in studies of metabolism and biological function in human and animal cells and tissues, bacterial cells and spores, fungi and plants. Similar types of measurements can be performed on environmental samples such as water, soils and sediment. NMR is able to elucidate atomic-resolution structure, conformation, molecular mechanism, dynamics and exchange processes (on timescales of picoseconds to seconds) in biomolecules, especially peptides, proteins and nucleic acids. NMR can be used for the observation, quantification and characterisation of ligand and drug binding to biomolecules, and for characterisation of ligand-protein, protein-protein and protein-nucleic acid interactions. NMR can be used for drug screening and it can acquire structural, binding and kinetic information for the design and discovery of new drugs. It can then monitor the absorption, distribution, metabolism and excretion (ADME) of administered drugs in pharmacokinetics studies. NMR can be used for the observation, quantification and kinetic characterisation of ion and small-molecule translocation across lipid bilayers and biological membranes, including those of cells, tissues and vesicles. Solid-state NMR in particular can investigate the interactions and effects of peptides, proteins and small molecules on the structure, phase behaviour and dynamics of lipid bilayers and biological membranes. Similarly, solid-state NMR can elucidate atomic-resolution structure, orientation and dynamics of transmembrane peptides and proteins in lipid bilayers and native biological membranes. Furthermore, the basic NMR experiment is the principle behind magnetic resonance imaging (MRI), which is one of the main established clinical techniques for in vivo imaging of the whole human body and specific organs and tissues. Only certain naturally-occurring nuclei have intrinsic properties that allow them to be used in NMR (and MRI) applications with biological and biomedical systems and samples. NMR active nuclei are those possessing a property called ‘spin’, whereby a charged nucleus spins about an axis and generates its own magnetic dipole moment. This property enables alignment of nuclei in an external magnetic field and absorption of radiofrequency radiation, which is the basis of the NMR experiment (Figure 1).

Figure 1. The basic NMR experiment with a spin-1/2 nucleus.

Nuclei that possess an even number of both neutrons and protons have no spin (spin quantum number = 0) and are not NMR-active, for example ²He, ¹²C, ¹⁶O, ³²S. Nuclei for which the number of neutrons plus the number of protons is an odd number have a half-integer spin (i.e. spin quantum number = 1/2, 3/2, 5/2, 7/2, 9/2), for example ¹H, ¹³C, ¹⁵N, ¹⁹F. Nuclei for which the number of neutrons and the number of protons are both odd have an integer spin (i.e. spin quantum number = 1, 2, 3), for example ²H, ⁶Li, ¹⁰B, ¹⁴N. In all nuclei for which the spin quantum number is greater than 1/2, the charge distribution of protons is asymmetric (Figure 2), which gives them an electric quadrupole moment in addition to their magnetic dipole moment.

Figure 2. Charge distributions in a spin-1/2 nucleus and in a quadrupolar nucleus.

A spin-1/2 nucleus has a spherical distribution of electric charge. A quadrupolar nucleus has an asymmetric distribution of nucleons, producing a non-spherical positive charge distribution. The nuclear charge distribution (black charges) interacts asymmetrically with electric field gradients (blue charges) in a molecule.

These ‘quadrupolar nuclei’, which constitute over two-thirds of all naturally occurring NMR active nuclei, can have very short longitudinal relaxation times (T₁) and produce broad NMR signals or none at all. Quadrupolar nuclei with an integer spin tend to produce much broader signals than those with a half-integer spin. Hence, the most useful nuclei for NMR applications are those with a half-integer spin, especially those with a spin number of 1/2. For a more comprehensive description of nuclear spin systems, the reader is referred to reference [1]. It is fortunate that some of the most common elements found in living organisms have an isotope that is spin-1/2 (i.e. ¹H, ¹³C, ¹⁵N, ³¹P) and these nuclei have prolific use in NMR applications with biological and biomedical systems and samples. The natural background of such nuclei can prove to be a problem for certain NMR studies, however. A surprisingly large number of other nuclei have also been used in published NMR (and MRI) applications with biological and biomedical systems and samples. Table 1 and Figure 3 give properties for 39 such nuclei from 33 different elements that will be covered in this article. Whilst some studies use natural abundance levels of the nucleus being analysed, others require enrichment with the nucleus (isotope labelling) to improve the sensitivity of detection. In the following sections of this article, each of the 39 nuclei is considered in order of increasing atomic number, with details and illustrated examples of published studies, as appropriate.

Element	Nucleus	Spin number (I)	Natural abundance (%)	Chemical shift range (ppm)	Sensitivity relative to ¹H (enriched)	Larmor frequency at 7.05 T (MHz)	Larmor frequency at 9.40 T (MHz)	Larmor frequency at 11.75 T (MHz)
Hydrogen	¹H	1/2	99.99	13	1.00000	300.13	400.13	500.13
	²H	1	0.01	13	0.00965	46.07	61.42	76.77
	³H	1/2	0.01	13	1.21000	320.13	426.80	533.46
Helium	³He	1/2	0.01	58	0.00348	228.64	304.82	380.99
Lithium	⁷Li	3/2	92.41	27	0.29400	116.64	155.51	194.37
Boron	¹⁰B	3	19.90	110	0.01990	32.25	42.99	53.73
	¹¹B	3/2	80.10	110	0.16500	96.29	128.38	160.46
Carbon	¹³C	1/2	1.11	200	0.01590	75.47	100.61	125.75
Nitrogen	¹⁵N	1/2	0.36	900	0.00104	30.42	40.56	50.70
Oxygen	¹⁷O	5/2	0.04	1160	0.02910	40.69	54.24	67.80
Fluorine	¹⁹F	1/2	100.00	700	0.08320	282.40	376.50	470.59
Sodium	²³Na	3/2	100.00	72	0.09270	79.39	105.84	132.29
Magnesium	²⁵Mg	5/2	10.00	70	0.00268	18.37	24.49	30.62
Aluminium	²⁷Al	5/2	100.00	400	0.20700	78.20	104.26	130.32
Silicon	²⁹Si	1/2	4.69	540	0.00786	59.63	79.50	99.36
Phosphorus	³¹P	1/2	100.00	430	0.06650	121.50	161.98	202.46
Sulphur	³³S	3/2	0.75	964	0.00227	23.04	30.71	38.39
Chlorine	³⁵Cl	3/2	75.76	1100	0.00472	29.41	39.20	49.00
	³⁷Cl	3/2	24.24	1100	0.00272	24.48	32.63	40.79
Potassium	³⁹K	3/2	93.26	65	0.00051	14.01	18.67	23.34
Calcium	⁴³Ca	7/2	0.14	70	0.00643	20.20	26.93	33.66
Vanadium	⁵¹V	7/2	99.75	1900	0.38400	78.94	105.25	131.55
Cobalt	⁵⁹Co	7/2	100.00	18000	0.27800	71.21	94.94	118.67
Copper	⁶⁵Cu	3/2	30.85	1100	0.11500	85.25	113.65	142.06
Zinc	⁶⁷Zn	5/2	4.10	2700	0.00287	18.78	25.04	31.29
Selenium	⁷⁷Se	1/2	7.63	3000	0.00703	57.24	76.31	95.38
Bromine	⁷⁹Br	3/2	50.69	600	0.07940	75.20	100.25	125.30
	⁸¹Br	3/2	49.31	600	0.09950	81.06	108.06	135.07
Krypton	⁸³Kr	9/2	11.50	220	0.00190	11.55	15.40	19.24
Rubidium	⁸⁷Rb	3/2	27.83	110	0.17700	98.20	130.92	163.65
Cadmium	¹¹¹Cd	1/2	12.80	650	0.00966	63.64	84.89	106.11
	¹¹³Cd	1/2	12.22	650	0.01110	66.61	88.80	111.00
Iodine	¹²⁷I	5/2	100.00	4200	0.09540	60.05	80.06	100.06
Xenon	¹²⁹Xe	1/2	26.40	500	0.02160	83.47	111.28	139.09
Caesium	¹³³Cs	7/2	100.00	160	0.04840	39.37	52.48	65.60
Platinum	¹⁹⁵Pt	1/2	33.83	6700	0.01040	64.52	86.02	107.51
Mercury	¹⁹⁹Hg	1/2	16.87	3500	0.00594	53.76	71.67	89.58
Thallium	²⁰⁵Tl	1/2	70.48	7000	0.20200	173.13	230.81	288.49
Lead	²⁰⁷Pb	1/2	22.10	11500	0.00906	62.79	83.71	104.63

Table 1. Properties of NMR active nuclei for biological and biomedical applications.

Natural abundances and Larmor frequencies are given to two decimal places. Sensitivities are given to five decimal places and include the first three non-zero numbers. Information was taken from Bruker NMR frequency tables, the Bruker NMR guide and from the NMR lab website of the Hebrew University of Jerusalem.

Figure 3. Properties of NMR active nuclei for biological and biomedical applications.

Natural abundances and receptivity values (sensitivity) were taken from Table 1 or calculated using values in Table 1.

2. NMR active nuclei

2.1. Hydrogen (¹H, ²H, ³H)

There are three NMR-active isotopes of hydrogen, the spin-1/2 protium (¹H), spin-1 deuterium (²H) and spin-1/2 tritium (³H). Whilst ³H is the most sensitive of all NMR active nuclei, it is radioactive (β-emitter), has a very low natural abundance (3 x 10^-16%) and is difficult and expensive to obtain or produce. ¹H is the most sensitive of all NMR active nuclei after ³H and has a natural abundance of 99.99%, it the most commonly used nucleus for NMR applications and is the nucleus to which all others are compared (Table 1). For example, the receptivity values of ²H, ³H and ¹³C relative to ¹H when enriched are 9.65 x 10^-3, 1.21 and 1.59 x 10^-2, respectively. Even though the chemical shift range for ¹H is relatively small (-1 to 12 ppm), it can produce very sharp and highly resolved signals, depending on sample properties and the NMR pulse sequence used.

2.1.1. Applications of protium (¹H)

Whilst ¹H is intrinsically involved in a large majority of NMR experiments for biological and biomedical applications, for example in those for structure determination of biomolecules, the large background of hydrogen in these samples means that direct ¹H detection has generally not been used. Exceptions include cases where samples have been prepared with various levels of deuteration in order to reduce or eliminate signals originating from background ¹H. In addition to removal of interfering ¹H signals from NMR spectra, deuteration also eliminates potential proton relaxation pathways and strong dipole-dipole interactions that would otherwise contribute to line broadening effects on the spectra. This is especially important when performing TROSY-type solution-state NMR experiments on detergent-solubilised membrane proteins where deuteration of both the protein and detergent may be essential [2–4]. An increasing number of studies have shown how sample deuteration and/or fast magic angle spinning (MAS) enable high-resolution proton-detected solid-state NMR spectra to be obtained for samples of biological and biomedical origin. Partial deuteration can reduce spectral congestion in ¹H, ¹³C, ¹⁵N solid-state NMR correlation spectra [5], thus making structural analysis amenable to larger and more complex biomolecules. Perdeuteration, however, can produce proton-detected solid-state NMR spectra of biomolecules with ultra-high resolution, even at low to moderate (5-30 kHz) MAS frequencies, as demonstrated with samples of amyloid fibrils and membrane proteins [6–8], for example, using the α-spectrin SH3 domain (Figure 4). Using highly deuterated samples, solid-state NMR methods have been developed for sensitivity enhancement by preserving water magnetisation [9] and for resonance assignment using dipolar-based interspin magnetisation transfers [10] and proton-detected 4D experiments [11]. Recent developments in MAS NMR technology have made it possible to spin solid samples up to a frequency of around 110 kHz [12], which improves significantly the feasibility for performing proton-detected measurements. Studies using fast MAS and proton detection have demonstrated resonance assignment procedures and assessments of sensitivity with different protein samples [13–16], quantified sugars in plant tissue [17], investigated structure and dynamics in measles virus nucleocapsids [18] and investigated the organic matrix and monitored structural and dynamic changes in bone [19,20]. Leading-edge technological advances for proton-detected solid-state NMR include fast MAS at ultra-high magnetic field [21] and high-resolution triple resonance micro-MAS NMR with nanolitre sample volumes [22]. All of these ongoing technological developments will make a larger range and complexity of samples of biological and biomedical origin amenable to chemical, structural and dynamic investigation by proton-detected NMR.

Figure 4. High-resolution proton-detected solid-state NMR spectrum of a perdeuterated biomolecule.

A. ¹H-detected ¹H,¹⁵N-correlation spectrum recorded with a perdeuterated α-spectrin SH3 sample that was recrystallised from a buffer containing 90% D₂O. B. Amide proton linewidths as a function of MAS rotation frequencies (8-24 kHz) for selected residues. This Figure was reproduced with permission from Reif 2012 [7]; copyright © 2012 by Elsevier Inc.

¹H-detected NMR is one of the main methods used for the study of metabolomics (and metabonomics), whereby metabolites in cells, biofluids, tissues or organisms are identified, quantified and changes monitored to reflect the underlying biochemical activity [23]. These can be affected by disease, drugs or environmental variation. The most common human body fluids to be analysed are blood (whole, plasma, serum) and urine [24,25], but also other fluids including cerebrospinal fluid, milk, prostatic fluids, saliva and sweat. Recent studies include ¹H and ¹H-¹³C HSQC NMR screening of urine in autism spectrum disorders [26], ¹H NMR metabolic profiling of five different brain regions in a mouse model of Alzheimer’s disease [27], ¹H NMR assessment of the lipoprotein profile in type 1 diabetes [28], ¹H NMR characterisation of serum metabolites in cervical cancer [29], use of ¹H NMR to identify urinary biomarkers of severe sepsis and septic shock in the Intensive Care Unit [30] and ¹H NMR analysis of metabolic profiles in ovarian tumour cyst fluid [31]. ¹H NMR-detected metabolomics is also emerging as a useful tool for assessment of cardiovascular disorders, including cardiac arrest [32,33]. Other types of samples that have been analysed for ¹H NMR-detected metabolomics are very diverse and include studies on metabolic profiling of reef-building corals [34] and the brains of sheep exposed to scrapie [35], effects of temperature and diet composition on the early developmental stages of cod larvae [36], predicting the optimum pH for the quality of chicken meat [37] and the detoxification mechanism of cucumber plants exposed to copper nanoparticles [38]. These are just a small number of recent examples, highlighting that a more comprehensive overview of ¹H NMR-detected metabolomics applications is beyond the scope of this current work. ¹H NMR is also the basis of the well-established and widespread clinical diagnostic tool magnetic resonance imaging (MRI), used for the non-invasive and non-destructive imaging of soft tissues such as brain, heart and muscles and for identifying and monitoring tumors in many organs. Related to both MRI and metabolomics is also the in vivo clinical tool proton magnetic resonance spectroscopy (¹H-MRS), which combines ¹H NMR-derived metabolic profiles with MRI images to diagnose and monitor a wide range of diseases and conditions.

2.1.2. Applications of deuterium (²H)

The quadrupolar properties of the deuterium nucleus (²H) can produce broad NMR signals of up to a few kHz, resulting in poor resolution. Direct ²H detection is therefore not routinely used for solution-state NMR, but it has found some useful solid-state NMR applications. The relatively low natural abundance of ²H (0.015%) means that ²H-enrichment of samples is usually required. One application is in investigating the structure and phase behaviour of biological membranes [39,40] and their interactions with drugs and antimicrobial peptides using lineshape analysis and relaxation measurements on static samples. This is made possible by the orientation dependence of the ²H electric quadrupolar interaction, which permits the study of molecular orientational order. For example, ²H solid-state NMR has recently been used to investigate the compositional distributions and lipid order profiles of raft model membranes comprising mixtures of site-specifically deuterated N-stearoylsphingomyelins, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol [41]. The application of ²H solid-state NMR for investigating deformation of lipid bilayers at the atomistic level in liquid-crystalline membranes has been reviewed recently [42]. The effects of commonly used cannabinergic agonists on the lipid membrane bilayer have been investigated using ²H solid-state NMR and hydrated bilayers of dipalmitoyl-phosphatidylcholine (DPPC) deuterated at the 2′ and 16′ positions of both acyl chains. The cannabinergic compounds lowered the phospholipid membrane phase transition temperature, increased the lipid sn-2 chain order parameter at the membrane interface and decreased the order at the centre of the bilayer. It was concluded that compounds can influence lipid membrane domain formation and this may contribute to their cannabinergic activities through lipid membrane microdomain related mechanisms [43]. ²H solid-state NMR studies on the effects of antimicrobial peptides have been performed in model bacterial membranes containing chain-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipids [44,45] and with membrane-deuterated whole Escherichia coli cells [46]. ²H solid-state NMR is also useful for studying the orientation and dynamics of the peptides themselves in lipid membranes [47]. A high impact example is observation of distinct orientation and dynamics for the drug amantadine in two different binding sites of the M2 proton channel from influenza A virus in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers (Figure 5) [48]. Differential binding of deuterated rimantadine enantiomers to the M2 proton channel has also been demonstrated [49].

Figure 5. ²H solid-state NMR analysis of amantadine binding to M2 proton channel from influenza A virus.

²H NMR spectra of d₁₅-amantadine in DMPC bilayers as a function of temperature and ratio of amantadine to M2 channel. A. No M2 channel. The calculated spectrum for 303 K reproduces the 1:3 frequency ratio and 4:1 intensity ratio of the two splittings. B. Amantadine/M2 channel ratio = 1:4. The sum spectrum reproduces the 303 K spectrum by 1:9 combination of the lipid-bound 303 K spectrum and peptide-bound 283 K spectrum. C. Amantadine/M2 channel ratio = 4:4. The sum spectrum uses a 1:3 combination of the M2-bound spectrum (II) and lipid-bound spectrum (I). D. Amantadine orientation in the M2 channel. E. One of two possible amantadine orientations in the lipid bilayer. This Figure was reproduced with permission from Cady et al. 2010 [48]; copyright © 2010 by Nature Publishing Group.

Another example is for the cardiac peptide phospholamban, where side-chain and backbone dynamics were measured by lineshape analysis on site-specific deuterated phospholamban in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers [50]. Dynamics in larger proteins have also been measured using ²H solid-state NMR. For example, in the signalling state of rhodopsin with 11-cis-retinal selectively deuterated at the methyl groups in aligned membranes [51] and in spider dragline silk fibre [52]. Using perdeuterated ubiquitin and Escherichia coli outer membrane protein OmpG as model systems, a suite of three-dimensional ²H-¹³C correlation experiments for high-resolution solid-state MAS NMR spectroscopy of large proteins were developed by exploiting the favourable lifetime of ²H double-quantum states. The ²H-¹³C correlation spectra were reminiscent of ¹H-¹³C correlations and allowed a substantial number of assignments for both proteins [53]. A further interesting application of ²H NMR has been in the study of water behaviour in bacterial spores, which can exhibit dormancy and thermal stability under extreme conditions. ²H magnetic relaxation dispersion measurements of water mobility in the core of Bacillus subtilis spores were in support of a gel scenario in which the core is a structured macromolecular framework permeated by mobile water [54]. A separate ²H NMR study suggested that the spore core is more rigid than expected for a gel-like state and that the gel core is inaccessible to external water [55].

2.1.3. Applications of tritium (³H)

Despite having the highest sensitivity of all NMR active nuclei, NMR applications of ³H are scarce, not least because it is radioactive. This is a pity because ³H certainly has some interesting and potentially useful properties. For example, the high gyromagnetic ratio of ³H allows measurement of long-range interatomic distances by solid-state NMR without structural modification of the molecule. Indeed, ³H MAS solid-state NMR has produced the largest NMR distance of 14.4 Å ever measured between two nuclei [56]. Measurement of distances using ³H labels incorporated at specific positions has potential to provide important structural information in samples of biological and biomedical origin. Five ³H NMR studies from the 1990s are described below, but no other significant studies appear to have been reported since then. ³H NMR was used to study anaerobic glycolysis in erythrocytes. Use of [1-³H]-glucose allowed monitoring of the disappearance of α and β tritons and the production of lactate, ¹H³HO and some intermediates. Spin-lattice relaxation times (T₁) were measured to avoid T₁ distortion of the spectral intensities. Formation of 1 mM ¹H³HO in the presence of 110 M H₂O was detected and this allowed the eventual fate of the label to be observed in vivo [57]. The conformation and dynamics of peptide inhibitor binding to a bacterial collagenase has been studied using ¹H and ³H NMR relaxation experiments. Specific ²H and ³H labelling of the succinyl part of the competitive inhibitor succinyl-Pro-Ala allowed measurement of cross-relaxation rates for individual ¹H or ³H spin pairs in the inhibitor-collagenase complex and also in the free inhibitor. Determination of order parameters in different parts of the inhibitor indicated that the succinyl and alanyl residues are primarily involved in interactions with the collagenase and that the succinyl moiety adopts a unique trans conformation in the bound state [58]. ³H NMR has been used to study anomeric specificity in complexes of ³H-labelled α- and β-maltodextrins with maltose-binding protein (MBP). At a temperature of 10 ºC, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose and longer maltodextrins had a ratio of affinities (K_dβ/K_dα) that was significantly greater (10- to 30-fold). Further interpretation of the spectra also revealed how MBP is able to bind both linear and circular maltodextrins [59]. ³H NMR has been used to study two nucleic acid molecules, an 8 kDa DNA oligomer and a 20 kDa ‘hammer-head’ RNA. ³H-¹H NOESY experiments allowed observation of through-space interactions in B-form DNA and an unexpected ‘antiphase’ cross-peak at the water frequency. ³H NMR spectra of the RNA molecule indicated conformational dynamics in the conserved region of the molecule in the absence of Mg²⁺ and spermine, which are two components necessary for cleavage [60]. ³H NMR was used to examine the complex formed by [4-³H]benzenesulphonamide and human carbonic anhydrase I (HCA I), showing that a 1:1 complex exists in solution. Interpretation of ³H relaxation behaviour and ³H-¹H NOEs showed that the rate of dissociation of the complex is 0.35 s^-1 and that the aromatic ring of the inhibitor undergoes rapid rotation whilst in the complex (Figure 6) [61].

Figure 6. ³H NMR analysis of the complex between [4-³H]benzenesulphonamide and human carbonic anhydrase I.

Comparisons of observed and calculated ³H T₁ relaxation behaviour in an inversion-recovery experiment (A), the transient ³H{¹H} NOE as a function of mixing time (B) and time development of the ³H{¹H} NOE (C) in a sample with HCA I at a concentration of 1.06 mM and a ratio of [4-³H]benzenesulphonamide to HCA I of 1.35:1. This Figure was reproduced with permission from Culf et al. 1997 [61]; copyright © 1997 by Kluwer Academic Publishers.

2.2. Helium (³He)

The spin-1/2 nucleus ³He is very sensitive when enriched, producing sharp signals and has a moderate chemical shift range (-50 to 8 ppm). Other properties of ³He mean the NMR applications of this nucleus are very limited, however. The chemistry of helium is limited to endohedral fullerenes and the resonance frequency of ³He falls outside the range of conventional NMR probes [62], so special equipment is required. ³He also tends to have long relaxation times, with the gas having a T₁ of around 1000 seconds. One medical application of ³He NMR that has emerged is the in vivo imaging of lung function. This is made possible because ³He nuclei can be hyperpolarised by spin-exchange optical pumping [63,64]. Thus, the appropriate wavelength of circularly polarised infrared laser light is used to excite electrons in an alkali metal, such as caesium or rubidium, inside a sealed glass vessel. The angular momentum is transferred from the alkali metal electrons to ³He gas nuclei through collisions, which aligns their nuclear spins with the magnetic field to enhance the NMR signal. The resultant hyperpolarised ³He gas can be stored at a pressure of 10 atm for up to 100 hours.

Gas mixtures containing hyperpolarised ³He can be inhaled and then imaged using an MRI scanner to produce pictures of lung and airway function. This method can be used to monitor conditions such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and radiation-induced lung injury (Figure 7), and also lung transplantation [65–68].

Figure 7. Hyperpolarised ³He-MRI analysis of human lungs.

A. Healthy 45 year old female with predicted forced expiratory volume in the first second of expiration (FEV1pred) 118%. B. COPD 79 year old male with FEV1pred 54%. C. Asthmatic 26 year old male (baseline, no provocation) with FEV1pred 77%. D. CF 23 year old female with FEV1pred 58%. This Figure was reproduced with permission from Fain et al. 2010 [68]; copyright © 2010 by Wiley-Liss, Inc.

2.3. Lithium (⁷Li)

There are two NMR-active isotopes of lithium, ⁶Li and ⁷Li, both of which are quadrupolar. ⁶Li (natural abundance 7.59%) is spin-1 and produces sharp signals, but has a relatively low quadrupolar moment and low sensitivity. ⁷Li (natural abundance 92.41%) is spin-3/2, has a higher quadrupolar moment than ⁶Li and produces broader signals, but it is highly sensitive. The principal use of lithium NMR is ⁷Li magnetic resonance spectroscopy analysis of the brain for studying and monitoring bipolar disorder, for which lithium and its salts are effective in both acute and prophylactic treatment [69,70]. A ⁷Li magnetic resonance spectroscopy study of the distribution and regional pharmacokinetics of lithium in rat brain suggested that lithium is most active in a region stretching from the anterior cingulate cortex and striatum to the caudal midbrain, with greatest activity in the preoptic area and hypothalamic region. Some activity was also seen in prefrontal cortex, but only minimal amounts in the cerebellum and metencephalic brainstem [71]. A ⁷Li and ¹H magnetic resonance spectroscopy analysis of the relationship between brain lithium levels and the metabolites N-acetyl aspartate and myo-inositol in the anterior cingulate cortex of older adults with bipolar disorder showed a direct association between brain lithium and higher levels of both metabolites. It was suggested that the higher levels of myo-inositol reflect increased activity of inositol mono-phosphatase [72]. A quantitative ⁷Li magnetic resonance spectroscopy study of the normal human brain measured the in vivo T₁ of ⁷Li as 2.1 ± 0.7 seconds. The mean brain ⁷Li concentration was 0.71 ± 0.1 mM, with no significant difference between grey and white matter, and the mean serum concentration was 0.9 ± 0.16 mM [73]. A later quantitative study on bipolar patients stable on long-term lithium treatment demonstrated a bi-exponential lithium T₂ relaxation in the majority of cases with an average short decay time of 5.3 ± 1.4 ms and an average long decay time of 68.2 ± 10.2 ms. In two of the patients, a strongly mono-exponential T₂ relaxation was observed with an average decay time of 47.4 ± 1.3 ms [74]. The compartmental distribution of lithium as a function of total lithium concentration in rat brain was studied using these biexponential ⁷Li T₂ decays. A linear interpolation using the biexponential T₂ values to estimate intracellular lithium from individual monoexponential T₂ decays were also assessed. The intracellular T₂ was 14.8 ± 4.3 ms and the extracellular T₂ was 295 ± 61 ms. The fraction of intracellular brain lithium ranged from 37.3 to 64.8% (mean 54.5 ± 6.7%) and did not correlate with total lithium concentration. The estimated intracellular lithium concentration ranged from 47 to 80% (mean 68.3 ± 8.5%) of the total brain lithium and was highly correlated with it [75]. A quantitative ⁷Li magnetic resonance spectroscopy study of brain lithium levels after six weeks of lithium therapy in patients with bipolar disorder revealed a significant association between central and peripheral lithium levels in remitters but not in non-remitters. It was therefore suggested that non-remitters may not transport lithium properly to the brain and this may underlie resistance to treatment with lithium. Also, brain lithium (but not plasma lithium) was inversely correlated with age, whilst plasma lithium did not correlate with any clinical outcome, lithium dosage or adverse effects [76]. The putative target protein for lithium therapy in bipolar disorder is inositol monophosphatase, which catalyses the hydrolysis of inositol monophosphate to inorganic phosphate and inositol. Using ⁷Li MAS solid-state NMR, including ¹³C-⁷Li dipolar recoupling experiments (Figure 8), the bound form of lithium in the active site of an inositol monophosphatase from Escherichia coli (SuhB) has been observed [77]. Lithium binds to site II in SuhB that is coupled to three aspartate residues (84, 87, and 212). The inositol monophosphatase activity of SuhB is strongly inhibited by lithium and SuhB shares significant sequence similarity with human inositol monophosphatase, including most of its key active-site residues.

Figure 8. ¹³C-⁷Li dipolar recoupling analysis of lithium binding to an Escherichia coli inositol monophosphatase (SuhB). Comparison of experimental CP-filtered, ⁷Li-detected REDOR data points (ΔS/S₀) of the 0.38 ppm ⁷Li signal from fully labelled SuhB inositol monophosphatase with Spinevolution simulations of the three Mg²⁺ binding sites from bovine inositol monophosphatase (PDB entry 2BJI). For sites I and II, three carboxyl carbons and their three nearest carbons were used in a seven-spin simulation. Site I: Glu-70 Cδ and Cγ, Asp-90 Cγ and Cβ, and Ile-92 C′ and Cα. Site II: Asp-90/93/220 Cγ and Cβ. For site III, simulations were performed using all four carbons within 5 Å of this site (Lys-36 Cε, Asp-41 Cγ, Glu-70 Cδ and Cγ). Typical ⁷Li spectra, corresponding to S and S₀ at the time point indicated by the arrow, are shown at the right along with the difference spectrum ΔS; the peak deconvolution of S₀ is shown inset. This Figure was reproduced with permission from Haimovich et al. 2012 [77]; copyright © 2012 by American Chemical Society.

2.4. Boron (¹⁰B, ¹¹B)

Boron has two NMR-active isotopes, spin-3 ¹⁰B (natural abundance 19.9%) and spin-3/2 ¹¹B (natural abundance 80.1%). ¹¹B is the preferred nucleus for NMR applications because it has a lower quadrupole moment and is more sensitive than ¹⁰B. A common problem encountered in boron NMR spectroscopy is broad background signals originating from regular NMR tubes (made of borosilicate glass) and from probe components. These can be avoided by using quartz tubes, which are more expensive, and/or by using a DEPTH pulse sequence, which increases the signal-to-background ratio [78]. The most common application of boron NMR is for the detection of ¹⁰B and ¹¹B in boron neutron capture therapy (BNCT). In this process of cancer treatment, boron-containing molecules enriched with ¹⁰B are targeted to the tumour and irradiated with thermal or epithermal neutrons. Capture of the neutrons by ¹⁰B nuclei generates cell-damaging radiation (production of an α-particle and a ⁷Li-particle) that is confined to single-cell dimensions. Boron NMR is used to study the metabolism and pharmacokinetics of the boron-containing molecules and for non-invasive in vivo mapping of the molecules [79,80]. Preliminary ¹H and ¹⁰B NMR relaxation studies have been performed in animal tissues and in living tumour cells to assess the suitability of ¹⁰B molecules tagged with a Gd(III) paramagnetic ion for BNCT. Such molecules may be useful as contrasting agents in MRI for mapping boron distribution in tissues [81]. ¹⁰B NMR has also been used to achieve the simultaneous analysis of ¹⁰B-p-boronophenylalanine (BPA), ¹⁰B-BPA-fructose complex and total ¹⁰B in blood for BNCT studies [82]. In an interesting application, ¹¹B MAS solid-state NMR has been used to assess pH levels in red coralline algae by measuring boron isotopic compositions. In this respect, 30% of boron in powdered bulk samples was present as boric acid [83]. The isotopic composition and elemental abundance of boron in marine carbonates is a useful tool for tracking changes in seawater pH and carbonate chemistry.

2.5. Carbon (¹³C)

The spin-1/2 nucleus ¹³C (natural abundance 1.11%) is the only NMR-active isotope of carbon. ¹³C has relatively low sensitivity and usually requires enrichment, but it produces sharp signals and has a wide chemical shift range (0 to 200 ppm) that allow good spectral dispersion. After ¹H, ¹³C is one of the most commonly used nuclei for biological and biomedical NMR applications, for example, in protein structure determination, observation and quantification of ligand and drug binding, characterisation of protein-ligand and protein-protein interactions and measurement of kinetics and dynamics. Despite the widespread involvement of ¹³C in NMR applications, direct ¹³C detection has only recently become useful for studying biomolecules. Developments include spin-state-selective methods that achieve homonuclear decoupling in the direct acquisition dimension of ¹³C detection and high-resolution methyl-selective ¹³C NMR experiments in both the solution- and solid-state [84,85]. Methods have also been developed for direct-detection ¹³C biomolecular NMR spectroscopy in living cells and other in vivo methods [86,87]. Hence, ¹³C has proved to be a very useful NMR-active nucleus for metabolomics, whereby the metabolic fluxes of ¹³C-enriched or natural abundance substrates can be monitored in vivo. A principal application of ¹³C NMR is in analysis of brain metabolism [88,89]. For example, the ratio of acetate-to-glucose oxidation in astrocytes has been measured from a single ¹³C NMR spectrum of cerebral cortex [90]. Along with ¹H magnetic resonance spectroscopy, ¹³C magnetic resonance spectroscopy is useful for monitoring the glutamate-glutamine cycle in the brain and central nervous system of healthy individuals and of those with neurological disorders in which this cycle is disturbed [91,92].

In addition to [¹⁸F]FDG-positron emission tomography (FDG-PET) [93], ¹³C magnetic resonance spectroscopy is a principal technique used to assess glucose transport and metabolism following traumatic brain injury (Figure 9) [94–96]. In a recent study, ¹³C NMR was used to explore neuron-astrocyte metabolic cooperation in the brains of diabetic mice with cognitive decline using intravenous infusions of [2-¹³C]-acetate and [3-¹³C]-lactate.

Figure 9. ¹³C magnetic resonance spectroscopy analysis of glucose transport and metabolism following traumatic brain injury. ¹³C-NMR spectra achieved by ex vivo NMR analysis of microdialysate after delivery of [3-¹³C]lactate and [1,2-¹³C₂]glucose to traumatic brain injury (TBI) patients.

Left panel: A and B. Examples of ¹³C NMR spectra of brain microdialysates from a TBI patient receiving perfusion with [3-¹³C]lactate (4 mM) by microdialysis catheters via a craniotomy; red stars indicate ¹³C signals for glutamine C4, C3 and C2 indicating metabolism via the TCA cycle. C. ¹³C NMR spectrum of the [3-¹³C] lactate substrate solution prior to perfusing. D. ¹³C NMR spectrum of brain microdialysate from an unlabelled patient. This Figure was reproduced with permission from Gallagher et al. 2009 [95]; copyright Ó 2009 by Gallagher et al. Right panel: Examples of ¹³C NMR spectra of brain microdialysates from patients receiving [1,2-¹³C₂]glucose (4 mM) perfused via a microdialysis catheter. The uninjured brain is a normal-appearing brain in a patient operated on for a benign tumour elsewhere in the brain. The TBI brain is from a patient with a diffuse injury. The part of the spectrum illustrated in each case is for the C3 carbon of lactate. Also present in this part of the spectrum is one of the signals due to the internal standard DSS (2,2-dimethyl-2-silapentane-5-sulfonate sodium salt). The remainder of the spectrum (including the main DSS signal at 0 ppm) is not shown. The C3 doublet indicated by red stars represents lactate doubly labelled with ¹³C, produced by glycolysis; the C3 signal for ¹³C is split into 2 peaks by coupling to ¹³C also present at the neighbouring C2 position within the same molecule. The C3 singlet indicated by green stars represents lactate singly labelled with ¹³C, produced via the pentose-phosphate pathway. This Figure was reproduced with permission from Carpenter et al. 2014 [96]; copyright © 2014 by Carpenter et al.

Relative to wild-type mice, the diabetic mice had significantly lower ¹³C labelling in neurotransmitters including glutamine, glutamate, and γ-aminobutyric acid after [2-¹³C]-acetate infusion, whilst infusion of [3-¹³C]-lactate resulted in increased ¹³C-enrichment of neurotransmitters in the diabetic mice. The results indicated a possible disturbance of neurotransmitter metabolism during the development of cognitive decline in diabetics [97]. The ¹³C nucleus plays important roles in a range of solid-state NMR applications. This includes structure determination of membrane proteins, which are major molecular targets for drug discovery. Structural and dynamic information on membrane proteins can be obtained from static oriented samples or from samples subjected to MAS in native lipid membranes [98]. Introduction of ¹³C labels at specific sites in the proteins allows measurement of highly accurate through-space distances using homonuclear (¹³C-¹³C) and heteronuclear (e.g. ¹³C-¹⁵N, ¹³C-¹⁹F) dipolar recoupling experiments such as rotational resonance [99] and rotational echo double resonance (REDOR) [100], respectively, and derivatives thereof. Under favourable sample and dynamic conditions, the ¹H-¹³C cross-polarisation magic-angle spinning (CP-MAS) experiment can be used to observe and quantify the binding of ¹³C-enriched ligands and drug molecules to wild-type and mutant membrane proteins in native membranes [101–104]. Measurement of through-space inter-atomic distances and torsion angles can then allow elucidation of atomic resolution structures, molecular conformations, positions and orientations of ligands and drugs in their binding sites [104–111]. For example, ¹³C-²H REDOR NMR was used to define the structure of the amantadine binding site of the M2 proton channel from influenza A virus in lipid bilayers (Figure 10) [48].

Figure 10. Solid-state NMR structure of amantadine-bound M2 proton channel in lipid bilayers. A. Side view showing Ser31, Val27, Gly34, His37, Trp41 and amantadine in the high-affinity luminal site. Ser31 Cα lies in the mid-plane between the two rings of deuterons. The instantaneous orientation of amantadine, which is slightly tilted from the channel axis, is shown. The time-averaged amantadine orientation is parallel to the channel axis. B. Top view showing the Ser31 and Val27 pore radii. This Figure was reproduced with permission from Cady et al. 2010 [48]; copyright © 2010 by Nature Publishing Group.

Such measurements can contribute important information for the design and discovery of drugs involving membrane-embedded targets [112–116]. ¹³C solid-state NMR has also provided chemical, structural and dynamic characterisation of a wide range of other types of samples of biological and biomedical origin. These include amyloid fibrils from the amyloid plaques that are a hallmark of Alzheimer’s disease [117–119], structural proteins such as elastin [120] and bone implants [121]. Characterisations of plant material include the structure and dynamics of pectic polysaccharides and the structure of cellulose and its interactions with matrix polysaccharides in plant primary cell walls [122,123], fungal degradation in wood by monitoring lignin and cellulose composition [124] and chemical composition in the biomass of a mushroom [125]. Variable-temperature ¹³C solid-state NMR has been used to study molecular structures of honey-bee wax and silk [126]. ¹H-¹³C CP-MAS solid-state NMR has also been used to study different types of soil samples and effects of environmental factors on their composition [127–129].

2.6. Nitrogen (¹⁵N)

There are two NMR-active isotopes of nitrogen, spin-1 ¹⁴N (natural abundance 99.63%) and spin-1/2 ¹⁵N (natural abundance 0.37%). ¹⁴N has medium sensitivity, but its quadrupolar properties produce broad signals that are usually unobservable by a high-resolution NMR spectrometer. ¹⁵N is very insensitive, but its sharp lines and wide chemical shift range (0 to 900 ppm) can produce well-dispersed spectra. Hence, ¹⁵N-enriched samples are widely used for determining the backbone structures of proteins by NMR. This includes the basic solution-state [¹⁵N,¹H]-HSQC and [¹⁵N,¹H]-TROSY experiments and the plethora of correlation experiments used for making resonance assignments and structural measurements. Because of the low gyromagnetic ratio of ¹⁵N, the direct detection of ¹⁵N has rarely been used in multidimensional NMR experiments on proteins. It has recently been shown how selection of the TROSY components of proton-attached ¹⁵N nuclei can produce high quality ¹⁵N-detected spectra in high field magnets (>600 MHz) by taking advantage of the slow ¹⁵N transverse relaxation and compensating for the inherently low ¹⁵N sensitivity. ¹⁵N-detected TROSY experiments theoretically produce the narrowest linewidths at a magnetic field of 900 MHz and sensitivity reaches a maximum at around 1.2 GHz [130]. Indeed, it was then demonstrated that ¹⁵N-detected TROSY produces comparable sensitivity to ¹H-detected TROSY for non-deuterated, large proteins under physiological salt conditions (Figure 11) [131]. The ¹⁵N nucleus is also used for solid-state NMR structural and dynamics measurements on proteins and other biomolecules in a similar manner to those with ¹³C.

Figure 11. High quality ¹⁵N-detected TROSY spectrum of a non-deuterated, large protein under physiological salt conditions. Comparison of a ¹⁵N-detected TROSY-HSQC spectrum (A) and a ¹H-detected TROSY-HSQC spectrum (B) of 0.5 mM non-deuterated maltose binding protein in complex with 2 mM β-cyclodextrin at 283 K. A. The ¹⁵N-detected TROSY- HSQC was recorded in 8.5 h, ns = 176, F1 = 128 pts (14 ms), F2 = 2048 pts (315 ms). B. The ¹H-detected TROSY-HSQC was recorded in 8.5 h, ns = 12, F1 = 2048 pts (315 ms), F2 = 800 pts (18 ms). The ¹H-detected TROSY-HSQC was transposed. ¹⁵N and ¹H projections of the 2D spectra are indicated without any multiplication. The regions that were expected to contain mainly the resonances from the structured region of the proteins (8.6 ppm in the ¹H dimension) are indicated by grey shading. The spectra were recorded at 283 K and 800 MHz, and the apparent sc of the system deduced from the TROSY for rotational correlation times (TRACT) experiment was 35 ns. This Figure was reproduced with permission from Takeuchi et al. 2016 [131]; copyright ©2016 by Springer Science+Business Media Dordrecht.

2.7. Oxygen (¹⁷O)

The only NMR active isotope of oxygen is the spin-5/2 ¹⁷O (natural abundance 0.038%). ¹⁷O-enrichment is generally required for NMR studies, which is very expensive, and the broad signals produced by ¹⁷O are partly compensated for by its wide chemical shift range (-40 to 1120 ppm). The quadrupolar moment of ¹⁷O can interact with local electric field gradients, resulting in extremely short T₁ and T₂ relaxation times in the order of several milliseconds. Solution-state NMR applications of ¹⁷O with biomolecules are very limited, one exception is a study that used ¹⁷O NMR for observing an oxidised cysteine residue in Cu,Zn-superoxide dismutase [132]. Solution-state ¹⁷O NMR has found a useful application in the study of brain function and cerebral bioenergetics, however. Cerebral blood flow can be studied by monitoring washout of the tracer H₂¹⁷O in brain tissue following an intravascular bolus injection of ¹⁷O-labelled water. The cerebral metabolic rate of oxygen utilisation can be measured by monitoring the dynamic changes of metabolically generated H₂¹⁷O in brain tissue from inhaled ¹⁷O-labelled oxygen gas [133,134]. Similarly, ¹⁷O-MRI has been developed for monitoring oxygen consumption in the heart, brain and in tumours by detecting metabolically generated H₂¹⁷O following injection of a suitable ¹⁷O-labelled tracer into blood or inhalation of ¹⁷O-labelled oxygen gas [135–137]. Experiments have also demonstrated that direct cerebral and cardiac ¹⁷O-MRI at 3 Tesla are feasible using natural abundance ¹⁷O. In the brain, a signal-to-noise ratio of 36 was obtained at a nominal resolution of 5.6 mm³ and with a T₂ relaxation time of 1.9 ± 0.2 ms; in the heart, ¹⁷O images were acquired with a temporal resolution of 200 ms [138]. A few studies have shown that it is feasible to apply high-field ¹⁷O solid-state NMR to biomolecules, including membrane-embedded peptides and proteins. The first example used a selectively ¹⁷O-labelled transmembrane peptide, ¹⁷O-[Ala12]-WALP23, in hydrated phospholipid vesicles, from which distance restraints were estimated [139]. A complete resolution of the eight non-equivalent oxygen sites in monosodium L-glutamate monohydrate was demonstrated by ¹⁷O NMR with ¹H-decoupled double angle rotation and multiple quantum experiments [140,141]. ¹⁷O MAS analysis of the tetrameric 28-residue phospholemman transmembrane domain, in which one glycine residue (Gly14) from each helix was enriched to <40% ¹⁷O, suggested that the tetramer is an asymmetric unit with either C2- or C1-rotational symmetry along the bilayer normal [142]. Hydrogen bonding in Alzheimer’s amyloid-β fibrils has been probed by ¹⁵N-¹⁷O REAPDOR solid-state NMR [143]. These studies have shown that sufficient resolution and sensitivity can be achieved in ¹⁷O solid-state NMR experiments with samples that contain either 100% or dilute ¹⁷O enrichment, therefore showing potential for wider biological and biomedical applications.

2.8. Fluorine (¹⁹F)

The spin-1/2 nucleus ¹⁹F (natural abundance 100%) is very sensitive, produces sharp signals and has a wide chemical shift range (-300 to 400 ppm). Indeed, ¹⁹F is of similar size and behaves similarly to ¹H and the receptivity of ¹⁹F relative to ¹H is x0.83, making ¹⁹F the third most sensitive of all NMR active nuclei after ³H and ¹H. ¹⁹F is therefore a useful NMR nucleus for biological and biomedical applications, where samples usually contain a very low background of fluorine or none at all. ¹⁹F solution-state NMR can be used to study interactions of peptides and proteins, protein conformation and dynamics, ligand and drug binding and for drug discovery. Such studies have been assisted by advances in biosynthetic methods and fluorine chemistry for introducing fluorine into peptides, proteins and small molecules with high precision, and by technological advances in NMR spectrometers and probe design for increasing the sensitivity of ¹⁹F detection [144–146]. For example, ¹⁹F solution-state NMR has been used to study conformational change in the dengue virus NS2B-NS3 protease, which is an antiviral target for drug development. ¹⁹F NMR measurements showed that low pH or the binding of bovine pancreatic trypsin inhibitor promote a conformation change from an open to a closed state, thus demonstrating an importance of charge forces in the interaction between NS2Bc and NS3p. A mutation (H51A) impaired the charge interaction and the pH dependence of the conformational changes and stabilised the open conformation, whilst the addition of the inhibitor still converted NS2B-NS3p from an open to a closed state [147]. The introduction of fluorine-containing amino acids into proteins allows ¹⁹F-observed NMR to be used in small molecule drug discovery, whereby the binding of molecules is detected by changes in the ¹⁹F signals [148]. For example, a fragment screening and druggability assessment of the CREB binding protein/p300 KIX domain was performed using protein-observed ¹⁹F NMR spectroscopy. Fluorination of aromatic side chains allowed the screening of 508 compounds and validation by ¹H-¹⁵N HSQC NMR spectroscopy led to identification of a minimal pharmacaphore for the MLL-KIX interaction site [149]. ¹⁹F NMR drug screening strategies do not always require isolation of the target protein, but can also be performed on intact living cells and cell extracts. For example, a ¹⁹F NMR-based assay called ‘n-FABS’ (n-fluorine atoms for biochemical screening) in living mammalian cells expressing the membrane protein fatty acid amide hydrolase has been developed. The method allows identification of both weak and potent inhibitors and measurement of their potency in a physiological environment [150]. The almost absence of ¹⁹F in the body makes ¹⁹F-MRI a useful tool where appropriate ¹⁹F tracers can be introduced. High-field ¹⁹F-MRI has been shown as potentially useful for the imaging of amyloid-β in senile plaques during the pathological development of Alzheimer’s disease. Three ¹⁹F tracers allowed detection of amyloid deposition in the brain of transgenic mouse models of Alzheimer’s disease; these are (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), 1,7-bis(4′-hydroxy-3′-trifluoromethoxyphenyl)-4-methoxy-carbonylethyl-1,6-heptadiene3,5-dione (FMeC1, Shiga-Y5) and 6-(3′,6′,9′,15′,18′,21′-heptaoxa-23′,23′,23′-trifluoro-tricosanyloxy)-2-(4′-dimethylaminostyryl)-benzoxazole (XP7, Shiga-X22) [151]. ¹⁹F-MRI can be used for in vivo cell tracking by using suitable ¹⁹F-compounds together with specifically adapted hardware and acquisition methods [152]. For example, the fate of ¹⁹F-labelled mesenchymal stem cells have been tracked in a mouse model [153] and ¹⁹F-labelled stromal vascular fraction cells have been tracked as part of a human phase I trial during treatment of radiation-induced fibrosis in breast cancer patients [154]. ¹⁹F-MRI can also be used for tracking the biodistribution of ¹⁹F-labelled drugs [155] and for real-time gastrointestinal tracking of ¹⁹F-labelled catheter devices [156]. ¹⁹F solid-state NMR has largely been used to measure distances, conformations and dynamics in site-specific ¹⁹F-labelled membrane peptides and proteins. The high gyromagnetic ratio of the ¹⁹F nucleus makes it suitable for measuring relatively long internuclear distances using dipolar recoupling experiments such as REDOR. For example, a modified REDOR sequence involving ¹H homonuclear decoupling and composite ¹⁹F pulses can measure ¹H-¹⁹F distances to approximately 8 Å and this was used to measure a [¹HN]Leu-[¹⁹F]Phe peptide distance of 7.7 Å [157]. ¹⁹F spin diffusion solid-state NMR experiments measured a distance of approximately 11 Å between nearest neighbour [¹⁹F]Trp41 residues in the tetrameric helical bundle of the M2 proton channel from influenza A virus, confirming its side-chain conformation [158]. By monitoring pH-dependent differences in ¹⁹F dipolar couplings and motionally narrowed chemical shift anisotropies of the [6-¹⁹F]Trp41 residue, further ¹⁹F solid-state NMR measurements demonstrated how Trp41 participates in the gating mechanism of the same channel (Figure 12) [159].

Figure 12. Side chain conformations of Trp41 and His37 in the transmembrane domain of the M2 proton channel from influenza A virus. A. The activated state at pH 5.3 for Trp41 (blue) (ø1, ø2) (-50°, +115°). His37 torsion angles are not defined. B. New coordinates for the inactivated state at pH 8.0 for His37 (green) (-175°, -170°) and Trp41 (-100°, +110°) based on ¹⁹F solid-state NMR measurements. This Figure was reproduced with permission from Witter et al. 2008 [159]; copyright © 2008 by American Chemical Society.

Using conformationally constrained ¹⁹F-labelled amino acids, it is possible to double the accessible ¹⁹F-¹⁹F internuclear distance range by combining a highly sensitive solid-state multipulse ¹⁹F-NMR scheme with favourable sample geometry. Two rigid 4F-phenylglycine labels placed into a helical antimicrobial peptide embedded in fluid oriented membrane samples and a modified Carr-Purcell-Meiboom-Gill sequence produced an intramolecular distance of 6.6 Å for the labels spanning one helix turn, and 11.0 Å when the labels spanned two turns [160]. ¹³C/¹⁵N-¹⁹F REDOR NMR has been used to study the interaction of HIV TAR RNA with the viral regulatory peptide Tat. A critical arginine in the peptide was uniformly ¹³C and ¹⁵N labelled and 5-fluorouridine was incorporated at the U23 position of the TAR RNA. [5-¹⁹F]U23-¹³C and [5-¹⁹F]U23-¹⁵N distances were in good agreement with distances obtained from solution-state NMR structures of partial complexes of Tat with TAR RNA [161]. ¹⁹F MAS solid-state NMR experiments have been performed on diacylglycerol kinase (DAGK) site-specific labelled with trifluoromethyl-phenylalanine in native lipid membranes. In comparison with solution-state NMR data of purified DAGK in detergent micelles, the MAS NMR data showed how ¹⁹F chemical shifts of residues at different membrane protein locations were influenced by interactions between membrane proteins and their surrounding lipid environments. Meanwhile, ¹⁹F side chain longitudinal relaxation values were likely affected by different interactions of DAGK with the planar lipid bilayer compared with globular detergent micelles [162]. In other types of samples, the molecular conformation, membrane alignment and dynamic behaviour of the cationic peptide [KIGAKI]₃, which serves as a model for amyloid-like β-sheet aggregation in membranes, were characterised by ¹⁹F solid-state NMR in DMPC lipid bilayers. The data demonstrated a concentration-dependent transition from monomeric β-strands to oligomeric β-sheet amyloid-like fibrils [163]. The orientation of 6-F-cholesterol in DMPC lipid bilayers was analysed by combined use of ¹⁹F chemical shift anisotropy, ²H NMR, and ¹³C-¹⁹F REDOR experiments. The data suggested that the conformational and dynamic properties of 6-F-cholesterol in DMPC lipid bilayers are similar to those of unmodified cholesterol [164].

2.9. Sodium (²³Na)

The only naturally occurring isotope of sodium is the spin-3/2 ²³Na, which is the second most abundant NMR-active nucleus in living tissues after ¹H. The two main uses of ²³Na in biological and biomedical NMR applications are measurement of sodium translocation across membranes and ²³Na-MRI. ²³Na NMR can be used to measure the translocation of sodium ions across membranes in cells, organelles and liposomes using a membrane-impermeable chemical shift reagent to resolve the signals originating from internal and external sodium. Commonly used shift reagents are dysprosium (III) tripolyphosphate [Dy(PPP)2(7-)] and thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis-(methylene phosphonate) [TmDOTP] [165,166]. In erythrocytes, measurement of sodium translocation by ²³Na NMR has been used as an assay of Na⁺/K⁺-ATPase activity [167], for measuring the maximal rate of active sodium ion efflux at 10.1 ± 1.0 mM/hour/litre of cells [168] and for studying Na⁺-K⁺ co-transport [169]. A ²³Na NMR study of the intracellular sodium concentration in the amoebae from the slime mold Dictyostelium discoideum showed that it remained low (0.6 to 3.0 mM) in the presence of external sodium (20 to 70 mM) and a large sodium gradient (20- to 40-fold) was maintained. Introduction of nystatin, an antibiotic known to perturb the ion permeability of membranes, increased the intracellular sodium concentration [170]. ²³Na NMR was to measure the intracellular sodium concentration and sodium transport in Escherichia coli cells under both aerobic and glycolytic conditions. Sodium efflux and maintenance of a stable low intracellular sodium concentration correlated with development and maintenance of the proton motive force, which is consistent with proton-driven Na⁺/H⁺ exchange as the mechanism of sodium transport [171]. ²³Na NMR measurements of sodium transport in cells of Bacillus subtilis defect in the Mrp (multiple resistance and pH) antiporter complex correlated to the inability of this strain to maintain a lower internal sodium concentration than an external one [172]. ²³Na-MRI can be used for imaging of sodium in the brain (Figure 13) and in the heart. See references [173, 174] for recent reviews.

Figure 13. ²³Na-MRI analysis of human brain. The total sodium content of the brain of subjects suffering from Huntington’s disease (HD) is increased compared with healthy controls (Con) of the same age and sex. This Figure was reproduced with permission from Reetz et al. 2012 [175]; copyright © 2012 by Elsevier Inc.

A lower sensitivity and significantly reduced multi-component relaxation behaviour (T₁ ~30 ms, T2 ~0.5-4 ms and 12-20 ms) of ²³Na compared with ¹H mean that use of sodium nuclei for imaging is more challenging. Hence, in vivo sodium imaging is feasible only at high magnetic field strength and by use of fast, specialised and ultra-short echo-time MRI pulse sequences. Also, intra- and extra-cellular sodium cannot be unambiguously resolved without the use of potentially toxic chemical shift reagents. ²³Na-MRI has demonstrated a correlation between sodium accumulation in brain and disability in relapsing-remitting multiple sclerosis. The study involved acquisition of three-dimensional imaging data at 3.0 T to quantify total sodium concentration levels within specific compartments (multiple sclerosis lesions, white matter and grey matter). Statistical mapping analysis showed confined increases in total sodium concentration inside the brainstem, cerebellum and temporal poles in early relapsing-remitting multiple sclerosis and widespread increases affecting the entire brain in advanced relapsing-remitting multiple sclerosis [176]. ²³Na-MRI has also demonstrated heterogeneity of total sodium concentration in hyperacute, acute and chronic lesions in multiple sclerosis [177].²³Na-MRI in vivo analysis at 3.0 T of human lumbar vertebral discs in healthy volunteers and in patients with low back pain showed decreases in intervertabral disc sodium content with disc degeneration [178]. A ²³Na-MRI study of the lower leg in acute heart failure patients during diuretic treatment showed that plasma sodium levels did not change during therapy, whilst sodium concentrations in muscle and skin decreased after furosemide therapy. Sodium concentrations in muscle and skin of patients before and after diuretic therapy were significantly higher than in healthy subjects [179].

2.10. Magnesium (²⁵Mg)

The only naturally occurring NMR-active isotope of magnesium is the spin-5/2 ²⁵Mg (natural abundance 10.0%), which has low sensitivity and produces moderately broad signals. The biological and biomedical applications of ²⁵Mg NMR are relatively scarce. ²⁵Mg NMR has been used to study magnesium binding to the erythrocyte constituents ATP, ADP, AMP, 2,3-bisphosphyoglycerate (DPG), and hemoglobin, thus demonstrating a possible method for studying the binding of magnesium within erythrocytes and other cells [180]. ²⁵Mg NMR was used to study interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase [181]. ²⁵Mg NMR was also used to study the divalent metal binding sites of of NAD⁺-dependent and NADP⁺-dependent isocitrate dehydrogenases from pig heart. Data showed that magnesium has a similar dissociation constant (1.8 mM) from NADP⁺-dependent isocitrate dehydrogenase as from the enzyme-isocitrate complex (1.1 mM). The extrapolated linewidth of bound magnesium increased from 674 Hz in the binary complex to 10,200 Hz in the ternary complex. Hence, the quadrupole coupling constant, calculated from relaxation rates, is larger in the ternary complex, which indicated greater distortion in the magnesium coordination sphere. Results were consistent with the metal sites having anisotropic octahedral symmetry [182]. ²⁵Mg NMR was used to study the polyelectrolyte behaviour of actin filaments. Data from F-actin solutions showed that the rotational correlation times of magnesium ions are independent of the overall rotational dynamics of the actin filaments, and competitive binding experiments demonstrated a facile displacement of F-actin-bound magnesium by [Co(NH₃)₆]³⁺. ATP also competed effectively with F-actin filaments for binding to magnesium ions. The results supported the hypothesis that magnesium ions bind loosely and non-specifically to F-actin filaments and therefore show behaviour typical of counterions in polyelectrolyte solutions [183]. ²⁵Mg NMR has contributed to a study that revealed the stacking of bacteriochlorophyll c macrocycles in chlorosome from Chlorobium limicola. The dimer-based stacking of the macrocycles was demonstrated by ²⁵Mg NMR, which exhibited a pair of signals showing different quadrupole coupling due to the presence or absence of a water molecule in the axial position [184]. ²⁵Mg solid-state NMR has been used to characterise magnesium binding to the DNA repair protein apurinic/apyrimidic endonuclease 1 (APE1). Low-temperature measurements and various mutants of APE1 were used to demonstrate that magnesium ions bind to APE1 and a functional APE1-substrate DNA complex with an overall stoichiometry of one magnesium ion per mole of APE1. The NMR spectra also showed that the single magnesium ion site is disordered and this is likely due to the arrangement of the protein-ligands (cis and trans isomers) about the magnesium ion [185].

2.11. Aluminium (²⁷Al)

The spin-5/2 nucleus ²⁷Al (natural abundance 100%) has relatively high sensitivity but produces broad signals. Biological and biomedical applications of ²⁷Al NMR are relatively rare and varied. The earliest of these used ²⁷Al NMR to investigate the binding of aluminium to [Leu5]-enkephalin. The spectra suggested that aluminium ions binds at two metal-binding sites, the first of which involves the Tyr1CO and Leu5COO^– groups to give a 2:1 species in a tetracoordinated structure. Binding of aluminium at the second site involves the NH₂terminal groups of the tyrosine moiety in a 2:2 species and ²⁷Al chemical shift values strongly suggested that this aluminium atom displays an octahedral environment [186]. A later study demonstrated the potential of ²⁷Al NMR for in vivo analysis and imaging of the human gastric lumen. This was based on following the dissolution kinetics of aluminium-containing drugs at physiological doses and their removal from the human stomach. Aluminium concentrations as low as 0.5 mg could be detected and the time course of gastric emptying was visualised with ²⁷Al-MRI under normal conditions and in the presence of an antimuscarinic agent, which reduces gastric motor function [187]. Gastric emptying and gastrointestinal transit times in mice and humans have also been monitored by ²⁷Al NMR. This study used orally administered aluminium bound to ion-exchange resin and perfluorononane as selective and specific markers for the stomach and the entire gastrointestinal tract, respectively [188]. ²⁷Al NMR has been used to study the transport of aluminium across yeast cell membranes using dysprosium (III) nitrate [Dy(NO₃)₃] as a chemical shift reagent to resolve the signals originating from internal and external aluminium. The results showed that aluminium enters the cells at 15 min and, over a period of 4 hours, equilibrium is reached between internal and external aluminium. Citrate does not favour aluminium entering the cells at pH 5.0 and addition of EDTA extracts out all of the aluminium that has entered the cells [189]. ²⁷Al NMR has been used to characterise the chemical forms of aluminium in xylem sap and to study the mechanism for detoxification of aluminium in roots of the tea plant (Camellia sinensis). The total concentration of aluminium in xylem sap was 0.29 mM and there were two signals in the ²⁷Al NMR spectrum. A larger signal at 11 ppm was consistent with the peak for an aluminium-citrate model solution, suggesting that this is the main complex in which aluminium exists in xylem [190]. In roots, the quantities of aluminium and aluminium-chelating compounds (fluoride, organic acids and catechins) were measured from plants cultivated in nutrient solutions containing 0, 0.4, 1.0 and 4.0 mM aluminium at pH 4.2 for approximately 10 weeks. The levels of soluble aluminium, water-soluble oxalate and citrate, but not fluoride, malate or catechins in young roots increased with an increase in the concentration of aluminium in the treatment solution. ²⁷Al NMR spectra of root tips and cell sap extracted from root tips that had been treated with aluminium were almost identical and had four signals, with two (11 and 16 ppm) apparently corresponding to the known chemical shifts of aluminium-oxalate complexes. In the spectra of cell sap, the resonances at 11 and 16 ppm increased with an increase in the aluminium contents. The results suggested that levels of aluminium-oxalate complexes increase in response to an increase in the aluminium level and that oxalate is a key aluminium-chelating compound in the mechanism of aluminium detoxification in the tea root [191]. Ionic aluminium is toxic for plant growth, but some plants are able to accumulate aluminium at high concentrations without showing toxicity symptoms. In this respect, ²⁷Al NMR was used to characterise aluminium tolerance and its accumulation in tartary and wild buckwheat. Both showed high aluminium tolerance comparable to common buckwheat, they secreted oxalate rapidly from the roots in response to aluminium in a time-dependent manner and accumulated greater than 1 mg/g aluminium in the leaves after short-term exposure to aluminium. The ²⁷Al NMR spectra revealed that aluminium was present in the form of aluminium-oxalate (1:3 ratio) in the roots and leaves, but in the form of aluminium-citrate (1:1 ratio) in the xylem sap in both species [192]. High field (19.6 T) ²⁷Al solid-state NMR with rapid MAS (17.8 kHz) was used for an in vitro study of aluminated human brain tissue, specifically, temporal lobe tissues exposed to 0.1 mM AlCl₃ and also retinal pigment epithelial cells grown in 0.1 mM AlCl₃. The spectra showed multiple aluminium binding sites, good signal-to-noise ratios and apparent chemical shift dispersions (Figure 14). Tentative assignments for ²⁷Al NMR signals in the brain tissue included those for: octahedral AlO₆ (phosphate and water) (-9 to -3 ppm); condensed AlO₆units (Al-O-Al bridges) (9 ppm); tetrahedral AlO₃N and/or octahedral aluminium-carbonate (24 ppm); more N-substituted aluminium and/or tetrahedral AlO₄ (35 ppm) [193].

Figure 14.²⁷Al solid-state NMR spectra of human brain tissue. 19.6 T ²⁷Al spectra for whole nuclear extracts obtained from human retinal pigment epithelial (ARPE) cells grown in media containing 0.10 mM Al³⁺ (A) and total RNA isolated human brain superior temporal lobe tissues exposed to 0.10 mM Al³⁺ then washed in neutral saline and lyophilised (B). This Figure was reproduced with permission from Bryant et al. 2004 [193]; copyright © 2004 by Elsevier Inc.

2.12. Silicon (²⁹Si)

The spin-1/2 nucleus ²⁹Si (natural abundance 4.68%) produces sharp lines and has a wide chemical shift range (-346 to 173 ppm), but has low sensitivity. ²⁹Si NMR spectra usually contain a broad background signal at around -110 ppm originating from the glass and quartz in the tube and probe, which can be suppressed by running a blank spectrum and subtracting this from the sample spectrum. ²⁹Si NMR has very little uses with biological and biomedical samples, but one use has been for characterisation of chemical composition and silicon uptake in marine diatoms, which are key indicators of marine environmental health. ²⁹Si MAS solid-state NMR was used for structural characterisation of biosilica deposits from four different species of diatom (Chaetoceros debilis, Chaetoceros didymum, Cylindrotheca fusiformis, Nitzschia angularis), specifically to determine the Q2:Q3:Q4 ratios. Whilst the analysis did not reveal any differences in the molecular architecture of the silica from the different diatom species, complete cells showed significantly smaller Q4:Q3 ratios (1.8-1.9) than extracted cell walls (2.5-2.8), indicating the existence of intracellular pools of less condensed silica [194]. Silicon uptake and metabolism of the marine diatom Thalassiosira pseudonana was studied by ²⁹Si solid-state NMR and confocal laser fluorescence microscopy, especially with respect to the presence and nature of an intracellular silicon-storage pool. Diatom cells were synchronised by silicon starvation and then frozen for NMR analysis to identify potential silica precursors. NMR spectra were assigned to the various developmental stages of the dividing diatom cells and suggested that the potential silicon-storage pool consists of four-coordinated, condensed silicon [195]. ¹H-²⁹Si CP-MAS solid-state NMR was used to monitor changes in silica and organic content in the diatoms Chaetoceros muelleri and Thalassiosira pseudonana grown at three different salinities (26, 36 and 46 practical salinity units). Data showed that the Q4:Q3 area ratios of C. muelleri, grown away from standard salinities, increased in response to the formation of more condensed and/or an increase in closely associated organic matter to the Q4 component of the diatoms, which was not observed for T. pseudonana. The results suggested that there is a strong relationship between diatom composition and salinity and that C. muelleri is more sensitive to its environment than T. pseudonana [196].

2.13. Phosphorus (³¹P)

The spin-1/2 nucleus ³¹P (natural abundance 100%) has medium sensitivity, produces sharp lines and has a wide chemical shift range (-180 to 250 ppm). ³¹P NMR spectra are usually acquired with ¹H decoupling, thus making them less crowded. The favourable properties of the ³¹P nucleus and its presence as a metabolite (inorganic phosphorus) and constituent of biomolecules (e.g. nucleotides, nucleic acids), phospholipid and biological membranes makes it one of the most commonly used for NMR studies with biological and biomedical systems. For example, ³¹P NMR can be used to probe phospholipid bilayers and biological membranes for structure, phase behaviour, dynamics and interactions with peptides, proteins, small molecules and drugs. A mention of the wide range of types of studies and samples that use ³¹P NMR is not possible here, so a selection of recent examples is given. ³¹P NMR has been used to study mitochondrial function, energy status and metabolism in resting and exercising skeletal muscle. For example, the rate of P_ito ATP exchange flux in resting muscle can be measured using saturation transfer [197] and the post-exercise recovery kinetics of pH and the concentrations of phosphocreatine, P_i and ADP contain valuable information about muscle mitochondrial function and cellular pH homeostasis in vivo [198]. Using ³¹P NMR it has been found that the recovery rate constant for phosphocreatine was significantly decreased in adults with Down’s syndrome compared to controls, which supports the theory of a global mitochondrial defect in Down’s syndrome [199]. Such measurements are also useful in forensic studies. Postmortem ³¹P NMR measurement of the α-ATP/P_i ratio in skeletal muscle showed a decrease over time from 0.445 to 0.032 over 24 hours, the method can therefore be used for determining the time of death [200]. ³¹P NMR has been used to measure energy status and metabolism in lesions occupying the intracranial space, where quantifying the concentrations of phosphomonoester, P_i, phosphodiester, γ-ATP, α-ATP, β-ATP with reference to phosphocreatine allowed a grading of lesions from infective to tumour [201]. ³¹P NMR has been used to study changes in brain bioenergetics following acute sleep deprivation. Phosphocreatine increased in grey matter after two nights of recovery sleep relative to acute sleep deprivation with no significant changes in white matter. Results also demonstrated that increases in phosphocreatine were associated with increases in electroencephalographic slow wave activity during recovery sleep. No significant changes in β-nucleoside triphosphates were observed [202]. ³¹P NMR analysis of phosphorus metabolites in brain regions has been used to study pathophysiology in schizophrenia [203]. In patients with gastric cancer, ³¹P NMR has been used to monitor premorphological alterations in gastric mucosa by measuring hypoxia levels [204]. ³¹P NMR measurement of the phosphomonoester/ phosphodiester ratio has been used to assess the response of chronic hepatitis C to antiviral therapy by interferon and ribavirin [205]. Other applications of solution-state ³¹P NMR include analysis of metabolism in dormant spores of Bacillus species by measurement of small molecules including 3-phosphoglyceric acid and ribonucleotides [206]. Also, analysis of phosphorus composition and speciation in sediments and soils, including those from Lake Erhai, southwestern China [207] and from wetland areas of eastern China [208]. ³¹P solid-state NMR has shown a correlation between the membrane-disruptive abilities of the β-hairpin antimicrobial peptide protegrin-1 and conformation and activity. Thus, ³¹P NMR lineshapes of uniaxially aligned membranes can be used as a diagnostic tool for understanding the peptide-lipid interactions of antimicrobial peptides [209]. The mechanisms of antimicrobial peptide-induced pore formation in lipid bilayers have also been investigated by oriented ³¹P solid-state NMR. In the cases of the peptides alamethicin and novicidin, a majority of the lipids remained in a planar bilayer conformation, but a number of lipids displayed reduced dynamics and these are involved in peptide anchoring. The results showed that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, whilst novicidin forms toroidal pores at high concentrations and produces more extensive disturbance of the membrane [210]. ³¹P-¹⁹F REDOR NMR has been used to measure distances between a trifluoromethyl group and a phosphodiester in nucleic acids [211]. A ³¹P solid-state MRI technique has been used to observe calcification in ex vivo atherosclerotic plaques [212]. ³¹P solid-state NMR has been used to study the interaction of amyloid-β peptide with lipid bilayers and gangliosides. Amyloid-β strongly perturbed the structure of DMPC bilayers to form a non-lamellar phase (most likely micellar) and the ganglioside GM1 potentiated the effect of amyloid-β. The difference of the isotropic peak intensity between DMPC/amyloid-β and DMPC/GM1/amyloid-β suggested a specific interaction between amyloid-β and GM1 [213]. In an interesting application, ³¹P solid-state MRI has been used for in vivo visualisation of bone mineral in human wrists. Using a 3 T scanner and a quadrature wrist ³¹P transmit/receive coil, it was possible to obtain three-dimensional ³¹P images for just bone material of the wrist (Figure 15) [214].

Figure 15. ³¹P solid-state MRI visualisation of bone mineral in human wrists. A. A ³¹P quadrature low pass birdcage transmit/receive coil for human wrist imaging. The enclosure containing the passive transmit/receive switch, quadrature hybrid, preamplifier and scanner interface is not shown. B-F. In vivo ³¹P solid-state MRI images of bone mineral in the wrist of a healthy 41 year old male: schematic view of the scanned region (A), posterior-anterior, lateral and transverse image slices (C-E), three-dimensional isosurface rendering of the full data set (F). This Figure was reproduced with permission from Wu et al. 2011 [214]; copyright © 2011 by Wiley-Liss, Inc.

2.14. Sulphur (³³S)

The only NMR-active isotope of sulphur is ³³S (natural abundance 0.76%), which is spin-3/2, has low sensitivity and produces very broad lines. Hence, ³³S has very few uses in high-resolution NMR and there is only one published study that applies ³³S NMR to biological samples. A 10 mm ³³S cryogenic NMR probe was developed that operates at 9-26 K with a cold preamplifier and a cold rf switch operated at 60 K. The ³³S NMR sensitivity of this cryogenic probe was up to 9.8 times greater than that of a conventional 5 mm broadband NMR probe. By application to biological samples such as human urine, bile, chondroitin sulphate and scallop tissue, it was demonstrated that the system can detect sulphur compounds having SO₄^2- anions and –SO₃^– groups, but other common sulphur compounds such as cysteine were still undetectable because the ³³S nuclei in these compounds are in asymmetric environments [215].

2.15. Chlorine (³⁵Cl, ³⁷Cl)

The two NMR-active isotopes of chlorine, ³⁵Cl and ³⁷Cl (natural abundance 75.6 and 24.24 %, respectively), are both spin-3/2 and produce relatively broad signals, but have a large chemical shift range (-50 to 1050 ppm). Of these, the more sensitive ³⁵Cl is used most commonly. Early ³⁵Cl NMR studies investigated the binding of chloride ions to zinc adenosine diphosphate complexes, zinc-pyruvate kinase complexes, carbonic anhydrases and human haemoglobin [216–219]. ³⁵Cl NMR measurements of chloride binding to carbonmonoxy- and deoxy-dromedary hemoglobin revealed the existence of two classes of chloride-binding sites, one of high and the other of low affinity. Whilst this also resembles the situation for human hemoglobin, the number of binding sites and the association equilibrium constant for chloride binding are significantly higher in the dromedary protein. It was suggested that this difference is due to the greater number of basic residues exposed to solvent and to the higher flexibility of dromedary haemoglobin [220]. ³⁵Cl NMR was used to reveal two new classes of chloride binding sites in the light-driven chloride pump halorhodopsin. One class exhibited low affinity (K_d much greater than 1 M) for chloride and bromide. The second class exhibited a higher affinity (K_d = 110 ± 50 mM) for chloride and also binds other anions according to the affinity series I^–, SCN^– > Br^–, NO^3- > Cl^– > F^–, citrate [221]. Another type of application of ³⁵Cl NMR has been for measuring the translocation of chloride ions across membranes, which includes the co-transport of chloride into vesicles [222,223]. To assist such measurements, cobalt ions (Co²⁺) have been used as a chemical shift reagent to resolve the signals originating from internal and external chloride in ³⁵Cl NMR measurements of chloride translocation with cells and vesicles [224]. The transport of chloride ions across the erythrocyte membrane has been studied by ³⁵Cl NMR. In this case, the signal for intracellular chloride was so broad that it was virtually undetectable (linewidth greater than 200 Hz), whilst the signal for extracellular chloride was relatively narrow (linewidth around 30 Hz). Transport was totally inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulphonate, which is a potent inhibitor of the erythrocyte band 3 protein [225]. A separate ³⁵Cl and ³⁷Cl NMR study was used to define a kinetic equation for the chloride transport cycle of band 3 in erythrocytes. The data supported a situation in which binding, dissociation and channel migration events are rapid compared to the translocation of bound chloride across the membrane. In this case, chloride binding to the transport site was described by a simple dissociation constant [K_d = K_off/K_on] rather than by a Michaelis-Menten constant [K_m = (K_off + K_{translocation})/K_on] [226]. ³⁵Cl and ³⁷Cl NMR relaxation measurements at various field strengths were used to study chloride binding to band 3 protein in the presence and absence of the transport inhibitor 4,4′-dinitrostilbene-2,2′-disulphonate. There were significant differences in NMR relaxation rates depending on whether the inhibitor was present or not. The results indicated that the rate of chloride association and dissociation at each external binding site occurs on a time scale of less than or equal to 5 ms, thus implying that the transmembrane flux is not limited by the rate of chloride binding to the external chloride binding site of band 3. The rotational correlation-time of chloride bound to band 3 was greater than 20 ns with a quadrupole coupling constant of approximately 2 MHz [227]. Chloride binding to band 3 in erythrocytes has also been detected by double-quantum-filtered ³⁵Cl NMR [228]. Other applications of ³⁵Cl and ³⁷Cl NMR include identification of a unique pair of zinc binding sites in the human alpha 2-macroglobulin tetramer. Zinc bound at these sites did not affect the ³⁵Cl NMR linewidth of free chloride. It was shown that additional lower affinity zinc sites exist that bind chloride weakly and cause broadening of the free chloride signal through fast exchange with bound chloride. Relaxation measurements demonstrated that chloride bound at these sites has an internal correlation time of 5.1 ns and a quadrupolar interaction of 4.2 MHz with zinc [229]. The anion binding selectivity of sarcoplasmic reticulum membranes has been studied by ³⁵Cl NMR. Titration experiments with a series of different anions revealed that multivalent, phosphate-like anions bind much stronger to sarcoplasmic reticulum vesicles than monovalent anions like halides, whilst oxalate has an intermediate position. The binding strength decreased with decreasing ionic radius according to the following sequence: vanadate > phosphate > sulphate >> iodide > oxalate > bromide > chloride >> fluoride [230]. A feasibility study for ³⁵Cl-MRI in humans has also been performed. It was found that ³⁵Cl-MRI at 7 T allows in vivo imaging of ³⁵Cl in human brain (Figure 16) and muscle in clinically feasible acquisition times (10-35 minutes) and voxel volumes (0.2-1.3 cm³). Pathophysiological changes of chloride homeostasis due to cancer or muscular ion channel disease could also be visualised [231].

Figure 16. ³⁵Cl-MRI analysis of human brain. Transverse, sagittal, and coronal sections of a ³⁵Cl MR imaging data set for a healthy human brain in a 67-year-old male. Two reference tubes were used (A, 103 mmol/L NaCl solution and 4% agar gel; B, 103 mmol/L NaCl solution without agar gel). An FOV of 225 x 225 mm² is shown. A. Cl₂ concentration (³⁵Cl MR imaging) of the total tissue: The signal intensities were measured with a short TE of 0.70 ms and a long TR of 65 ms. Normalisation was performed with respect to reference tube B. Because brain tissue and reference tube B have different relaxation properties, the Cl₂ concentration of brain tissue is underestimated by a factor of 1.12, when considering the measured relaxation times of brain tissue and saline solution. The Cl₂ concentration was determined to be 100 mmol/L in the CSF (circle 1) and 1.12 x 32 mmol/L = 36 mmol/L in the entire tissue (circle 2). B. Differences in T1 can be taken advantage of to suppress signal from CSF (³⁵Cl IR MR imaging). Signal intensities were normalised to reference tube A. Signal from reference tube B is well suppressed in ³⁵Cl IR MR imaging. Colour bar = measured Cl₂/Na⁺ concentration in mmol/L liter or measured signal intensity normalised to reference tube A. This Figure was reproduced with permission from Nagel et al. 2014 [231]; copyright © 2014 by Radiological Society of North America.

2.16. Potassium (³⁹K)

There are three NMR-active isotopes of potassium, spin-3/2 ³⁹K (natural abundance 93.26%), spin-4 ⁴⁰K (natural abundance 0.01%) and spin 3/2 ⁴¹K (natural abundance 6.73%), which all produce broadened signals over a relatively small chemical shift range (-30 to 35 ppm). ³⁹K is usually preferred for NMR applications because it has the highest sensitivity and produces sharper signals than ⁴¹K. Whilst ⁴⁰K produces sharper signals than ³⁹K, it has very low sensitivity and is not really used at all. Transport of potassium ions in yeast cells and in human erythrocytes has been measured using ³⁹K NMR with chemical shift reagents to resolve the signals originating from internal and external potassium [232,233]. Similarly, a TmDOTP shift reagent was used to assist ³⁹K NMR measurement of intracellular potassium during ischemia in the perfused guinea pig heart [234]. A number of studies have demonstrated the usefulness of ³⁹K NMR for in vivo measurement of potassium levels and its transport in a range of different rat tissues including blood, brain, muscle, kidney, liver, testes and mandibular salivary gland [235–238]. A number of feasibility studies have also been performed for ³⁹K-MRI analysis of human muscle (Figure 17) and brain [239–241].

Figure 17. ³⁹K-MRI analysis of human muscle. ¹H (left), ²³Na (middle) and ³⁹K (right) MR images of the right healthy thigh muscle in a 26-year old female. ²³Na and ³⁹K MR imaging were performed with a nominal spatial resolution of 3.75 x 3.75 x 10.14 mm³ and 8 x 8 x 16 mm³, respectively. Acquisition times were approximately 30 minutes. Reference tubes containing 4% agarose gel with 20 mmol/L NaCl and 103 mmol/L KCl solutions are marked in the ¹H image by 1 and 2, respectively. The femur is clearly visible on the ²³Na and ³⁹K MR images as areas of reduced signal intensity. The territory of the femoral artery and vein (arrow) exhibits high signal intensity in the ²³Na MR image but reduced signal intensity in the ³⁹K MR image. Fatty tissue that surrounds the ischiadic nerve (circles) exhibits low ³⁹K signal intensity, while subcutaneous fat shows no ³⁹K MR imaging signal. The mean estimated Na⁺ and K⁺ concentrations of muscle tissue (white ROI) are [Na⁺] = 18 mmol/L ± 1 and [K⁺] = 116 mmol/L ± 4. Colour bars = Measured Na⁺ and K⁺ concentrations in the ²³Na and ³⁹K MR images, respectively. This Figure was reproduced with permission from Umathum et al. 2013 [239]; copyright © 2013 by Radiological Society of North America.

2.17. Calcium (⁴³Ca)

The NMR-active isotope of calcium is the spin-7/2 ⁴³Ca (natural abundance 0.135%), which has moderate sensitivity and produces moderately sharp signals over a relatively small chemical shift range (-35 to 35 ppm). Early ⁴³Ca NMR experiments studied calcium binding to a range of different proteins including pancreatic prophospholipase A2 [242], calmodulin, parvalbumin and troponin C [243], pancreatic phospholipase A2 and its zymogen [244], prothrombin fragment 1 [245] bone γ-carboxyglutamic acid protein [246] and later lysosymes and alpha-lactalbumins [247]. Early ⁴³Ca NMR experiments also studied binding of calcium to DNA [248,249]. A later ⁴³Ca NMR study on calcium binding to DNAs involved chemical shift and linewidth measurements obtained during titration with ⁴³CaClO₄, which demonstrated the existence of at least two classes of bound ⁴³Ca²⁺ ions. Binding to C. perfringens DNA (31% GC) was dominated by a delocalised, non-specific interaction. Binding to M. lysodeikticus DNA (72% GC) suggested that a small fraction of the ⁴³Ca²⁺ experiences significant motional retardation and/or an increase in the electric field gradient when associated with the DNA, and therefore appeared to be locally bound to discrete sites on the DNA. Hence, the results demonstrated that higher GC content correlates with an increase in favorable Ca²⁺ binding environments [250]. More recently, ⁴³Ca solid-state NMR experiments have been designed to study the structures of calcium binding sites in biological materials including metalloproteins and bone [251,252]. Indeed, natural abundance ⁴³Ca MAS solid-state NMR has been used to obtain structural information about the coordination environment of calcium in bone using powdered bovine cortical bone samples (Figure 18) [253].

Figure 18. ⁴³Ca MAS experiments probing the interaction of osteocalcin bone protein with calcium sites in 5% carbonated apatite. A. One-dimensional ⁴³Ca MAS spectrum of ⁴³Ca-enriched carbonated apatite containing osteocalcin protein at room temperature (solid black line), simulated spectrum of 5% carbonated apatite (dashed red line) and simulated spectrum of cortical bone (dashed blue line). B. Two-dimensional MQMAS spectrum correlating the ⁴³Ca chemical shift and a triple-quantum frequency of ⁴³Ca-enriched carbonated apatite powder sample containing osteocalcin protein at room temperature. The lengths of the excitation and conversion radio frequency pulses were 5 and 10 μs, respectively. The 2D spectrum is the resultant of 16 t1 experiments, 61,440 scans, a 0.5 s recycle delay and a 10 kHz sample spinning frequency. The 1D spectrum was obtained using a single pulse sequence with a 90° pulse length of 2 μs, 512,000 scans, a recycle delay of 250 ms and a 10 kHz sample spinning frequency. C. The two calcium coordination environments in carbonated apatite. This Figure was reproduced with permission from Xu et al. 2010 [253]; copyright © 2010 by American Chemical Society.

2.18. Vanadium (⁵¹V)

There are two NMR-active isotopes of vanadium, spin-6 ⁵⁰V (natural abundance 0.25%) and spin-7/2 ⁵¹V (natural abundance 99.75%), which produce relatively broad signals but have a wide chemical shift range (-1900 to 0 ppm). ⁵¹V is the nucleus of choice because it is more sensitive and produces sharper signals than ⁵⁰V. ⁵¹V NMR has been applied to a number of biological systems and samples.

One of the earliest applications of ⁵¹V NMR was in the study of vanadium metabolism in wild-type and respiratory-deficient strains of Saccharomyces cerevisiae [254]. ⁵¹V NMR has been used to study the binding of vanadium, vanadate and vanadium compounds to a number of different proteins including ribonuclease-T1 [255], phosphoglycerate mutase [256], chloroplast CF1-ATPase [257], transferrins [258] and prostatic acid phosphatase [259]. ⁵¹V NMR was used to study interactions of vanadate oligomers with sarcoplasmic reticulum Ca²⁺-ATPase. Addition of sarcoplasmic reticulum caused narrowing of the linewidth of the tetrameric vanadate signal in the presence of ATP and Ca²⁺, whilst there was a broadening of the signal for monomeric vanadate. Hence, ATP decreased the affinity of the enzyme for tetravanadate, whilst inducing the interaction with monomeric vanadate. In the presence of Ca²⁺, tetrameric and decameric vanadate were bound to sarcoplasmic reticulum ATPase, whilst monomeric vanadate only bound to sarcoplasmic reticulum when ATP was present. It was found that an increase of vesicular Ca²⁺ concentration counteracts monovanadate inhibition of sarcoplasmic reticulum Ca²⁺-ATPase activity but it does not significantly affect decavanadate inhibition [260]. ⁵¹V NMR was used to investigate the binding of vanadate to human erythrocyte ghosts. Results suggested that monomeric and polymeric vanadate species bind to the anion binding sites of band 3 protein of the erythrocyte membrane, where monomeric vanadate binds with a dissociation constant of around 0.23 mM [261]. ⁵¹V MAS solid-state NMR has been used to study the coordination environment in the 67.5-kDa protein vanadium chloroperoxidase. This was possible despite the low concentration of vanadium sites in the protein (one per molecule). Density functional theory calculations of the NMR spectra for an extensive series of active site models suggested that the vanadate cofactor is most likely to be anionic with one axial hydroxo- group and an equatorial plane consisting of one hydroxo- and two oxo- groups [262]. Correlations between ⁵¹V solid-state NMR parameters and the chemical structures of vanadium complexes have been interpreted as models for related metalloproteins. These included the isotropic chemical shift and the chemical shift anisotropy correlated with specific structural features such as the coordination number of the vanadium atom, the number of coordinating nitrogens, the number of oxygen atoms and the chemical environment of the complex. Quantitative correlations between the solid-state NMR parameters and specific bond angles and bond lengths were also obtained [263]. Further methods for analysing ⁵¹V solid-state NMR spectra of complex systems have also been developed [264].

2.19. Cobalt (⁵⁹Co)

The naturally occurring isotope of cobalt, ⁵⁹Co (natural abundance 100%), is NMR-active with a spin of 5/2. ⁵⁹Co has high sensitivity and produces broad signals, but it does have an extremely wide chemical shift range (-4000 to 14000 ppm). ⁵⁹Co NMR has found some applications with biological and biomedical systems and samples. A method using ⁵⁹Co NMR was developed for measuring the volume of intracellular water space. This is based on the signal for an inert, stable and membrane-impermeable cobalt(III) compound such as [Co(CN)6]³⁻ or [Co(imidazole)6]³⁺. In its first application, variation of the intracellular water space in human erythrocytes as a function of osmolality was measured [265]. The method was later used in studies of experimental acute pancreatitis, whereby interstitial and total water volumes of the pancreas were estimated by measuring the distribution of [Co(CN)6]³⁻. In the healthy pancreas, the interstitial compartment comprised 35% of the tissue volume. In the diseased pancreas, the penetration volume of [Co(CN)6]³⁻ reached 90% of the total tissue volume, which was indicative of extensive membrane injury [266]. The method has been used for measuring intracellular water volumes in a range of different cell suspensions and perfused organs [267]. ⁵⁹Co NMR has been used for in vivo thermometry with liposomes containing cobalt complexes, which is useful for making localised temperature measurements in tissue during hyperthermia treatment of cancer. The method uses the complex tris(ethylenediamine) cobalt(III) trichloride as a temperature sensor by determining the temperature dependence of its ⁵⁹Co NMR chemical shift (Figure 19). Encapsulation within liposomes allows targeting of the agent to the reticuloendothelial system and temperature changes in the order of 0.1 ºC have been measured in vivo in rats [268].

Figure 19. ⁵⁹Co NMR in vivo thermometry with liposomes containing a cobalt complex. A. Three representative ⁵⁹Co spectra showing the change in chemical shift as a function of temperature. B. Resonant frequency shift of the cobalt (III) nucleus in the Co(en)₃C1₃ complex as a function of temperature for a solution in water. The standard error for temperature measurements using the Luxtron fibre-optic thermometer was 0.1 ºC and the standard error for the measured frequency shift was also 0.1 ºC. This Figure was reproduced with permission from Webb et al. 1995 [268]; copyright © 1995 by Taylor & Francis.

⁵⁹Co NMR has been used to probe interactions of [Co(NH₃)6]³⁺ with helical B-DNA (Braunlin et al., 1987) and for chiral recognition of deoxyoligonucleotides by δ- and λ-tris(ethylenediamine)cobalt(III) [269]. ⁵⁹Co solid-state NMR has also been used to probe metal binding sites in polynucleotides. This included elucidation of magnesium-nucleic acid binding sites by using [Co(NH₃)6]³⁺ as surrogate for [Mg(H₂O)6]²⁺ [270].

2.20. Copper (⁶⁵Cu)

The two NMR-active isotopes of copper, ⁶³Cu (natural abundance 69.15%) and ⁶⁵Cu (natural abundance 30.85%), are both spin-3/2 and produce broad signals, but have a large chemical shift range (-300 to 800 ppm). ⁶³Cu is the more sensitive nucleus, whilst ⁶⁵Cu produces slightly narrower signals. There are very few applications of ⁶³Cu or ⁶⁵Cu with biological samples. Low temperature ⁶⁵Cu NMR measurements have been performed on the blue copper protein azurin in the reduced copper I state. Measurements at18.8 T and 10 K produced a strongly second order quadrupole perturbed spectrum, which gave a ⁶⁵Cu quadrupole coupling constant of ± 71.2 ± 1 MHz, corresponding to an electric field gradient of ± 1.49 atomic units at the copper site, and an asymmetry parameter of approximately 0.2 [271].

2.21. Zinc (⁶⁷Zn)

The only NMR-active isotope of zinc is spin-5/2 ⁶⁷Zn (natural abundance 4.102%), which is low sensitivity and produces moderately broad signals. ⁶⁷Zn NMR applications with biological samples are scarce. ⁶⁷Zn solid-state NMR has been used to study the minimal DNA binding domain of human nucleotide excision repair protein XPA [272] and to investigate the zinc binding site in the zinc protein rubredoxin [273]. Residue ionisation in the zinc-dependent deacetylase of bacterial lipid A synthesis (LpxC) has been directly observed by ⁶⁷Zn solid-state NMR. The pH-dependence of ⁶⁷Zn solid-state NMR lineshapes were measured for both wild-type and mutant (H265A) forms of Aquifex aeolicus LpxC, each in the absence of substrate (resting state). The spectrum of wild-type LpxC at pH 6 contained two overlapping quadrupole lineshapes with C q values of 10 and 12.9 MHz, whilst the spectrum for a sample prepared at pH 9 was dominated by the appearance of a third species with a C q of 14.3 MHz. Spectra of the mutant were pH-independent, where a C q of 9.55 MHz was sufficient to describe both low and high pH data. Quantum mechanical/molecular mechanical modelling of the H265A mutant suggested that over this pH range water is bound to the zinc ion whilst Glu78 is protonated (Figure 20) [274].

Figure 20. Residue ionisation in Aquifex aeolicus LpxC observed by ⁶⁷Zn solid-state NMR. QM/MM optimised quantum regions of WT LpxC with doubly protonated His265, water bound to zinc and a proton added to Glu78 Oε1 and Oε2 in (A) and (B), respectively, and H265A LpxC with water bound to zinc and (C) Glu78 Oε1H and (D) Glu78 Oε2H. This Figure was reproduced with permission from Lipton et al. 2008 [274]; copyright © 2008 by Journal of the American Chemical Society.

2.22. Selenium (⁷⁷Se)

The NMR-active isotope of selenium, spin-1/2 ⁷⁷Se (natural abundance (7.63%), has low sensitivity but produces narrow signals over a very wide chemical shift range (-1000 to 2000 ppm). ⁷⁷Se NMR applications with biological samples are relatively scarce. An early study demonstrated the feasibility of using ⁷⁷Se NMR for observing selenium covalently attached to proteins. The disulphide bonds of ribonuclease-A and lysosyme were reductively cleaved under denaturing conditions and the resulting sulphydryl groups were treated with the reagent 6,6′-diselenobis(3-nitrobenzoic acid) to give proteins containing covalently attached selenium in the form of selenenyl sulphides. In high-resolution ⁷⁷Se NMR spectra of these proteins under denaturing conditions, five to six signals within a chemical shift range of 14-15 ppm were observed for each protein and were compared to the chemical shifts of several model selenenyl sulphides derived from cysteine [275]. A later study investigated the NMR relaxation properties of ⁷⁷Se-labelled proteins. Cysteine residues in the proteins bovine hemoglobin, reduced ribonuclease A and glutathione were labelled with ⁷⁷Se by reaction with [⁷⁷Se]6,6′-diselenobis(3-nitrobenzoic acid) and the resultant species contained Se-S linkages with ⁷⁷Se NMR signals in the range 568-580 ppm. It was concluded that when there are no complications from protein aggregation or chemical exchange, the chemical shift anisotropy values anticipated to exist in selenoproteins should result in signals with linewidths in the range 27 to 170 Hz, depending on field strength. Such signals should therefore be observable in the intact protein, if ⁷⁷Se-enriched material is available [276]. ⁷⁷Se NMR was used to characterise ⁷⁷Se-labelled ovine erythrocyte glutathione peroxidise. Lambs, maintained on a selenium-deficient diet supplemented with 94 atom % Na₂⁷⁷SeO₃, were used as the source of ⁷⁷Se-enriched erythrocyte glutathione peroxidise, for which the content of ⁷⁷Se reached 88%. Monthly bleeding of two animals produced approximately 20 mg of ⁷⁷Se-enriched glutathione peroxidase in pure form [277]. More recently, ⁷⁷Se NMR has been used for site-specific pK_a determination of selenocysteine residues in seleno-vasopressin [278].

2.23. Bromine (⁷⁹Br, ⁸¹Br)

The two naturally occurring isotopes of bromine, ⁷⁹Br (natural abundance 50.69%) and ⁸¹Br (natural abundance 49.31%), are both NMR-active with spin-3/2 and produce broad signals, but have a wide chemical shift range (-500 to 100 ppm). ⁸¹Br is the more sensitive nucleus and produces slightly narrower signals than ⁷⁹Br. Because ⁷⁹Br has a frequency very similar to that of ¹³C, is observable using a carbon specific probe and produces clear quadrupolar spinning sidebands in the solid-state, it is widely used in MAS solid-state NMR for adjusting the magic angle. ⁷⁹Br and ⁸¹Br NMR applications with biological samples are very scarce. An early study used ⁷⁹Br and ⁸¹Br NMR to probe the reversible association of α-chymotrypsin and a mercury-containing substrate (4-bromomercuriocinnamic acid, BrHgCin), which rapidly exchanges bromide ions. It was found that the rate of bromide exchange is diffusion-limited and faster than the rate of reorientation of the BrHgCin-α-chymotrypsin complex. The rapid rate of bromide exchange with the complex was not compatible with the side chain of BrHgCin being entirely buried in a nonpolar pocket on the enzyme but compatible with the side chain being exposed to the solution [279].

2.24. Krypton (⁸³Kr)

The NMR-active isotope of krypton, spin-9/2 ⁸³Kr (natural abundance 11.50%), has low sensitivity but produces narrow signals over a chemical shift range of 40 to 180 ppm. The main use of ⁸³Kr has been as a contrast agent for ⁸³Kr-MRI analysis of lungs. The feasibility for using hyperpolarised ⁸³Kr for imaging was first tested in canine lung tissue by using krypton gas with natural abundance isotopic distribution [280]. ⁸³Kr-MRI has been used for ex vivo imaging of rat lungs using natural abundance krypton gas [281] and to detect tobacco smoke deposition on a model glass surface coated with bovine lung surfactant extract [282]. A recent study used ⁸³Kr-MRI with a surface quadrupolar relaxation (SQUARE) contrast method for imaging of alveolar degradation in a rat model of emphysema [283].

2.25. Rubidium (⁸⁷Rb)

There are two NMR-active isotopes of rubidium, spin-5/2 ⁸⁵Rb (natural abundance 72.17%) and spin-3/2 ⁸⁷Rb (natural abundance 27.83%), which produce relatively broad signals in the chemical shift range -80 to 30 ppm. ⁸⁷Rb has the highest sensitivity and produces less broad signals than ⁸⁵Rb. The main applications of ⁸⁷Rb NMR originate from the fact that rubidium can be used to replace potassium in biological systems and ⁸⁷Rb is more sensitive than ³⁹K. Indeed, ⁸⁷Rb NMR measurements in human erythrocytes, using the chemical shift reagent dysprosium(III) triethylenetraamine-N,N,N’,N”,N”‘,N”‘-hexaacetic acid (DyTTHA^3-) to resolve internal and external signals, demonstrated that rubidium transport proceeds via Na⁺,K⁺-ATPase as there was inhibition of uptake in presence of ouabain [284]. Both ⁸⁷Rb NMR and ⁸⁷Rb-MRI have been used for studies of potassium transport and energetics in mammalian cells, organs and in vivo. This includes studies on isolated perfused rat and pig hearts and also on kidney, skeletal muscle, salivary gland and red blood cells. The method has been used for measuring the kinetics of unidirectional rubidium uptake and efflux and steady-state rubidium levels [285]. For example, 3D ⁸⁷Rb-MRI analysis of isolated pig hearts allowed the effects of regional ischemia on rubidium uptake to be investigated. In the control group, distribution of rubidium in the left ventricle and the intensities of the three-dimensional ⁸⁷Rb images were uniform. In the ischemic group, images showed a reduced ⁸⁷Rb intensity, corresponding to decreased rubidium content (33 ± 11% of normal) [286]. ⁸⁷Rb NMR was used to measure mitochondrial potassium fluxes in whole rat hearts [287] and potassium transport in mouse hearts [288]. ⁸⁷Rb-MRI has been used to investigate ischemia and infarction in blood-perfused pig hearts [289] and to measure potassium transport and energetics in Kir6.2 channel knockout mouse hearts [290].

2.26. Cadmium (¹¹¹Cd, ¹¹³Cd)

There are two NMR-active isotopes of cadmium, ¹¹¹Cd (natural abundance 12.80%) and ¹¹³Cd (natural abundance 12.22%), which are both spin-1/2 and produce narrow signals over a wide chemical shift range (-650 to 0 ppm). ¹¹³Cd is the more commonly used nucleus because it is slightly more sensitive than ¹¹¹Cd. A large majority of the applications of ¹¹¹Cd and ¹¹³Cd NMR have been for probing the binding properties and structures of metal binding sites in metalloproteins and in proteins that have metals as ligands or substrates, including metal transporters. The relatively few applications of ¹¹¹Cd NMR include a study of the binding of novel N-hydroxybenzene-sulphonamide carbonic anhydrase inhibitors to native and ¹¹¹Cd-substituted carbonic anhydrase [291] and characterisation of zinc(II) binding to the peptide amyloid-β1-16 linked to Alzheimer’s disease [292]. There are a number of ¹¹¹Cd NMR studies with metallothioneins, which are small (6-7 kDa) cysteine- and metal-rich proteins. The domain specificity of silver and copper binding to metallothionein was studied by ¹¹¹Cd NMR. For example, titration of ¹¹¹Cd₇MT with silver indicated that silver ions bind preferentially to the β-domain of the protein to form the metal hybrid species (Cd₄)α(Ag₆)βMT. Once the β-domain is filled, additional silver ions displace cadmium from the α-domain to form (Ag₆)α(Ag₆)βMT. The metal displacement reaction is cooperative and the two domains react independently of one another. Copper reacts with Cd₇MT in a manner similar to silver, except that, unlike silver, copper appears to produce intermediate species that may contain mixed-metal clusters. The differential affinities of the two domains for monovalent and divalent metal ions along the availability of facile pathways for metal exchange may be features that enable metallothionein to function simultaneously in the metabolism of different metal ions [293]. Whilst it was already known that in mammalian metallothionein, zinc is exclusively coordinated with Cys-thiolate to form clusters in which the metal is thermodynamically stable but kinetically labile, little was known about coordination in prokaryotic metallothionein. Hence, ¹¹¹Cd NMR measurements with a prokaryotic maetallothionein showed how metal coordination involves the imidazole moieties of histidine residues [294]. ¹¹¹Cd NMR was also used to demonstrate histidine coordination in a plant zinc-metallothionein. Two conserved histidine residues participated in metal binding in a novel cluster composition that was different compared to those from mammals or cyanobacteria [295]. ¹¹³Cd NMR has been used in a large number of studies with metallothioneins, including correlation of the ¹¹³Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry and structure determination of the metal binding sites and whole proteins [296,297]. ¹¹³Cd NMR was used to determine the structures of the multiple metal-binding sites in the two major isoproteins of metallothionein from mammalian livers (rabbits, calf and human) and from giant mud crab hepatopancreas. This was achieved by metal replacement with ¹¹³Cd and then analysis of ¹¹³Cd NMR spectra to provide structures for the binding site clusters [298]. Two-dimensional heteronuclear ¹H-¹¹³Cd correlated spectroscopy was used for obtaining through-bond connectivities between the metals and sequentially assigned cysteine residues to assist spatial structure determination of rabbit liver metallothionein-2. The protein comprised two domains enclosing a three-metal cluster and a four-metal cluster. The metal coordination within these clusters was different from that of (Cd₅, Zn₂)-MT-2 from rat liver as determined by X-ray crystallography. In the two structures different metal-cysteine combinations were apparent for 5 of the 12 connectivities in the three-metal cluster and for 14 of the 16 connectivities in the four-metal cluster [299]. Chinese hamster metallothioneins with mutations in invariant cysteine residues had altered metal binding capacity, cadmium resistance and ¹¹³Cd NMR spectra. Analysis of the ¹¹³Cd NMR spectra for the mutant metallothioneins provided an indication as to the structural basis for the effects of each mutation on metal binding [300]. The three-dimensional structure of metallothionein-1 from blue crab was determined by solution-state NMR, including heteronuclear ¹H-¹¹³Cd correlation experiments. The protein binds six divalent metal ions in two separate metal-binding clusters, which reside in two distinct domains. Structure determination involved measurement of 24 metal-to-cysteine connectivities from the ¹H-¹¹³Cd correlation experiments. The only element of regular secondary structure in either of the two metal binding domains was a short segment of helix in the C-terminal α-domain between Lys42 and Thr48. The folding of the polypeptide backbone chain in each domain, however, produced several type I beta turns [301]. The effect of nitric oxide on metal release from metallothionein-3 [302] and the reaction of human metallothionein-3 with the platinum-based anticancer drugs cisplatin and transplatin [303] have also been investigated by ¹¹³Cd NMR. ¹¹³Cd NMR has been used in applications with a large number of other types of proteins. One of the earliest studied the effect of pH, bicarbonate and cyanide on carbonic anhydrases [304]. Other examples are studies of the metal binding sites in blue copper proteins [305], a yeast superoxide dismutase [306], human retinoic acid receptor-β DNA binding domain [307], a bacterial phosphotriesterase [308] , bovine and human α-lactalbumin and equine lysozyme [309] and cabbage histidinol dehydrogenase [310]. ¹¹³Cd NMR was also used to dentify the coordinating residues in the DNA binding domain of the glucocorticoid receptor [311]. The forms of cadmium in the leaves of Thlaspi caerulescens and in xylem sap of Arabidopsis halleri, which are both cadmium hyperaccumulators, have been identified by ¹¹³Cd NMR. In the former case it was a cadmium-malate complex [312] and in the latter case it was Cd(NO₃)₂ [313]. ¹¹³Cd NMR has been used to study interactions between cadmium(II) and extracellular organic matter released by the green alga Selenastrum capricornutum [314] and to characterise interactions between cadmium and models of the main components of soil organic matter, specifically exopolysaccharides, exudates of roots (polygalacturonic acid) and soil bacteria [315]. ¹¹³Cd solid-state NMR has been used to probe metal binding to the Escherichia coli zinc exporter ZitB with amplified expression in native membranes. Direct observation of ¹¹³Cd binding to ZitB allowed competitive titrations with a range of other metals. These indicated that ZitB is able to bind both nickel and copper, as well as zinc [316].

2.27. Iodine (¹²⁷I)

The only naturally occurring isotope of iodine, ¹²⁷I, is NMR-active with a spin of 5/2 and has medium to high sensitivity. Whilst ¹²⁷I has a very wide chemical shift range (-100 to 4100 ppm), it also tends to produce very broad signals. Hence, applications of ¹²⁷I NMR with biological systems and samples are very scarce. ¹²⁷I NMR was used to study the binding of iodide by lactoperoxidase. The ¹²⁷I NMR signal for iodide binding showed no competition with cyanide and spectra revealed that the binding of iodide is facilitated by protonation of an ionisable group with a pKa value of 6.0-6.8, which was presumed the distal histidyl residue [317]. ¹²⁷I NMR was also used to study iodide binding to Arthromyces ramosus peroxidise. This showed that the binding of iodide depends on protonation of an amino acid residue with a pKa of around 5.3, which was presumed to be the distal histidine (His56), which is 7.8 Å away from the iodide ion [318].

2.28. Xenon (¹²⁹Xe)

There are two NMR-active isotopes of xenon, spin-1/2 ¹²⁹Xe (natural abundance 26.40%) and spin-3/2 ¹³¹Xe (natural abundance 21.23%), which have low to medium sensitivity and a very wide chemical shift range (-5700 to 100 ppm). ¹²⁹Xe produces the sharpest signals and is used most commonly. The hyperpolarisation of ¹²⁹Xe gas has allowed a large number of NMR applications with biological and biomedical systems and samples [319]. The two main approaches that have emerged are ¹²⁹Xe NMR-based biosensors and ¹²⁹Xe-MRI. Xenon-encapsulating molecules called cryptophanes emerged as probes for ¹²⁹Xe NMR that could be used in biosensors. The 1.70 Å resolution crystal structure of a cryptophane-derivatised benezenesulphonamide complexed with human carbonic anhydrase II showed how an encapsulated ¹²⁹Xe atom can be directed to a specific biological target. The structure indicated that the cryptophane cage does not strongly interact with the surface of the protein, which may enhance the sensitivity of ¹²⁹Xe NMR spectroscopic measurements in solution (Figure 21) [320]. More recently, the (+) and (-) enantiomers for a cryptophane-7-bond-linker-benzenesulphonamide biosensor (C7B) have been synthesised and biosensor binding to carbonic anhydrase II was characterised for both enantiomers by hyperpolarised ¹²⁹Xe NMR [321]. A biosensor based on the non-selective grafting of cryptophane precursors on holo-transferrin have been assessed, where cell uptake of the sensor was accompanied by hyperpolarised ¹²⁹Xe NMR detection [322].

Figure 21. Cryptophanes as xenon-encapsulating molecules as probes for a ¹²⁹Xe NMR biosensor of carbonic anhydrase II.

A. MoMo and PoPo enantiomers of the cryptophane-A-derived carbonic anhydrase biosensor. The benzenesulphonamide moiety serves as an affinity tag that targets the Zn²⁺ ion and the R1 substituents contain triazole propionate moieties that enhance aqueous solubility. B. Stereoview of a simulated annealing omit map showing 1-MoMo (blue) and 1-PoPo (red) bound in the active site (1.9 σ contour, teal). A Bijvoet difference Fourier map (2.0 σ, black) confirms encapsulation of Xe (yellow). This Figure was reproduced with permission from Aaron et al. 2008 [320]; copyright © 2008 by American Chemical Society.

Targeting and delivery of a peptide-modified cryptophane to cells expressing αvβ3 integrin receptor, which is overexpressed in many human cancers, was reported for a ¹²⁹Xe NMR biosensor. Cryptophane was functionalised with cyclic RGDyK peptide and ¹²⁹Xe NMR revealed a single resonance at 67 ppm for the ¹²⁹Xe-cryptophane-cyclic RGDyK biosensor. Introduction of αIIbβ3 integrin receptor in detergent solution generated a new ‘bound’ ¹²⁹Xe biosensor signal that was shifted 4 ppm downfield from the ‘free’ ¹²⁹Xe biosensor [323]. A ‘clickable’ and highly water-soluble PEGylated cryptophane has been developed as a potential universal platform for ¹²⁹Xe NMR biosensors. The molecule is easily functionalised by Huisgen cycloaddition and exhibits excellent xenon-encapsulation properties (Figure 22) [324]. Molecular imaging of cancer cells using a bacteriophage-based ¹²⁹Xe NMR biosensor has been achieved [325] and an antibody-based, modular biosensor for ¹²⁹Xe NMR molecular imaging of cells has been developed using cryptophanes as the chemical host for hyperpolarised nuclei [326]. Hyperpolarised ¹²⁹Xe chemical exchange saturation transfer (Hyper-CEST) NMR techniques allow the ultrasensitive (1 picomolar) detection of xenon in cryptophane host molecules. Hyper-CEST ¹²⁹Xe NMR has been used to detect Bacillus anthracis and Bacillus subtilis spores in solution and to interrogate the layers that comprise their structures. ¹²⁹Xe-spore samples were selectively irradiated with radiofrequency pulses, the depolarised ¹²⁹Xe was returned to aqueous solution and depleted the ¹²⁹Xe-water signal, which provided a measurable contrast. Removal of the outermost spore layers in B. anthracis and B. subtilis (the exosporium and coat, respectively) enhanced ¹²⁹Xe exchange with the spore interior [327].

Figure 22. ‘Clickable’ and highly water-soluble PEGylated cryptophanes for ¹²⁹Xe NMR biosensors. ¹²⁹Xe NMR spectra obtained at 11.7 T and 293 K for compound 1 (9 mm) in D₂O (A) and compound 11 (37 mm) in D₂O (B). Inset: subspectra obtained by a succession of 32 selective Gaussian pulses of 500 ms centered at the Xe@cryptophane frequency and acquisitions with a 50 ms repetition time. This Figure was reproduced with permission from Delacour et al. 2013 [324]; copyright © 2013 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hyper-CEST ¹²⁹Xe NMR has also been used for the simultaneous detection of lead, zinc and cadmium ions at nanomolar concentration [328]. An alternative method called ultra-fast Z-spectroscopy has been developed as a powerful means to detect low concentrations of ¹²⁹Xe NMR-based sensors and to measure the in-out xenon exchange [329]. Furthermore, optical hyperpolarisation and NMR detection of ¹²⁹Xe has been achieved on a microfluidic chip (Figure 23). The chip was capable of ¹²⁹Xe polarisations of greater than 0.5% at flow rates of several microlitres per second, compatible with typical microfluidic applications. In situ optical magnetometry was employed to sensitively detect and characterise the ¹²⁹Xe polarisation at magnetic fields of 1 μT. Because the device was constructed using standard microfabrication methods, it may enable implementation of highly sensitive ¹²⁹Xe NMR in compact, low-cost, portable devices [330].

Figure 23. A microfluidic chip ¹²⁹Xe polariser. A. A gas mixture containing 400 Torr N₂ and 200 Torr Xe in natural isotopic abundance (26.4% ¹²⁹Xe content) flows from a bulk gas manifold into the inlet chamber, through the pump and probe chambers and out of the outlet chamber. The chip is loaded with 2 mg of ⁸⁷Rb metal. Unpolarised ¹²⁹Xe atoms entering the pump chamber become polarised through spin exchange with optically pumped ⁸⁷Rb. The ¹²⁹Xe then moves downstream, passes through a microchannel into the probe chamber and eventually exits the device through the output chamber. Optical characterisation of the ¹²⁹Xe polarisation in the pump and probe chambers is carried out using the ensemble of ⁸⁷Rb atoms in each chamber as in situ magnetometers. B. The silicon chip footprint is 3 cm x 1 cm, with a thickness of 1 mm. The dimensions of the pump and probe chambers are 5 mm x 5 mm x 1 mm and 3 mm x 3 mm x 1 mm, respectively, whereas the channel connecting the pump and probe chambers is 1 mm x 0.3 mm x 0.3 mm. Two tall, narrow grooves are etched from the middle of the chip to provide thermal isolation between the two sides of the device. C. Pumping and probing sequence for ¹²⁹Xe. Continuous pumping is carried out in presence of a longitudinal field Bz = 0.8 mT. Every 10-20 s, a transverse DC field of magnitude 5.3 mT is switched on for 4 ms to tip the ¹²⁹Xe atoms onto the x-y plane and initiate ¹²⁹Xe precession about the longitudinal axis. This Figure was reproduced with permission from Jiménez-Martínez et al. 2014 [330]; copyright © 2014 by Nature Publishing Group.

Because of the generally inert nature of xenon gas and the absence of a xenon background in biological tissues, hyperpolarised ¹²⁹Xe-MRI has emerged as a viable clinical technique, especially for imaging of lung function and associated conditions such as asthma and COPD. Indeed, studies in humans using techniques for measuring ventilation, diffusion and partial pressure of oxygen have demonstrated results for hyperpolarised ¹²⁹Xe-MRI comparable to those obtained using hyperpolarised ³He-MRI, even though ¹²⁹Xe produces reduced signals due to its smaller magnetic moment. Xenon also has the advantage of readily dissolving in lung tissue and blood following inhalation, which makes hyperpolarised ¹²⁹Xe particularly attractive for investigating some characteristics of lung function, such as gas exchange and uptake, which cannot be accessed using ³He [331]. A quantitative comparison of hyperpolarised ³He- and ¹²⁹Xe-MRI in healthy individuals and patients with COPD was performed, in which images were obtained within five minutes of each other. In patients with COPD, ventilation defect percentages obtained with ¹²⁹Xe were significantly greater than those obtained with ³He. This suggested incomplete or delayed filling of lung regions that may be related to the different properties of ¹²⁹Xe gas and physiologic and/or anatomic abnormalities in COPD [332]. Human lung structure and physiology in healthy individuals were quantified by hyperpolarised ¹²⁹Xe-MRI. Values obtained from xenon uptake (averaged over all individuals) included: surface-area-to-volume ratio (210 ± 50 cm^-1); total septal wall thickness (9.2 ± 6.5 μm); blood-air barrier thickness (1.0 ± 0.3 μm); hematocrit (27 ± 4%); pulmonary capillary blood transit time (1.3 ± 0.3 s). All were in good agreement with literature values from invasive experiments [333]. A regional mapping study of gas uptake by blood and tissues (lung parenchyma and plasma) in the human lung was performed using hyperpolarised ¹²⁹Xe-MRI. A three-dimensional, multi-echo, radial-trajectory pulse sequence was used to obtain ventilation (gaseous ¹²⁹Xe), tissue and red blood cell images in healthy subjects, smokers and asthmatics. Signal ratios (total dissolved ¹²⁹Xe to gas, tissue-to-gas, red blood cell-to-gas and red blood cell-to-tissue) were calculated from the images. Healthy subjects demonstrated generally uniform values within coronal slices and a gradient in values along the anterior-to-posterior direction (Figure 24). In contrast, images and associated ratio maps in smokers and asthmatics were generally heterogeneous and exhibited values mostly lower than those in healthy subjects. Whole-lung values of total dissolved ¹²⁹Xe-to-gas, tissue-to-gas, and red blood cell-to-gas ratios in healthy subjects were significantly larger than those in diseased subjects [334].

Figure 24. Hyperpolarised ¹²⁹Xe-MRI mapping of human lung. Coronal gas, tissue and RBC images (left) and corresponding tissue-to-gas, RBC-to-gas and RBC-to-tissue ratio maps (right) of whole lung from a healthy individual. While dissolved-phase signal intensities and ratio values were generally uniform within each coronal slice, the tissue and RBC images, as well as tissue-to-gas and RBC-to-gas ratio maps, showed an anterior-to-posterior gradient associated with the gravity-dependent gradient in lung tissue density in the supine position. This Figure was reproduced with permission from Qing et al. 2014 [334]; copyright © 2013 by Wiley Periodicals, Inc.

Hyperpolarised ¹²⁹Xe-MRI has also been used for the imaging of other organs and tissues. Using a rat permanent right middle cerebral artery occlusion model, it was demonstrated that hyperpolarised ¹²⁹Xe-MRI can be used for the in vivo detection of the hypoperfused area of focal cerebral ischemia [335]. Another study used hyperpolarised ¹²⁹Xe-MRI for detection of brown adipose tissue (BAT) and thermogenic activity in mice. There was a greater than 15-fold increase in xenon uptake by BAT during stimulation of BAT thermogenesis, which enabled acquisition of background-free maps of BAT in both lean and obese mice (Figure 25).

Figure 25. Hyperpolarised ¹²⁹Xe-MRI maps of interscapular BAT in lean and obese mice and comparison with [¹⁸F]FDG-PET/CT. The figure shows detection of BAT by hyperpolarised ¹²⁹Xe-MRI, [¹⁸FDG]-PET, and fat fraction ¹H-MRI in lean (right) and obese (left) mice. Hyperpolarised dissolved-phase xenon images are displayed as a false colour overlay on the corresponding 1-mm-thick axial proton reference image at baseline (A and B) and during stimulation of BAT thermogenesis (C and D) in the same animals. E and F. [¹⁸F]FDG-PET/CT images in a lean (F) and obese (E) animal during stimulation of BAT thermogenesis. G and H. Fat fraction ¹H-MRI measurements in a lean (H) and obese (G) mouse. The inset in G delineates interscapular BAT in the obese mouse. This Figure was reproduced with permission from Branca et al. 2014 [336]; copyright © 2014 by Branca et al.

The study also demonstrated in vivo MRI thermometry of BAT by hyperpolarised ¹²⁹Xe gas and the linear temperature dependence of the chemical shift of xenon dissolved in adipose tissue was used to directly measure BAT temperature and to track in vivo thermogenic activity [336]. Because inhaled xenon atoms are transferred from the lung via the bloodstream to the brain, this offers an opportunity to image the brain using hyperpolarised ¹²⁹Xe-MRI. Also, the longitudinal relaxation time (T₁) of hyper-polarised ¹²⁹Xe is inversely proportional to the pulmonary oxygen concentration in the lung because oxygen molecules are paramagnetic. In contrast, the T₁ of ¹²⁹Xe is proportional to the pulmonary oxygen concentration in the blood, because the higher pulmonary oxygen concentration will result in a higher concentration of diamagnetic oxyhemoglobin. Hence, there should be an optimal pulmonary oxygen concentration for a given quantity of hyperpolarised ¹²⁹Xe in the brain. These relationships have been explored in theoretical and in vivo experiments for improving the ¹²⁹Xe signal in brain (Figure 26) [337].

Figure 26. Typical dynamic spectra of the hyperpolarised ¹²⁹Xe-MRI signal in rat brain. The hyperpolarised ¹²⁹Xe signal decreased with increasing interval time. The inset shows assignments of the visible dissolved ¹²⁹Xe peaks in the brain, and the signals at 189 ppm and 195 ppm originate from the jaw muscle and grey matter, respectively. The other signals at 192 ppm and 197 ppm likely originate from the white matter and plasma, respectively. This Figure was reproduced with permission from Li et al. 2016 [337]; copyright © 2016 by John Wiley & Sons, Ltd.

2.29. Caesium (¹³³Cs)

The only naturally occurring isotope of caesium, ¹³³Cs, is NMR-active with a spin number 7/2. ¹³³Cs has medium sensitivity and produces relatively narrow signals over a chemical shift range of -30 to 130 ppm. The main applications of ¹³³Cs NMR wih biological and biomedical systems and samples are based on the fact that caesium can be used as a substitute for potassium [338]. Indeed, caesium is accumulated in the intracellular space primarily through the action of the Na⁺-K⁺-ATPase and dietary loading of ¹³³Cs can be used as a potassium substitute in NMR studies of tissues [339]. An early study used ¹³³Cs NMR to measure uptake of caesium ions by human erythrocytes and perfused rat heart. Spectra exhibited two sharp resonances originating from intra- and extracellular caesium, separated by a chemical shift of 1.0-1.4 ppm. Hence, resonances can be resolved without the addition of paramagnetic shift reagents that are required to resolve resonances of the other metal ions. The uptake of caesium ions by erythrocytes occurred at approximately one-third of the reported rate for potassium ions and was reduced by a factor of 2 upon addition of ouabain [340]. ¹³³Cs NMR was also used to compare caesium uptake into human erythrocytes from abstemious individuals and alcoholic patients, but no discernible differences were observed [341]. ¹³³Cs NMR was used as an in vivo probe for studying subcellular compartmentation and ion uptake in maize root tissue. Three ¹³³Cs-NMR signals were observed in spectra of CsCl-perfused and CsCl-grown maize seedling root tips. Two relatively broad lower field resonances were assigned to the subcellular, compartmented caesium ions in the cytoplasm and vacuole, respectively. ¹³³Cs NMR spectra of excised, maize root tips and excised top sections of the root adjacent to the kernel, each grown in 10 mM CsCl showed a difference in the relative areas of the resonance corresponding to the distinct cytoplasm/vacuole volume ratio of the different sections of the root. The results suggested that ¹³³Cs NMR might be useful for studying ionic strength and osmotic pressure associated chemical shifts and transport properties of caesium ions, and hence potassium ions, in subcellular compartments of plant tissues [342]. Using both excised and in situ tissues from rats fed and administered CsCl, it was demonstrated how ¹³³Cs NMR can be used as a probe of intracellular space. Chemical shifts, relaxation properties, sensitivity and detectability of caesium in tissues were investigated. Two ¹³³Cs NMR resonances, representing intra- and extracellular caesium, were detected in blood, whilst one resonance was detected in brain, kidney and muscle tissue [343]. Studies of ion transport in perfused and septic rat heart have been performed using ¹³³Cs as an NMR-active substitute for potassium [344,345]. ¹³³Cs NMR measurements with porcine aortic endothelial cells have demonstrated that nitric oxide radicals, through stimulation of guanylate cyclase, cause a reduction Na⁺,K⁺-ATPase activity [346]. ¹³³Cs NMR measurements on isolated perfused hepatocytes from caesium-fed rats demonstrated two distinct intracellular environments, evident as compartments with different ¹³³Cs chemical shifts and containing different proportions of total detected caesium. The chemical shifts of the two intracellular compartments were 2.44 ± 0.07 and 1.21 ± 0.18 ppm, relative to the ¹³³Cs signal from the perfusate [347]. Similarly, in vivo ¹³³Cs NMR measurements of caesium uptake into cells of Arabidopsis thaliana exposed to a caesium stress revealed that intracellular caesium was distributed in two kinds of compartment [348]. Sterically stabilised superparamagnetic iron oxide nanoparticles (SPIONs) were explored as potential MRI contrasting agents by ¹³³Cs NMR. Thus, SPIONs were incubated with fresh human erythrocytes and the chemical shift and linewidth of ¹³³Cs resonances from inside and outside the erythrocytes in ¹³³Cs NMR spectra were monitored as a function of time. Results suggested that the SPIONS did not enter the erythrocytes and that their relaxation properties showed promise for use as MRI contrasting agents [349].

2.30. Platinum (¹⁹⁵Pt)

There is one NMR-active isotope of platinum, spin-1/2 ¹⁹⁵Pt (natural abundance 33.83%), which has medium sensitivity and produces relatively narrow signals over a very wide chemical shift range (-6500 to 200 ppm). There are a few applications of ¹⁹⁵Pt NMR with biological samples. In a study on the human metabolism of the anticancer drug iproplatin, a ¹⁹⁵Pt NMR analysis of urine from patients receiving a high dose of iproplatin showed that platinum species existed as divalent complexes and not quadrivalent complexes [350]. ¹⁹⁵Pt NMR has also been used for in vivo monitoring of the local disposition kinetics of the anticancer drug carboplatin after subcutaneous injection in rats. This used a surface coil of 2 cm diameter tuned to 18.3 MHz that was placed over the injection site (back of the neck of the animals) and experiments were optimised by in vitro ¹⁹⁵Pt NMR measurements on model solutions of potassium tetrachloroplatinate(II), carboplatin, and cisplatin with different solvents including H₂O, DMSO and DMF. In vivo ¹⁹⁵Pt NMR spectra showed a broad resonance at -1715 ± 8 ppm and the elimination rate constant of local disposition of carboplatin was 0.017 min^-1 [351].

2.31. Mercury (¹⁹⁹Hg)

There are two NMR-active isotopes of mercury, spin-1/2 ¹⁹⁹Hg (natural abundance 16.87%) and spin-3/2 ²⁰¹Hg (natural abundance 13.18%), which have a very wide chemical shift range (-3000 to 500 ppm). ¹⁹⁹Hg has low sensitivity, but produces sharp signals and is preferred over ²⁰¹Hg, which produces very broad signals. There have been a few applications of ¹⁹⁹Hg NMR with biological samples. Interactions of mercury chloride with membranes have been studied by ¹⁹⁹Hg NMR. There was evidence for complexation with phosphatidylethanolamine (PE), phosphatidylserine (PS) and human erythrocyte membranes, whilst Hg(II) did not form complexes with egg phosphatidylcholine (PC) membranes. Interaction with PE and PS model membranes was described by the presence of two mercury sites, one labile and the other unlabile, on the NMR time scale. Calculated thermodynamic data indicated that PE is a better complexing agent than PS and mercury complexation with ligands or membranes was completely reversed by addition of decimolar NaCl solutions [352]. ¹⁹⁹Hg NMR was used to obtain structural information about the metal receptor site of the MerR metalloregulatory protein alone and in a complex with the regulatory target, DNA. One- and two-dimensional NMR data were consistent with a trigonal planar mercury-thiolate co-ordination environment consisting only of cysteine side chains and they resolved structural details about metal ion recognition and the allosteric mechanism [353]. ¹⁹⁹Hg NMR was used to confirm that in the structure of the mercury-bound form of the bacterial mercury detoxification system MerP, Hg(II) is bicoordinate with cysteine side chains [354].

2.32. Thallium (²⁰⁵Tl)

There are two NMR-active isotopes of thallium, ²⁰³Tl (natural abundance 29.52%) and ²⁰⁵Tl (natural abundance 70.48%), which are both spin-1/2. ²⁰⁵Tl is the preferred nucleus and there have been a number of applications with biological systems and samples. ²⁰⁵Tl NMR was used to monitor the binding of thallium ions to gramicidins A, B and C in aqueous dispersions of lysophosphatidylcholine. For 5 mM gramicidin dimer in the presence of 100 mM lysophosphatidylcholine, only 50% or less of the gramicidin appeared to be accessible to thallium. Analysis of the ²⁰⁵Tl chemical shift as a function of thallium ion concentration over the range 0.65-50 mM indicated that only one thallium ion can be bound by the gramicidins under these conditions. The thallium equilibrium binding constants were 582 ± 20 M^-1, 1949 ± 100 M^-1 and 390 ± 20 M^-1 for gramicidins A, B and C, respectively [355]. The interaction between thallous ions and gramicidin A in DMPC vesicles was also studied by ²⁰⁵Tl NMR. Results showed that only multiple-channel occupancy can account for the observed chemical shifts and the data were analysed to give the equilibrium association constants of 450-600 and 5-20 M^-1 for the binding of the first and the second ions, respectively, at a temperature of 34 ºC [356]. ²⁰⁵Tl NMR analysis of enzyme-bound thallium was used to characterise the structure of the monovalent cation activator binding site of S-adenosylmethionine synthetase from Escherichia coli. The chemical shift of the enzyme-thallium complex is 176 ppm downfield from aqueous thallium. The ²⁰⁵Tl resonance shifts upfield to 85 ppm in the enzyme-Mg(II)-thallium complex, to 38 ppm in the enzyme-thallium-AdoMet complex and to 34 ppm in the enzyme-thallium-AdoMet-Mg(II) complex. The NMR data suggested that the substrates or products of the enzyme do not coordinate to the monovalent cation activator, implying that monovalent cation activation results from alterations in protein conformation [357]. ²⁰⁵Tl NMR was used to probe the monovalent cation binding sites of bovine plasma activated protein C and des-1-41-light-chain-activated protein C [358]. ²⁰⁵Tl NMR has been used to study the binding of thallium ions to human serotransferrin and chicken ovotransferrin in the presence of carbonate and oxalate. With carbonate as the synergistic anion, two ²⁰⁵Tl NMR signals were observed that originate from the bound metal ion in the two high-affinity iron-binding sites of each protein. Titration experiments showed that thallium ions are bound with a greater affinity at the C-terminal site of serotransferrin, whilst there was no site preference for ovotransferrin. When oxalate was used as the anion instead of carbonate, the ²⁰⁵Tl NMR signals originating from the bound metal ion in the sites of ovotransferrin were shifted downfield and became almost degenerate [359]. Conformational changes in wild-type and mutant forms of yeast pyruvate kinase have been investigated by ²⁰⁵Tl NMR [360,361]. ²⁰⁵Tl NMR methods have also been developed for the characterisation of monovalent cation binding to nucleic acids, including the first ¹H-²⁰⁵Tl scalar couplings observed in a biological system [362]. ²⁰⁵Tl solid-state NMR has been used to characterise cation binding in Na⁺,K⁺-ATPase using thallium ions as a substitute for potassium ions. ²⁰⁵Tl signals originating from occluded thallium and from non-specifically bound thallium were detected and distinguished (Figure 27).

Figure 27. ²⁰⁵Tl MAS NMR spectra of Tl⁺ interacting with Na+, K±ATPase from shark rectal glands. In all five samples, the protein concentration was about 30 mg/mL in the rotor. For experiment A, the sample contains about 9 mol Tl⁺ per mol enzyme, and the integrated intensity corresponds to about 7 mol Tl⁺ per mol enzyme. In experiments B-E, the sample contains 3.3 mol Tl⁺ per mol enzyme. For experiment B, the integrated intensity corresponds to about 0.8 mol Tl⁺ per mol enzyme. Experiment C is identical to B, except that ¹H decoupling was employed during data collection, and the integrated intensity was 3.2 mol Tl⁺ per mol enzyme. Experiments D and E are similar to B and C but with the addition of a 100-fold excess of K⁺ (330 mol K⁺ per mol enzyme). The integrated signal intensities correspond to 2.9 (D) and 3.4 mol Tl⁺ per mol enzyme (E). The experiments were performed at 17 °C at pH 6.9 in a 20 mM histidine and 1.3 mM CDTA buffer. Spectra B-E have the same vertical scale, and spectrum A has been reduced 2.2-fold in order to ease comparison. This Figure was reproduced with permission from Jakobsen et al. 2006 [363]; copyright © 2006 by American Chemical Society.

The effects of dipole-dipole coupling between ¹H and ²⁰⁵Tl in the occlusion sites showed that the thallium ions are rigidly bound, rather than just occluded. A low chemical shift also suggested that occlusion site geometries have a relatively small contribution from carboxylate and hydroxyl groups. The non-specific binding of thallium ions were characterised by rapid chemical exchange, in agreement with an observed low binding affinity [363].

2.33. Lead (²⁰⁷Pb)

Lead has one NMR-active isotope, the spin-1/2 ²⁰⁷Pb (natural abundance 22.1%), which has medium sensitivity and produces narrow signals over an extremely wide chemical shift range (-5500 to 6000 ppm). There appears to be only one published application of ²⁰⁷Pb NMR with a biological sample. ²⁰⁷Pb NMR has revealed that Pb(II) coordinates with the thiol-rich biomolecules glutathione and tris cysteine zinc finger proteins with trigonal pyramidal geometry (PbS3). Thus, glutathione binds Pb(II) with PbS3 geometry at pH 7.5 or higher with a 1:3 molar ratio of Pb(II) to glutathione. The zinc binding domain from HIV nucleocapsid protein (HIV-CCHC) appeared to bind Pb(II) in two PbS3 structures (5790 and 5744 ppm), one of which was more stable at high temperatures. This observation was consistent with previous studies suggesting that the HIV-CCHH peptide does not fold properly to afford a PbS2N motif, because histidine does not bind to Pb(II) [364].

3. Conclusions

This article has provided an overview of the properties of 39 NMR active nuclei and their applications in NMR measurements with biological and biomedical systems and samples. The measurements include solution- and solid-state NMR, magnetic resonance spectroscopy and magnetic resonance imaging. Examples have been provided for the wide range of analyses and types of information that can be obtained by NMR measurements using different NMR active nuclei. The current and ongoing technological developments in all types of NMR measurements will continue to push the boundaries for achieving higher resolution and higher sensitivity chemical, structural, dynamic and imaging information, thus expanding the types and increasing the complexities of biological and biomedical systems and samples amenable to NMR investigation. This will provide information for metabolic profiling in greater detail, a better understanding of the structures, functions and molecular mechanisms of biomolecules, the design and pharmacokinetics of new drugs, and the detection and monitoring of human diseases and their therapies.

Conflict of interest

The author confirms that this article content has no conflict of interest.

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Key Reference: 1, 2, 7, 48, 68, 131, 214, 231, 334, 336

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Article information

Corresponding author: Simon G. Patching.

Copyright: © 2016 Patching SG. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited.

How to Cite: Patching SG. NMR active nuclei for biological and biomedical applications. J. Diagn. Imaging Ther. 2016;3 (1): 7-48 (https://dx.doi.org/10.17229/jdit.2016-0618-021).

Article history: Received 9 May 2016; Accepted 25 May 2016; Published online 17 June 2016.

Archive link: JDIT-2016-0618-021

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NMR active nuclei for biological and biomedical applications

Abstract

1. Introduction

2. NMR active nuclei

2.1. Hydrogen (1H, 2H, 3H)

2.1.1. Applications of protium (1H)

2.1.2. Applications of deuterium (2H)

2.1.3. Applications of tritium (3H)

2.2. Helium (3He)

2.3. Lithium (7Li)

2.4. Boron (10B, 11B)

2.5. Carbon (13C)

2.6. Nitrogen (15N)

2.7. Oxygen (17O)

2.8. Fluorine (19F)

2.9. Sodium (23Na)

2.10. Magnesium (25Mg)

2.11. Aluminium (27Al)

2.12. Silicon (29Si)

2.13. Phosphorus (31P)

2.14. Sulphur (33S)

2.15. Chlorine (35Cl, 37Cl)

2.16. Potassium (39K)

2.17. Calcium (43Ca)

2.18. Vanadium (51V)

2.19. Cobalt (59Co)

2.20. Copper (65Cu)

2.21. Zinc (67Zn)

2.22. Selenium (77Se)

2.23. Bromine (79Br, 81Br)

2.24. Krypton (83Kr)

2.25. Rubidium (87Rb)

2.26. Cadmium (111Cd, 113Cd)

2.27. Iodine (127I)

2.28. Xenon (129Xe)

2.29. Caesium (133Cs)

2.30. Platinum (195Pt)

2.31. Mercury (199Hg)

2.32. Thallium (205Tl)

2.33. Lead (207Pb)