Cerebral creatine kinase deficiency influences metabolite levels and morphology in the mouse brain: a quantitative in vivo 1H and 31P magnetic resonance study

Creatine kinase (CK)-catalysed ATP-phosphocreatine (PCr) exchange is considered to play a key role in energy homeostasis of the brain. This study assessed the metabolic and anatomical consequences of partial or complete depletion of this system in transgenic mice without cytosolic B-CK (B-CK-/-), mitochondrial ubiquitous CK (UbCKmit-/-), or both isoenzymes (CK -/-), using non-invasive quantitative magnetic resonance (MR) imaging and spectroscopy. MR imaging revealed an increase in ventricle size in a subset of B-CK-/- mice, but not in animals with UbCKmit or compound CK mutations. Mice lacking single CK isoenzymes had normal levels of high-energy metabolites and tissue pH. In the brains of CK double knockouts pH and ATP and Pi levels were also normal, even though PCr had become completely undetectable. Moreover, a 20-30% decrease was observed in the level of total creatine and a similar increase in the level of neuronal N-acetyl-aspartate compounds. Although CKs themselves are not evenly distributed throughout the CNS, these alterations were uniform and concordant across different brain regions. Changes in myo-inositol and glutamate peaks did appear to be mutation type and brain area specific. Our results challenge current models for the biological significance of the PCr-CK energy system and suggest a multifaceted role for creatine in the brain.     

Inhibition of NMDA-type glutamate receptors induces arousal from torpor in hibernating arctic ground squirrels (Urocitellus parryii)

J. Neurochem. (2012) 122, 934-940. ABSTRACT: Hibernation is an adaptation to overcome periods of resource limitation often associated with extreme climatic conditions. The hibernation season consists of prolonged bouts of torpor that are interrupted by brief interbout arousals. Physiological mechanisms regulating spontaneous arousals are poorly understood, but may be related to a need for gluconeogenesis or elimination of metabolic wastes. Glutamate is derived from glutamine through the glutamate-glutamine cycle and from glucose via the pyruvate carboxylase pathway when nitrogen balance favors formation of glutamine. This study tests the hypothesis that activation of NMDA-type glutamate receptors (NMDAR) maintains torpor in arctic ground squirrel (arctic ground squirrel (AGS); Urocitellus parryii). Administration of NMDAR antagonists MK-801 (5?mg/kg, i.p.) that crosses the blood-brain barrier and AP5 (5?mg/kg, i.p.) that does not cross the blood-brain barrier induced arousal in AGS. Central administration of MK-801 (0.2, 2, 20 or 200?μg; icv) to hibernating AGS failed to induce arousal. Results suggest that activation of NMDAR at a peripheral or circumventricular site is necessary to maintain prolonged torpor and that a decrease in glutamate at these sites may contribute to spontaneous arousal in AGS.     

In vivo hepatic lipid quantification using MRS at 7 Tesla in a mouse model of glycogen storage disease type 1a

The assessment of liver lipid content and composition is needed in preclinical research to investigate steatosis and steatosis-related disorders. The purpose of this study was to quantify in vivo hepatic fatty acid content and composition using a method based on short echo time proton magnetic resonance spectroscopy (MRS) at 7 Tesla. A mouse model of glycogen storage disease type 1a with inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc^−/−) mice and control mice were fed a standard diet or a high-fat/high-sucrose (HF/HS) diet for 9 months. In control mice, hepatic lipid content was found significantly higher with the HF/HS diet than with the standard diet. As expected, hepatic lipid content was already elevated in L-G6pc^−/− mice fed a standard diet compared with control mice. L-G6pc^−/− mice rapidly developed steatosis which was not modified by the HF/HS diet. On the standard diet, estimated amplitudes from olefinic protons were found significantly higher in L-G6pc^−/− mice compared with that in control mice. L-G6pc^−/− mice showed no noticeable polyunsaturation from diallylic protons. Total unsaturated fatty acid indexes measured by gas chromatography were in agreement with MRS measurements. These results showed the great potential of high magnetic field MRS to follow the diet impact and lipid alterations in mouse liver.     

Measurements of the anaplerotic rate in the human cerebral cortex using 13C magnetic resonance spectroscopy and [1-13C] and [2-13C] glucose

Recent studies in rodent and human cerebral cortex have shown that glutamate-glutamine neurotransmitter cycling is rapid and the major pathway of neuronal glutamate repletion. The rate of the cycle remains controversial in humans, because glutamine may come either from cycling or from anaplerosis via glial pyruvate carboxylase. Most studies have determined cycling from isotopic labeling of glutamine and glutamate using a [1-(13)C]glucose tracer, which provides label through neuronal and glial pyruvate dehydrogenase or via glial pyruvate carboxylase. To measure the anaplerotic contribution, we measured (13)C incorporation into glutamate and glutamine in the occipital-parietal region of awake humans while infusing [2-(13)C]glucose, which labels the C2 and C3 positions of glutamine and glutamate exclusively via pyruvate carboxylase. Relative to [1-(13)C]glucose, [2-(13)C]glucose provided little label to C2 and C3 glutamine and glutamate. Metabolic modeling of the labeling data indicated that pyruvate carboxylase accounts for 6 +/- 4% of the rate of glutamine synthesis, or 0.02 micromol/g/min. Comparison with estimates of human brain glutamine efflux suggests that the majority of the pyruvate carboxylase flux is used for replacing glutamate lost due to glial oxidation and therefore can be considered to support neurotransmitter trafficking. These results are consistent with observations made with arterial-venous differences and radiotracer methods.     

Aging alters the multichemical networking profile of the human brain: an in vivo (1)H-MRS study of young versus middle-aged subjects

In our most recent study of normal aging, we found decreased concentration of multiple chemicals in the brain of middle-aged subjects, as compared with younger subjects using in vivo proton magnetic resonance spectroscopy ((1)H-MRS). We hypothesized that these age-dependent differences in brain chemistry changes might be a reflection of the multichemical-networking-profile (MCNP) changes during aging. Using (1)H-MRS and correlation analysis, we examined the patterns of regional chemical levels and MCNP within and across multiple brain regions for all nine chemicals of (1)H-MR spectra. The brain chemistry changes and MCNP patterns were compared between 21 young (19--31-year-old) and 31 middle-aged (40--52-year-old) normal volunteers. Middle-aged subjects demonstrated a significant decrease of chemical levels in the prefrontal cortex and sensorimotor cortex (SMC), as compared with the young age group. Of these, neurotransmitters GABA and glutamate in the dorsolateral prefrontal cortex (DLPFC) were altered the most. We also found a significant increase of overall chemical correlation strength in MCNP within and across all studied brain regions with increased age. These changes were caused by alterations in the pattern of negative chemical connectivity across brain regions, which become weaker (less negative) in middle-aged subjects. The interregional chemical connectivity for the cingulate cortex, SMC and the thalamus was changed the most with increased age. Increased levels of chemical correlation strength across brain regions in aging were found for most chemicals studied (including neurotransmitters GABA and glutamate), and not for N-acetyl aspartate. These age-related differences in the connectivity of neurotransmitters were not region dependent. The results suggest that aging is associated with changes of the regional brain chemistry and the brain MCNP. The latter process may reflect an adaptive or compensatory response (possibly related to the elongation of dendrites with aging) to reduced levels of regional brain chemicals. The (1)H-MRS approach proposed here can be used as a valuable tool in the study of the brain chemistry, MCNP and their relationships in normal and abnormal aging.     

Classification of Brain Tumors by Ex Vivo 1H NMR Spectroscopy

Abstract: Ex vivo biopsy samples (n = 42) from human brain tumors and normal brain have been examined by high-resolution proton magnetic resonance spectroscopy. Parameters from one-dimensional ¹H spectra, two-dimensional COSY spectra, and transverse relaxation time (T₂) data were used to classify the tumors according to the histopathological diagnoses. The ratio of the area between 3.4 and 3.1 ppm to that between 1.5 and 1.1 ppm distinguished glioblastomas from astrocytomas and normal brain, and appeared to be indicative of malignant potential. In support of the one-dimensional data, cross-peaks in the COSY spectra of brain specimens classified glioblastomas and metastases into one group and the more benign tumors, meningiomas, astrocytomas, and normal brain into a second group. The transverse relaxation of the resonance at 1.3 ppm was fitted by a model with two T₂ values. The longer T₂ value could be used to distinguish glioblastomas from normal brain, the latter having a much longer long T₂ value. Astrocytomas showed a continuum of T₂ values between glioblastomas and normal brain, with the grade of the astrocytoma correlating roughly with the value of the long T₂ component.     

Identification of mobile cholesterol compounds in experimental gliomas by (1)H MRS in vivo: effects of ganciclovir-induced apoptosis on lipids

This letter presents a novel identification and analysis of mobile cholesterol compounds in an experimental glioma model by (1)H MRS in vivo. The cholesterol compounds turned out to comprise as much as 17 mol% of MRS visible total lipids. The results also imply partly associated accumulation of (1)H MRS detectable cholesterol compounds and unsaturated lipids during gene therapy-induced apoptosis, and indicate that the contribution of cholesterol compounds cannot be bypassed in spectral lipid analysis. The introduced (1)H MRS approach facilitates a non-invasive follow-up of mobile cholesterol compounds, paving way for studies of tumour cholesterol metabolism in vivo.     

High-field (9.4 T) 1H magnetic resonance microscopy of mouse brain

The FLASH and STEAM pulse sequences were used to perform the microimaging and localized spectroscopy of brain of living and dead mice, respectively. The phase-shift presaturation approach was used to sup-press water NMR signal. The experimental results show that the differences in localized spectra and MR images of brain between live and dead mice can be observed by means of magnetic resonance microscopy.     

Magnetic resonance imaging and spectroscopy (MRI, MRS) of seasonal patterns of body composition: A methodological pilot study in White Storks (Ciconia ciconia)

Summary Here we present a systematic application of magnetic resonance imaging (in the following called MRI) and magnetic resonance spectroscopy (MRS) to the White Stork. The main aim was to demonstrate the annual cycle of fat deposition in the same individuals for comparison to wild conspecifics, to clarify the energy metabolism of this migratory species. To obtain sharp, high-contrast images of the interior of the body, the birds were kept still by enclosing them in simple plastic tubes with additional fixation of legs and head, avoiding the problematic sedation with drugs. Altogether 12 test birds (young storks) were monitored systematically for 15 months, to follow seasonal changes in the internal organs (mainly breast muscles) and tissues (mainly fat depots). At each examination 22 high-contrast pixel images representing serial dorsoventral sections through the body were generated with the computer program MatLab, after which the pixels per section image were converted to tissue components in cm² and the distances between consecutive sections used to calculate the tissue volumes in cm³. To measure the fat in the breast muscle spectroscopy was used to determine the fat : water ratio, from which changes in fat content could be derived. The study revealed pronounced seasonal changes in the visceral and cutaneous/subcutaneous fat depots, which precisely paralleled the annual variation in body weight of the birds (see also the preceding paper, Berthold et al. 2001). The breast muscles exhibited the prolonged growth typical of the juveniles of large species but no conspicuous change at the migration periods. In this project MRI and MRS proved to be successful methods that show great promise.

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Magnet-Resonanz-Tomographie und -Spektroskopie der jahreszeitlichen Muster der Körperzusammensetzung: Eine methodische Pilotstudie am Weißstorch (Ciconia ciconia)

Zusammenfassung In der vorliegenden Arbeit stellen wir eine systematische Anwendung der Magnet-Resonanz-Tomographie (Kernspin-Tomographie, im Folgenden MRT) und der Magnet-Resonanz-Spektroskopie (im Folgenden MRS) am Weißstorch vor. Hauptaufgabe war es, die Jahresperiodik der Fettdeposition an denselben Individuen zu ermitteln, um Aufschluss über den Energiehaushalt dieser Zugvogelart durch Vergleiche mit freilebenden Vögeln zu bekommen. Die erforderliche Ruhigstellung der Vögel zum Erreichen scharfer kontrastreicher Bilder des Körperinneren war in einfachen Plastikröhren mit zusätzlicher Fixierung von Beinen und Kopf möglich, so dass auf die problematische Sedierung mit Narkotika verzichtet werden konnte. Insgesamt 12 Versuchsvögel (Jungstörche) wurden 15 Monate lang systematisch auf jahresperiodische Veränderungen von inneren Organen (v. a. Brustmuskeln) und Geweben (v. a. Fettdepots) untersucht. Aus je 22 seriellen kontrastreichen dorsoventralen Schnittbildern durch den Vogelkörper ließen sich mit dem Computerprogramm MatLab Pixelbilder erstellen und dann die Pixel pro Schnittbild in Gewebeanteile in cm² umrechnen und anschließend aus den aufeinander folgenden Schnittbildern die Gewebevolumina in cm³ ermitteln. Für die Fettbestimmung im Brustmuskel wurde durch Spektroskopie das Verhältnis von Fett: Wasser bestimmt, aus dem Veränderungen des Fettgehalts abgeleitet wurden. Die Studie ergab ausgeprägte jahresperiodische Änderungen der viszeralen und kutanen/subkutanen Fettdepots, die genau parallel zum Jahresgang des Körpergewichts der Vögel verliefen (s. auch die vorangehende Arbeit, Berthold et al. 2001). Für die Brustmuskeln ergab sich ein für Jungvögel großer Arten typisches lang anhaltendes Wachstum, aber keine auffallende Veränderung zu den Zugperioden. MRT und MRS erwiesen sich in dieser Arbeit als erfolgreich und vielversprechend.

     

In vivo brain macromolecule signals in healthy and glioblastoma mouse models: 1H magnetic resonance spectroscopy,post‐processing and metabolite quantification at 14.1 T

In ¹H magnetic resonance spectroscopy, macromolecule signals underlay metabolite signals, and knowing their contribution is necessary for reliable metabolite quantification. When macromolecule signals are measured using an inversion‐recovery pulse sequence, special care needs to be taken to correctly remove residual metabolite signals to obtain a pure macromolecule spectrum. Furthermore, since a single spectrum is commonly used for quantification in multiple experiments, the impact of potential macromolecule signal variability, because of regional differences or pathologies, on metabolite quantification has to be assessed. In this study, we introduced a novel method to post‐process measured macromolecule signals that offers a flexible and robust way of removing residual metabolite signals. This method was applied to investigate regional differences in the mouse brain macromolecule signals that may affect metabolite quantification when not taken into account. However, since no significant differences in metabolite quantification were detected, it was concluded that a single macromolecule spectrum can be generally used for the quantification of healthy mouse brain spectra. Alternatively, the study of a mouse model of human glioma showed several alterations of the macromolecule spectrum, including, but not limited to, increased mobile lipid signals, which had to be taken into account to avoid significant metabolite quantification errors.     

Aging alters regional multichemical profile of the human brain: an in vivo 1H-MRS study of young versus middle-aged subjects

Age-related differences in the multichemical proton magnetic resonance spectroscopy (1H-MRS) profile of the human brain have been reported for several age groups, and most consistently for ages from neonates to 16-year-olds. Our recent 1H-MRS study demonstrated a significant age-related increase of total chemical concentration (relative to creatine) in the prefrontal and sensorimotor cortices within young adulthood (19-31-year-olds). In the present study we test the hypothesis that the level of brain chemicals in the same cortices, which show increased chemical levels during normal development, are reduced with normal aging after young adulthood. The multichemical 1H-MRS profile of the brain was compared between 19 young and 16 middle-aged normal subjects across multiple brain regions for all chemicals of 1H-MRS spectra. Chemical concentrations were measured relative to creatine. Over all age groups the total relative chemical concentration was highest in the prefrontal cortex. Middle-aged subjects demonstrated a significant decrease of total relative chemical concentration in the dorsolateral prefrontal (F = 54.8, p < 10(-7), ANOVA), orbital frontal (F = 3.7, p < 0.05) and sensorimotor (F = 15.1, p < 0.0001) cortices, as compared with younger age. Other brain regions showed no age-dependent differences. The results indicate that normal aging alters multichemical 1H-MRS profile of the human brain and that these changes are region-specific, with the largest changes occuring in the dorsolateral prefrontal cortex. These findings provide evidence that the processes of neuronal maturation of the human brain, and neurotransmitters and other chemical changes as the marker of these neuronal changes are almost finished by young adulthood and then reduced during normal aging toward middle age period of life. The present data also support the notion of heterochronic regressive changes of the aging human brain, where the multichemical brain regional profile seems to inversely recapitulate cortical chemical maturation within normal development.     

Function and energy metabolism of isolated hearts obtained from hyperthyroid spontaneously hypertensive rats (SHR)

It was the aim of this study to evaluate the effects of hyperthyroidism on heart function and cardiac energy metabolism of spontaneously hypertensive (SHR) rats. Hyperthyroidism was induced by daily injections of T₃ (0.2 mg/kg s.c.) for 14 days. The hearts were then isolated and perfused in the Langendorff mode. ATP, phosphocreatine (PCr), and inorganic phosphate (Pi) were measured continuously by means of³¹P-nuclear magnetic resonance (NMR) spectroscopy. Work load was altered by varying stepwise the Ca⁺⁺ concentration in the perfusion fluid from 0.5 to 1.0, 1.5, and 2.0 mM, respectively. At every elevation of the Ca⁺⁺ concentration, the increase in left ventricular developed pressure (LVDP) was higher in the hyperthyroid SHR than in the untreated SHR hearts. The ATP and PCr concentrations were lower in the hyperthyroid SHR compared to the untreated SHR hearts throughout the perfusion period. PCr decreased at every Ca⁺⁺ elevation in both the untreated and hyperthyroid SHR hearts. The PCr/ATP ratio was not altered at any Ca⁺⁺ concentration neither in the untreated SHR nor in the hyperthyroid SHR hearts. The Ca⁺⁺-induced stepwise elevation in LVDP was higher at any given PCr/Pi ratio in the hyperthyroid SHR than in the untreated SHR hearts. Thus, the Ca⁺⁺-inducible contractile reserve was greater in the hyperthyroid SHR heart.     

Changes of characteristics of preoptic neurons and NA metabolism in hypothalamus of ground squirrel (Citelleus Dautieus) in different seasons and hibernating phases

The unit firing activities of neurons in the preoptic area (POA) of ground squirrel hypothalamic tissue slices were recorded and the metabolism of NA in hypothalamus was measured with high performance liquid chromatography (HPLC). Thermosensitivity, proportions, the critical temperature (Tc) and the lowest temperature (TL) of firing activity of the above-mentioned neurons, and NA metabolism in hypothalamus were compared in different seasons and hibernating phases. In comparison with that in summer euthermar, it was shown that (i) the percentage and thermosensitivity of the POA neurons varied respectively in the hibernating phases; (ii) TL and Tc of the POA neurons in winter, both euthermar and hibernation, were markedly decreased; (iii) the POA neurons in hibernation became much more sensitive to NA, and the response of cold-sensitive neurons to NA changed from inhibiting pattern in summer to exciting one in hibernation; (iv) the contents and metabolism of NA in hypothalamus decreased significantly in the entering phase and deep hibernation phase, while the metabolism of NA increased remarkably in the arousal phase. These changes might explain the regulatory mechanism how ground squirrel actively decreases body temperature (Tb) in entering into hibernation and quickly recovers body temperature in arousal phase. Project supported by the National Natural Science Foundation of China (Grant Nos. 39230060 and 39570100)     

Brain magnetic resonance study of Mecp2 deletion effects on anatomy and metabolism

Rett syndrome, a neurodevelopmental X-linked disorder, represents the most important genetic cause of severe mental retardation in the female population and results from a mutation in the gene encoding methyl-CpG-binding protein 2 (MECP2). We report here the first characterization of Mecp2-null mice, by in vivo magnetic resonance imaging and spectroscopy, delineating the cerebral phenotype associated with the lack of Mecp2. We performed a morphometric study that revealed a size reduction of the whole brain and of structures involved in cognitive and motor functions (cerebellum and motor cortex). Significant metabolic anomalies, including reduced N-acetylaspartate, myo-inositol, and glutamine plus glutamate, and increased choline levels were evidenced. These findings indicate that not only neuronal but also glial metabolism is affected in Mecp2-null mice. Furthermore, we uncovered an important reduction of brain ATP level, a hitherto undetected anomaly of energy metabolism that may reflect and contribute to cerebral injury and dysfunction.     

Changes of characteristics of preoptic neurons and NA metabolism in hypothalamus of ground squirrel (Citelleus Dautieus) in different seasons and hibernating phases

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The effect of short-term fasting on liver and skeletal muscle lipid, glucose, and energy metabolism in healthy women and men

Fasting promotes triglyceride (TG) accumulation in lean tissues of some animals, but the effect in humans is unknown. Additionally, fasting lipolysis is sexually dimorphic in humans, suggesting that lean tissue TG accumulation and metabolism may differ between women and men. This study investigated lean tissue TG content and metabolism in women and men during extended fasting. Liver and muscle TG content were measured by magnetic resonance spectroscopy during a 48-h fast in healthy men and women. Whole-body and hepatic carbohydrate, lipid, and energy metabolism were also evaluated using biochemical, calorimetric, and stable isotope tracer techniques. As expected, postabsorptive plasma fatty acids (FAs) were higher in women than in men but increased more rapidly in men with the onset of early starvation. Concurrently, sexual dimorphism was apparent in lean tissue TG accumulation during the fast, occurring in livers of men but in muscles of women. Despite differences in lean tissue TG distribution, men and women had identical fasting responses in whole-body and hepatic glucose and oxidative metabolism. In conclusion, TG accumulated in livers of men but in muscles of women during extended fasting. This sexual dimorphism was related to differential fasting plasma FA concentrations but not to whole body or hepatic utilization of this substrate.     

Biochemical characterization of breast tumors by in vivo and in vitro magnetic resonance spectroscopy (MRS)

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) have evolved as sensitive tools for anatomic and metabolic evaluation of breast cancer. In vivo MRS studies have documented the presence of choline containing compounds (tCho) as a reliable biochemical marker of malignancy and also useful for monitoring the tumor response to therapy. Recent studies on the absolute quantification of tCho are expected to provide cut-off values for discrimination of various breast pathologies. Addition of MRS investigation was also reported to increase the specificity of MRI. Further, ex vivo and in vitro MRS studies of intact tissues and tissue extracts provided several metabolites that were not be detected in vivo and provided insight into underlying biochemistry of the disease processes. In this review, we present briefly the role of various ¹H MRS methods used in breast cancer research and their potential in relation to diagnosis, monitoring of therapeutic response and metabolism.     

In vivo assessment of nodularin-induced hepatotoxicity in the rat using magnetic resonance techniques (MRI, MRS and EPR oximetry)

Acute nodularin-induced hepatotoxicity was assessed in vivo, in rats using magnetic resonance (MR) techniques, including MR imaging (MRI), MR spectroscopy (MRS), and electron paramagnetic resonance (EPR) oximetry. Nodularin is a cyclic hepatotoxin isolated from the cyanobacterium Nodularia spumigena. Three hours following the intraperitoneal (i.p.) administration of nodularin (LD50), a region of 'damage', characterized by an increase in signal intensity, was observed proximal to the porta hepatis (PH) region in T2-weighted MR images of rat liver. Image analysis of these regions of apparent 'damage' indicated a statistically significant increase in signal intensity around the PH region following nodularin administration, in comparison with controls and regions peripheral to the PH region. An increase in signal intensity was also observed proximal to the PH region in water chemical shift selective images (CSSI) of nodularin-treated rat livers, indicating that the increased signal observed by MRI is an oedematous response to the toxin. Microscopic assessment (histology and electron microscopy) and serum liver enzyme function tests (aminotransferase (ALT) and aspartate ALT (AST)) confirmed the nodularin-induced tissue injury observed by MRI. In vivo and in vitro MRS was used to detect alterations in metabolites, such as lipids, Glu+Gln, and choline, during the hepatotoxic response (2-3 h post-exposure). Biochemical assessment of perchloric acid extracts of nodularin-treated rat livers were used to confirm the MRS results. In vivo EPR oximetry was used to monitor decreasing hepatic pO2 (approximately 2-fold from controls) 2-3 h following nodularin exposure. In vivo MR techniques (MRI, MRS and EPR oximetry) are able to highlight effects that may not have been evident in single end point studies, and are ideal methods to follow tissue injury progression in longitudinally, increasing the power of a study through repeated measures, and decreasing the number of animals to perform a similar study using histological or biochemical techniques.     

Determination of 3J(C,P) and 3J(H,P) coupling constants in nucleotide oligomers with FIDS-HSQC

Summary A method for measuring J(C,P) and J(H,P) coupling constants is presented, based on fitting a target multiplet containing the heteronuclear coupling to a reference multiplet that lacks the heteronuclear coupling. In DNA and RNA oligonucleotides, information on backbone torsion angles can be obtained from these couplings. Experimental multiplets are obtained from ³¹P-coupled and ³¹P-decoupled ¹H, ¹³C HSQC spectra of Rp-cyclic methylphosphonate. The accuracy to which the heteronuclear coupling constants can be determined depends on the signal-to-noise ratio of the experimental data and is analyzed in detail.Dedicated to Prof. R.R. Ernst on the occasion of his 60th birthday.