In Vivo NMR, Applications, Other Nuclei*

In Vivo NMR, Applications, Other Nuclei Jimmy D Bell, E Louise Thomas, and K Kumar Changani, Hammersmith Hospital, London, UK & 1999 Elsevier Ltd. All rights reserved. This article is reproduced from the previous edition, volume 2, pp 857–866, & 1999, Elsevier Ltd.

Introduction In vivo nuclear magnetic resonance spectroscopy (or in vivo MRS as it has become known) is increasingly being used to provide direct, localized biochemical information on animal and human metabolism in health and disease. Dedicated systems for animal studies use magnetic fields ranging from 2.0 to 9.4 T. Most human MRS systems range from 1.5 to 2.0 T, although 4 T systems are available. Clearly these systems offer spectra with a quality and sensitivity no better than that obtained with high-resolution NMR systems from 30 years ago. However, even within these constraints, in vivo MRS has contributed greatly to our understanding of the metabolic processes associated with a wide range of clinical and metabolic disorders by providing researchers with information that could not be obtained previously even with tissue biopsies. The application of in vivo MRS has expanded rapidly in recent years and its value as a research tool depends on the organ under investigation as well as the nuclei being utilized. This article reviews the work carried out to date in in vivo MRS using 13C, 19F, 15 N, 23Na and other less common nuclei. A list of nuclei currently being used for in vivo biomedical research is shown in Table 1. Although many of these nuclei are routinely used in high-resolution

NMR characteristics of some nuclei of biomedical

Table 1 interest Nucleus

Spin

Relative sensitivity

Natural abundance (%)

Observation frequency for 1H observation at 100 MHz

2

1

7

3 2 3 2 1 2 1 2 5 2 1 2 3 2 5 2 1 2 3 2 1 2

9.65  103 2.93  101 1.65  101 1.59  102 1.03  103 2.91  102 8.3  101 9.25  101 2.06  101 7.84  103 1.7  101 2.12  102

1.5  102 92.58 80.42 1.11 0.37 0.04 100 100 100 4.70 27.85 26.44

15.35 38.86 32.08 25.14 10.13 13.56 94.08 26.45 26.06 19.86 32.72 27.66

H Li 11 B 13 C 15 N 17 O 19 F 23 Na 27 Al 29 Si 87 Rb 129 Xe

in vitro NMR spectrometers, their implementation in in vivo MRS systems required substantial technical development and many are available only to a handful of research groups around the world. This is partly due to the considerable cost associated with developing multinuclear capabilities into commercial systems. Accordingly, most commercial MR systems have very restricted spectroscopic capabilities, with 1H and 31P being the nuclei generally available. This has, of course, restricted the application of less common nuclei in biomedical research. A summary of the main areas of research and application of some of these nuclei is shown in Table 2. Results of many of these studies have made considerable contributions to important areas of clinical and biochemical research, while other studies have concentrated on demonstrating technical feasibility, although they suggest the prospect of major areas of future research.

In Vivo

13

C MRS

Whole-body 13C MRS is potentially the most powerful tool available for the study of human metabolism in vivo. The nuclide’s low natural abundance (1.1%) allows the study of important biological molecules such as glycogen and lipids, while at the same time allowing the use of 13 C-enriched compounds in dynamic measurements of intermediate metabolism. However, several technical issues need to be addressed before its implementation in vivo, including a second transmit channel, optimum localization and proton decoupling. The latter has been one of the major stumbling blocks for the application of in vivo 13C MRS, because of the potential problem of power deposition during proton decoupling. This problem has now been addressed by the use of the newer pulse train decoupling sequences, which allow full decoupling of signals with low power deposition. The need for a second transmit channel with frequency synthesizer, phase modulator and power amplifier, together with the need for specifically designed 1H/13C coil systems, means that the actual cost of a standard in vivo scanner is substantially increased, hence limiting its availability. Thus, although many of the technical problems associated with the use of 13C MRS in vivo have been overcome, it is still not routinely available to the great majority of research groups

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1100 Table 2

In Vivo NMR, Applications, Other Nuclei In vivo applications of NMR spectroscopy

Nucleus

Application

Organ/tissue

13

Glycogen Amino acids, glucose Lipids Drug metabolism Anaesthetics Phospholipids Intracellular Ca2þ

Liver, muscle Brain

C

19

F

15

N

23

Na

2

H (D)

27

Al Xe 17 O 87 Ru 7 Li 129

11

B Si

29

Glucose metabolism Glutamine metabolism Electrolyte balance D2O, methionine, glucose Choline Gastric emptying Lung volume Blood flow Naþ/Kþ-ATPase Psychiatric disorders Therapy agents Breast implants

Application of In Vivo Metabolism

13

Adipose tissue, liver Liver, brain, muscle Brain Tumour Brain, kidney, spleen Brain Brain Muscle, brain, liver, heart Brain, adipose tissue, liver Kidney Gut Lungs Brain, liver Muscle Brain, muscle Liver, brain, tumour Liver

C MRS to Carbohydrate

Carbohydrate metabolism is one area of research that has significantly benefited from in vivo 13C MRS since signals from sugar carbons occur in a region of the spectrum that is relatively unpopulated by other resonances. Using specific carbon labelling on different substrates, various metabolic pathways have been studied. Further information has been gained by labelling two adjacent carbon atoms within the same molecule; this produces a characteristic 13C–13C spin–spin coupling, helping to elucidate important information about particular enzyme pathways

Liver

The first in vivo experiments, performed in 1981, explored the potential application of 13C MRS to glucose/glycogen metabolism. Following infusion of 13Cenriched glucose in rats, well-resolved resonances from both glucose and glycogen could be observed, enabling the time course of the relative changes in concentration to be established simultaneously. One area of debate at the time was the degree of glycogen visibility in liver. It was initially thought that glycogen, being such a large macromolecule, would be only partly NMR ‘visible’. However, the most recent consensus is that glycogen is in fact 100% visible and that 13C MRS can therefore provide reliable noninvasive measurements of hepatic glycogen concentrations over physiological ranges.

Glycogen repletion studies have been carried out in animals and humans. Infusions of [1-13C]glucose and [3-13C]alanine to fasted rats suggested that 30% of the total glycogen formed originated from the direct conversion of glucose in the short term. However, over the longer term, active glycogen synthesis from [3  13C]alanine occurred for a longer period of time, with a rate constant threefold higher than from [1  13C]glucose, implying greater efficiency of glycogen synthesis from the indirect route. These studies have also highlighted potential problems associated with substrate cycling, since following entry into the Krebs cycle the label may be assimilated into other pathways and then reintroduced into the gluconeogenic pathway. Definitive mechanisms of glycogen repletion therefore become difficult to assess in vivo, unless measurements are performed immediately following administration. Human studies have concentrated on direct observation of glycogen repletion using natural abundance 13C or after administration of [1-13C]glucose. Early work compared pre- and post-meal hepatic glycogen repletion rates in individuals starved for 30 h showing repletion rates 3.8 times greater in the post-meal states compared with the fasted basal rate. Hepatic gluconeogenesis in healthy subjects and those with non-insulin-dependent diabetes mellitus (NIDDM) was also investigated by 13C MRS and conventional methods. Concentration of glycogen was lower in NIDDM patients compared to controls (Figure 1). Furthermore, following infusion of [6-3H]glucose, gluconeogenesis in the NIDDM subjects was found to be 60% greater than in control subjects and accounted for 88% of the total glucose production compared with 70% in the normal subjects. This suggests that increased gluconeogenesis accounts for the increase in whole-body glucose production in overnight-fasted NIDDM subjects. 13 C MRS has also been applied to determine the metabolic alterations caused by glycogen storage diseases. Patients with glycogen type IIIA storage disease were shown to have increased levels of hepatic glycogen compared with normal volunteers. This disease manifests owing to the inactivation of amylo-1,6-glucosidase, which is required for glycogen hydrolysis. It was concluded that in vivo 13C MRS could play an important role in the assessment of other glycogen storage diseases where current clinical practice requires needle biopsies to be performed. 13 C MRS has also been used to assess enzyme capacities and kinetics in animals. Following infusion of [1-13C]butyrate (90% enriched), rates of ketogenesis could be monitored by measuring the formation of acetoacetate and b-hydroxybutyrate within the carbonyl region of the 13C MR spectrum. Further studies of 13 C-labelled fatty acids, such as [1,3-13C]butyrate, assessed the metabolic pathways of ketone body production in

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Figure 1 Typical in vivo 1H-decoupled 13C MRS spectra at 22.5 MHz of the C-1 position of liver glycogen from a control subject (a) and from a type II diabetic patient (b) 4 hours after a liquid meal. Reproduced with permission from Magnusson I, Rothman DL, Katz LD, Shulman RG, and Shulman GI (1992) Increased rate of gluconeogenesis in type II diabetes mellitus. Journal of Clinical Investigation 90: 1323–1327.

fasted and diabetic rats. Increased b-hydroxybutyrate production was seen in diabetic rats; it was thought that a large fraction of butyrate evaded b-oxidation to acetylCoA and was converted directly into acetoacetyl-CoA. Different physiological states can therefore be monitored by in vivo 13C MRS since labelling patterns following 13Cenriched metabolite administration would be different. In vivo 13C MRS has also been applied to monitor the pharmokinetics of 13C-labelled xenobiotic products in hepatic metabolism. One such study used a phenacyl imidaolium compound that effects glycogen synthesis. It was shown that glycogen synthesis increases twofold in drug-treated rats and it was suggested that augmentation of the indirect pathway is the probable mechanism. Further studies have shown that long-term monitoring of drug concentrations is possible using 13C-labelled compounds, which may be important for monitoring hepatic tumour treatments. Muscle

Most human 13C MRS studies in muscle have concentrated on the study of glycogen synthesis and its role in NIDDM, glycogen storage diseases and muscle myopathies. Studies of insulin action on muscle glycogen synthesis have shown that there appears to be an impairment of muscle’s ability to either take up glucose or assimilate it as glycogen in NIDDM. Studies have used in vivo 13C MRS to study muscles following intense exercise. This, together with interleaved 31P MRS, has provided important information on the phosphorylation states of glycogen synthase that could be correlated with post-exercise glycogen synthesis rates. Studies of this kind could provide important information on enzyme pathways that may be defective in particular muscle myopathies. Acetate metabolism has also been assessed in rabbit muscles to determine rates of anaplerotic flux (i.e. determination of label of glutamate and glutamine) compared with basal flux of the Krebs

cycle. Different muscle types (muscle fibre/oxidative or nonoxidative) have dissimilar anaplerotic flux rates, which may again be relevant for particular disease types. Heart

In vivo 13C MRS of the heart is difficult owing to continuous contraction of the heart. For this reason, together with the relative insensitivity of the 13C nucleus, which requires a large number of averages, in vivo MRS studies are few. Consideration of the metabolites within blood must also be made so that the spectral acquisitions contain information purely reflecting heart metabolism. The bulk of studies on the heart have therefore been conducted using isolated, perfused organs. However, there have been a few in vivo studies that have looked at glycogen synthesis rates following infusions of acetate and glucose. These studies can provide information about physiological conditions that may effect carbohydrate utilization in favour of fatty acid metabolism, with implications for coronary heart disease. Brain

In vivo 13C MRS of the brain has been used to determine in vivo levels of glutamate, glutamine, g-aminobutyrate (GABA), and lactate following intravenous infusions of enriched precursors. Brain tumours in rats have been studied to investigate carbohydrate metabolism following administration of [1-13C]glucose. Rat gliomas produced lactate and glutamate/glutamine as well as glycogen. Further studies used combinations of labelled substrates such as glucose and acetate to determine the relative flux through the tricarboxylic acid cycle together with their relative conversion rates into the cerebral amino acids, important for neurotransmission. Both these studies suggested that in vivo 13C MRS is ideal for studying the individual carbons of different metabolites in the brain. Furthermore, cerebral compartmentation of glial and neuronal cells may also be studied because of their

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different metabolism. Two separate glutamate pools and two separate tricarboxylic acid cycles, one preferentially metabolizing acetate and the other mainly using unlabelled acetyl-CoA and a predominant production of GABA in the glutamate pool lacking glutamine synthase, have been identified, accounted for by either the glial or neuronal cells. A further application of in vivo 13C MRS has been to study the metabolite changes associated with mild or severe hypoxia as well as electroshock. Changes associated with lactate and the cerebral amino acids were correlated with both electroshock and degree of hypoxia. This work may be useful in characterizing the metabolic state following epilepsy, stroke, trauma, tumours and other pathological conditions in humans. In humans, total brain glutamate concentration has been assessed following [1-13C]glucose infusions, while in stroke it has helped to determine metabolic turnover. Some of these studies will benefit from the introduction of protonobserved carbon-edited (POCE) techniques, which can provide better relative sensitivity and resolution. This technique has been very useful, for example, in determining the effects on brain metabolism of drugs such as inhibitors of succinate dehydrogenase and the GABAtransaminase inhibitor vigabatrin.

Application of In Vivo

13

C MRS to lipids

The main application of in vivo 13C MRS to the field of lipids has been the study of adipose tissue composition. Although the natural abundance of the 13C isotope is low, the high content of lipids in adipose tissue has enabled acquisition of 13C MR spectra in vivo in a matter of minutes, with or without proton decoupling. This has allowed the use of this technique on subjects (e.g. neonates and in pregnancy) where power deposition from decoupling, though small, is still unacceptable. Adipose tissue

Adipose tissue is principally composed of triglycerides and it has been shown to play an important role in major diseases, including NIDDM and cardiovascular disease. Preliminary studies in the early 1980s showed that in vivo 13 C MRS could be used to detect differences in the lipid composition of adipose tissue and liver in rats fed a diet high in polyunsaturated fatty acids. The first human studies showed that linoleic acid (18:2n  6), usually the principal polyunsaturated fatty acid present in human adipose tissue, dominated the polyunsaturated signal observed in vivo, allowing estimation of this stored essential fatty acid. Further work showed that fatty acid composition of adipose tissue closely correlated with dietary fat content, with subjects on long-term vegan diet showing significantly higher levels of polyunsaturated

fatty acids and reduced saturated fatty acids compared to omnivores (general public) and vegetarians. Indeed, there was no difference in the adipose tissue composition of vegetarians compared to omnivores, suggesting that the former have substituted the animal fat (mainly saturated) with saturated fat in dairy products. In vivo 13C MRS has also been used to study adipose tissue composition in babies. The work showed that the influence of maternal diet on infant adipose tissue composition and changes due to gestational age and during postnatal development could all be detected noninvasively by in vivo 13C MRS. Preterm infants had significantly lower levels of unsaturated adipose tissue fatty acids than the infants born at full term. Furthermore, the level of unsaturated fatty acids continued to increase as the infants matured (from birth to 6 weeks, to 6 months). Interestingly, the authors also found that infants breast-fed by vegan mothers had 70% more polyunsaturated fatty acids than infants breast-fed by omnivore mothers, the longterm consequences of which (beneficial or harmful) are unknown. The same authors showed that in vivo 13C MRS could also measure the effect of intensive exercise on adipose tissue composition of adult human volunteers. A decrease in polyunsaturated fatty acids was observed, suggesting that exercise is an independent factor for adipose tissue composition. In addition, in vivo 13C MRS has been applied to the study of adipose tissue composition in disease. Children with cystic fibrosis were shown to have lower levels of polyunsaturated adipose tissue fatty acids than healthy children, possibly owing to a disorder in essential fatty acid metabolism that may be partly responsible for the development of the disease. Further studies with in vivo 13 C MRS in disease have shown a significant increase in saturated adipose tissue fatty acids following transplantation and subsequent weight gain in malnourished patients with liver cirrhosis. It was suggested that this increase in saturated fatty acids may be secondary to a general repletion of membrane polyunsaturated fatty acids or the use of essential fatty acids for biosynthesis of eicosanoids in the post-operative period. Liver 13

C MRS has also been used for noninvasive quantification of hepatic triglyceride content. Diagnosis of hepatic steatosis (fatty liver) is important because this is a reversible condition and early detection can help to prevent irreversible liver damage. The condition is normally diagnosed by a liver biopsy, but the invasive nature of this procedure and the problems associated with biopsies make it difficult to monitor patients at risk of developing hepatic steatosis. In a study of 15 patients with varying degrees of fatty infiltration it was shown that by quantifying the intensity of the CH2 resonances from the in vivo 13C MR spectrum of the liver it was possible to

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1103

determine hepatic lipid content. Excellent correlation with conventional liver biopsy measurements was observed. As the authors suggested, this technique will in future become a valuable tool in the diagnosis and follow-up in patients with hepatic steatosis. Brain

The application of in vivo 13C MRS to the study of brain lipids has been rather limited, partly because most of the lipids in the brain are present in the form of membranes. This gives rise to broad and uninformative signals. An interesting possibility for improving the discriminatory power of MRS, as applied to lipids, is the use of 13 C-enriched fatty acids. However, at present such studies would be prohibitively expensive as the subjects would be required to consume gram quantities of 13C-enriched fatty acid.

In Vivo

19

F MRS

Fluorine-19 is a highly NMR-sensitive and naturally abundant nucleus. An important advantage of 19F MRS is the fact that it allows direct observation of fluorinated compounds and their metabolites in the human body without background signal from tissue. In vivo 19F MRS has been used to detect noninvasively anticancer and psychoactive drugs and other fluorinated compounds and to study their metabolism. It has also been used to monitor the effects of anaesthetics and to measure intracellular pH and calcium levels. One of the major applications of in vivo 19F MRS has been in the field on oncology, detecting and monitoring the metabolism of fluorine-containing anticancer drugs. An important chemotherapeutic compound is the fluorinated drug 5-fluorouracil (5-FU). Much of the work with 19F MRS has concentrated on determining optimal drug administration schedules and studying the effects of this drug on both tumour and healthy tissue. 19F MRS has allowed detection and serial measurement of 5-FU and its metabolites in patients undergoing chemotherapy, increasing the understanding of the variation in efficacy of chemotherapy in different subjects (Figure 2). For example, levels of 5-FU ‘trapping’ by tumours following intravenous administration of the drug have been measured. The results reveal that patients whose tumours trapped the drug had a significantly better response to chemotherapy than those patients whose tumours did not trap 5-FU. This differential in response to therapy could not be ascertained from the standard plasma concentration measurements. From this study it was concluded that 19 F MRS can be used to identify patients who are likely to respond to chemotherapy with 5-FU and can therefore aid selection of the optimal treatment for individual patients. Studies of 5-FU metabolism in patients with

Figure 2 (a) Liver in vivo19F MR spectrum at 60 MHz following administration of 5-fluorouracil (5-FU). (b) Time course of 5-FU and its major catabolite a-fluoro-b-alanine (FBAL). (FUranuc ¼ 5-fluorouracil nucleoside þ nucleotides). Reproduced with permission from Semmler W, Bacher-Baumann P and Guckel F (1990) Real time follow-up of 5-fluorouracil metabolism in the liver of tumor patients by means of F-19 MR spectroscopy. Radiology 174: 141–145.

protein calorie malnutrition (a common condition in patients with cancer) have shown increased toxicity from this drug. Therefore, identifying alterations in 5-FU metabolism may be an important tool in reducing toxicity during chemotherapy. Interestingly, like 31P MRS, in vivo 19F MRS may also be used to measure pH noninvasively. This may be useful for 19F MRS studies of tumours, as the uptake and retention of 5-fluorouracil is dependent on tumour pH. An important area of research using in vivo 19F MRS has been the study of the pharmacokinetics of fluvoxamine and fluoxetine, drugs used to treat obsessive– compulsive disorder and depression, respectively. Several groups have shown that it is possible to detect these compounds noninvasively in the brain. Brain fluvoxamine levels were shown to reach a steady state 30 days after consistent daily dosing, substantially more rapidly than reported for fluoxetine, which was shown to plateau after 6–8 months of treatment. Brain concentrations of both drugs were shown to correlate with plasma levels, though the absolute concentrations were substantially higher in

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the brain. Comparison of the in vivo measurement of fluoxetine with in vitro samples taken at autopsy suggests that fluorinated drugs may not be 100% visible in vivo. These are based on the in vitro findings from just one brain that had been fixed in formaldehyde, so further studies would be required to confirm these findings. Some of the other applications of in vivo 19F MRS to the study of fluorinated drugs have included the study of fluorinated antibiotics and antifungal agents in animals and humans. Orally administered fleroxacin (a fluoroquinolone antibiotic agent) could be detected in the liver and calf muscle by in vivo 19F MRS. Furthermore, its washout and metabolism were studied over a 24-h period. There are important implications of this work, and it will in future be possible to ensure that drugs reach their target site in appropriate concentrations. In vivo 19F MRS has also been used to study the pharmacokinetics and metabolism of anaesthetic agents such as halothane, isoflurane and desflurane in experimental animals. It has also been shown that anaesthetic agents can be observed noninvasively in the brains of patients immediately following surgery. The principal aim of these studies was to elucidate the mechanism of action of these compounds and eventually help in the development of more effective and selective anaesthetics. Unfortunately, factors such as sensitivity and localization of the region of interest (which are particularly relevant to the study of anaesthetics) have greatly limited the impact of this research. Animal studies have shown that in vivo 19F MRS can be used in metabolic studies. It has been shown that 3-fluoro3-deoxy-D-glucose in the brain is metabolized primarily in the aldose reductase sorbitol pathway, suggesting that 19F MRS may be a useful tool for the further elucidation of this pathway. Further studies have shown that it is also possible to study the metabolism of fluorinated galactose, tryptophan and protein in vivo and determine intracellular calcium levels. The concentration of this cation was shown to be the same in the kidney, spleen and brain of rats, at approximately 200 nM. These measurements were obtained by infusing the calcium indicator 5F-BAPTA ( ¼ 5,5-difluoro-1,2-bis(o-amino-phenoxy)ethane-N,N,N1, N1-tetraacetic acid) intravenously or intraventricularly into the animal, and so the potential application to human subjects appears limited. It has also been possible to follow the time course of recovery from nerve injury in skeletal muscle using in vivo 19F MRS. Muscle injury leads to significant decreases in the energy state and local circulation dynamics, which appear to reverse during recovery. While 31P MRS was used to determine the energy status of the cells, 19F MRS was utilized to determine local circulation dynamics by measuring blood volume. The results indicate that the recovery processes of circulation precede those of energy state.

Many of the above studies have shown that in the future 19F MRS is likely to become a valuable tool for monitoring drug metabolism at the site of action, though at the moment this is limited by the availability of in vivo MR scanners with 19F capabilities.

Less Common Nuclei Over the past decade or so there has been a significant increase in the number of ‘less common’ nuclei that are being utilized for in vivo MRS (see Table 1). The use of some of these nuclei was initially limited by technical constraints and/or inherent NMR characteristics. However, recent studies have shown that these nuclei can be used to increase our understanding of some important physiological processes. A number of studies have utilized in vivo MRS to assess the extent of delivery of pharmacological substances to target tissue. Nitrogen-15 MRS in combination with intravenous 15NH4þ infusion into animal models of hepatic encephalopathy (Figure 3) enabled the determination of glutamine synthetase activity in vivo. The results show that in situ the activity of this enzyme is kinetically limited by the levels of some substrates and cofactors, and that the activity of phosphate-activated glutaminase was maintained at a low level by a suboptimal concentration of Pi and the strong inhibitory effect of glutamine. The introduction of the heteronuclear multiple quantum coherence (HMQC) transfer technique is likely to further these studies by probing the kinetics of glutamine synthesis at physiological concentrations. Similarly, lithium-7 MRS has been used to examine the pharmacokinetics of lithium, a drug that is widely used in the treatment of a number of psychiatric disorders. Initial studies showed that there was a slow accumulation of lithium in the brain, which may be responsible for the delay in therapeutic response that is often seen following the initiation of therapy. Subsequent studies showed that lithium levels in the brain and muscle were lower than the average serum concentration. From the ratio of these in vivo measurements, the authors hypothesized that the minimal effective concentration of brain lithium level for maintenance treatment of bipolar disorder could be determined. However, in a similar study, researchers found that in a subgroup of patients there was only a weak correlation between serum and brain lithium levels, which may account for the failure of lithium therapy in some patients with serum lithium levels within the therapeutic range. This suggests that measurement of in vivo brain lithium concentration by MRS may be more clinically relevant than simply assessing serum concentration.

In Vivo NMR, Applications, Other Nuclei

Figure 3 In vivo 15N spectra at 24.3 MHz from cerebral region of (a) a control rat given low-rate 15NH4þ infusion, showing cerebral [g-15N]glutamine at a near physiological concentration; (b) a portacaval-shunt rat showing cerebral and blood 15NH4þ, in addition to [g-15N]glutamine; (c) a control rat showing cerebral and blood [15N]urea and lower [g-15N]glutamine. Peaks are downgoing following NMR convention for 15N spectroscopy. Reproduced with permission from Kanamori K, Parivar F and Ross BD (1993) A 15N NMR study of in vivo cerebral glutamine synthesis in hyperammonemic rats. NMR in Biomedicine 6: 21–26.

Boron-11 MRS has been used to assess the delivery of boron neutron capture therapy (BNCT) agents to a target lesion. Uptake and retention of these compounds was determined in vivo in humans and animals and the results show that there is differential retention between normal and abnormal tissue. Furthermore, 1H-observed 10 B-edited MRS has been shown to provide much higher sensitivity and could be utilized to monitor the distribution and excretion of boron agents noninvasively in patients about to undergo BNCT. Sodium-23 MRS has been used to yield information on electrolyte balance, which appears to reflect physiological changes associated with cell function. In myotonic dystrophy, an inherited multisystem disease that leads to muscle malfunction, 23Na MRS was shown to be a sensitive method for quantification of disease progression. Quantification of intracellular and extracellular sodium by MRS has also generated important information on organ function following transplantation or injury. In heart transplantation experiments in rats, 23Na MRS showed that there was a steady increase in intracellular sodium 3 days prior to rejection, followed by a sharp

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increase after rejection. In the liver, intracellular sodium levels were shown to be a sensitive indicator of hepatic dysfunction after major systemic injury. In the kidney, sodium compartmentation (intracellular, vascular and intraluminal) was determined using the shift reagent TmDOTP5, while in the brain this shift reagent was shown to be a useful tool in determining compartmental sodium concentration and measuring blood flow kinetics. The activity of ion transport systems, such as sodiumand potassium-activated adenosine triphosphatase, has been studied in vivo by the use of 87Rb MRS. This has been possible because rubidium has been shown to substitute for potassium in a number of transmembrane transport systems, accumulating in the intracellular space. Standard in vitro methods of determining Naþ/ Kþ-ATPase activity, which also use rubidium, give highly variable measurements and in some cases contradictory results. Many of these problems appear to have been overcome by the use of in vivo 87Rb MRS, especially when sequential measurements are required. In a longitudinal study of spontaneously hypertensive rats, 87Rb MRS showed that skeletal muscle rubidium rose at a faster rate in hypertensive rats than in control animals, which is consistent with a marked increase in Naþ/KþATPase activity. This type of experiment emphasizes the value of in vivo MRS since rubidium kinetics can be determined sequentially on the same animal, minimizing inter/intra-subject variability as well as the number of animals required in the study. In vivo deuterium (2H) MRS has been used to characterize amino acid metabolism, body iron content, brain and kidney metabolism and body fat utilization rates in rodents. These studies rely on the use of deuterium labelling or the existence of natural-abundance deuterium in water or lipids. For example, deuterium-labelled methionine was used to confirm the dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess methionine, while deuterium-labelled glucose was used to show that systemic glucose level influences brain metabolic recovery following partial ischaemia. The renal distribution and metabolism of methyl groups was assessed by the use of deuterium-labelled choline. The results show that the concentration of trimethylamines, metabolic products of choline, was higher in the cortex and inner medulla than in the outer medulla, but that the inner medullary fraction was more liable to diuresis. By combining D2O dilution techniques with 2H MRS it has been possible to study fat turnover in vivo. The rate of loss of deuterium from body fat, after a short period of D2O administration, has been shown to reflect utilization of body fat. This technique was used successfully to demonstrate that induction of diabetes in mice did not affect the utilization of fat as a metabolic fuel. Given that D2O administration is regularly used with human subjects, this opens up the possibility, for the first time, of

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determining fat turnover noninvasively in human subjects. A number of studies have used nuclei such as oxygen17, aluminium-27 and xenon-129 in conjunction with in vivo MRS for structural or dynamic measurements in different organs. 129Xe signals were detected in the lung and head from human volunteers following inhalation of hyperpolarized 129Xe gas, allowing the determination of the temporal evolution of the gas-phase and dissolvedphase components. Most of this work, however, was used to demonstrate the feasibility of obtaining xenon images of the lung. 129Xe imaging now allows the possibility of imaging the lung at resolution equivalent to that of conventional imaging of normal tissue. 27Al MRS has been applied to the visualization of gastric emptying and measurement of gastric phospholipids, while 17O MRS has been used to explore cerebral blood flow. Although these studies have again concentrated mainly on their technical feasibility, they do open up important areas of clinical and biochemical research.

Conclusions The application of in vivo multinuclear MRS to biological sciences is an ever-expanding area of research. Present-day clinical NMR systems routinely allow the use of 1H and 31P MRS in clinical and scientific research; however, the use of other nuclei is still restricted to a relatively small number of research groups around the world. This is principally for commercial reasons rather than scientific or technical considerations. Demands for an increase in the multinuclear capability of commercial systems is of course increasing as the usefulness of nuclei such as 13C, 19F and 15N continues to be demonstrated both in animal models and in human subjects. It is conceivable that in the near future these technical capabilities will be routinely available in most clinical systems as they are today available in most in vivo animal systems. This will no doubt greatly help to further elucidate metabolic mechanisms in health and disease and in the development and monitoring of the efficacy of novel clinical treatments, including gene therapy. See also: 1H MAS NMR Spectroscopy of Tissues, 13C NMR, Methods, Drug Metabolism Studied Using NMR Spectroscopy, 19F NMR, Applications, Solution State, Heteronuclear NMR Applications (B, AI, Ga, In, Tl), Hyperpolarized Gases in NMR, Methods and Applications, In Vivo 1H MRS Applications, In Vivo 1H

MRS Methods, In Vivo NMR, Applications, 31P, In Vivo NMR Methods, Overview of Techniques, Labelling Studies in Biochemistry Using NMR, Nitrogen NMR, NMR Spectrometers, Nuclear Overhauser Effect, Perfused Organs Studied Using NMR Spectroscopy, Solvent Suppression Methods in NMR Spectroscopy, Xenon NMR Spectroscopy.

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