13C isotopomer analyses in intact tissue using {13C}homonuclear decoupling

Entry of ¹³C-enriched acetyl-CoA into the citric acid cycle results in scrambling of ¹³C into the various carbon positions of all intermediate pools. The eventual result is that the ¹³C resonances of all detectable intermediates or molecules exchanging with those intermediates appear as multiplets due to nearest neighbor spin-spin couplings. We have previously shown that an isotopomer analysis of the glutamate ¹³C multiplets provides a history of ¹³C flow through the cycle pools and that relative substrate utilization and relative anaplerotic flux can be quantitated (C.R. Malloy, A.D. Sherry, and F.M.H. Jeffrey, Am. J. Physiol. 259, H987–H995 (1990)). A major limitation of the method for in vivo applications is spectral resolution of multiline resonances required for a complete isotopomer analysis. We now show that (¹³C)homonuclear decoupling of the glutamate C3 resonance collapses nine-line C4 and C2 resonances into three-line multiplets. We demonstrate that these three line ¹³C multiplets are well resolved in isolated, perfused rat hearts and present steady-state equations that allow an isotopomer analysis from data obtained in intact tissue. This advancement offers for the first time the possibility of extending ¹³C isotopomer methods to complex metabolic conditions in vivo.     

13C spectroscopic imaging. A simple approach to in vivo 13C investigations

13C spectroscopic imaging (SI) is implemented and tested on a Siemens whole-body MR instrument for in vivo localized 13C spectroscopy. The method provides spectroscopic maps of extended regions of interest. It is found that 13C SI is an ideal field of application for the spectroscopic imaging technique since most other localization methods suffer from chemical-shift or offset artifacts for this nucleus. Applications of 13C SI with a single 13C rf channel as well as in combination with polarization transfer experiments and heteronuclear broadband decoupling are shown.     

Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose: a simple analysis by 13C NMR of urinary menthol glucuronide.

Menthol glucuronide was isolated from the urine of a healthy 70-kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U-(13)C]glucose. Glucuronide (13)C-excess enrichment levels were 4-6% and thus provided high signal-to-noise ratios (SNRs) for confident assignment of (13)C-(13)C spin-coupled multiplet components within each (13)C resonance by (13)C NMR. The [U-(13)C]glucuronide isotopomer derived via direct pathway conversion of [U-(13)C]glucose to [U-(13)C]UDP-glucose was resolved from [1,2,3-(13)C(3)]- and [1,2-(13)C(2)]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U-(13)C]glucose. In a second study, a group of four overnight-fasted patients (63 +/- 10 kg) with severe heart failure were given peppermint oil and infused with [U-(13)C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady-state plasma [U-(13)C]glucose enrichment of 4.6% +/- 0.6%. Menthol glucuronide was harvested and glucuronide (13)C-isotopomers were analyzed by (13)C NMR. [U-(13)C]glucuronide enrichment was 0.6% +/- 0.1%, and the sum of [1,2,3-(13)C(3)] and [1,2-(13)C(2)]glucuronide enrichments was 0.9% +/- 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% +/- 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% +/- 10% of GP.     

Multiple bond 13C-13C spin-spin coupling provides complementary information in a 13C NMR isotopomer analysis of glutamate.

Most 13C nuclear magnetic resonance (NMR) isotopomer analyses relate a metabolic index of interest to populations of 13C isotopomers as reported by one-bond 13C-13C spin-spin couplings. Metabolic conditions that produce highly enriched citric acid cycle intermediates often lead to 13C NMR spectra of metabolites such as glutamate that show extra multiplets due to long-range couplings. It can be demonstrated from 13C NMR spectra of hearts perfused with mixtures of acetate plus propionate that multiplets in glutamate C2 arising from 3J25 coupling provide a direct readout of acetyl-CoA fractional enrichment (FC1 and FC3), while multiplets in glutamate C5 arising from 2J35 and 3J25 couplings quantitatively reflect enrichment of the anaplerotic substrate.     

Natural-abundance 13C NMR of brain

In natural-abundance 13C NMR spectrum of excised rat brain 55 resonances were resolved. The chemical shifts of most of the resonances were well correlated with those of pure brain metabolites, determined under identical experimental conditions. These resonances were also found in the cytosol fraction and perchloric acid extract of the brain. Some brain resonances were not observed in the perchloric acid extract but only in the microsomes or cytosol.     

13C NMR investigation of synovial fluids

¹³C NMR spectra of synovial fluids from 20 patients suffering from rheumatic diseases have been recorded. Structural changes in hyaluronic acid, the main carrier of the viscoelastic properties of synovial fluid, could be observed in the NMR spectra of the native biological fluid. By comparing these spectra with those of purified hyaluronic acid, a rough estimation of the degree of depolymerization of synovial hyaluronlic acid was possible. The patients with rheumatoid arthritis appeared to have a lower degree of polymerization compared to patients with osteoarthrosis. Thus, ¹³C NMR spectroscopy prolvides useful information about biophysical properties of sartorial fluid.     

Which prior knowledge? Quantification of in vivo brain 13C MR spectra following 13C glucose infusion using AMARES

The recent developments in high magnetic field ¹³C magnetic resonance spectroscopy with improved localization and shimming techniques have led to important gains in sensitivity and spectral resolution of ¹³C in vivo spectra in the rodent brain, enabling the separation of several ¹³C isotopomers of glutamate and glutamine. In this context, the assumptions used in spectral quantification might have a significant impact on the determination of the ¹³C concentrations and the related metabolic fluxes. In this study, the time domain spectral quantification algorithm AMARES (advanced method for accurate, robust and efficient spectral fitting) was applied to ¹³C magnetic resonance spectroscopy spectra acquired in the rat brain at 9.4 T, following infusion of [1,6‐¹³C₂] glucose. Using both Monte Carlo simulations and in vivo data, the goal of this work was: (1) to validate the quantification of in vivo ¹³C isotopomers using AMARES; (2) to assess the impact of the prior knowledge on the quantification of in vivo ¹³C isotopomers using AMARES; (3) to compare AMARES and LCModel (linear combination of model spectra) for the quantification of in vivo ¹³C spectra. AMARES led to accurate and reliable ¹³C spectral quantification similar to those obtained using LCModel, when the frequency shifts, J‐coupling constants and phase patterns of the different ¹³C isotopomers were included as prior knowledge in the analysis. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

抗癌药物紫杉醇的手性C13侧链的合成

目的方便快捷的合成抗癌药物紫杉醇的C13侧链。方法以一种可回收的小分子手性配体催化反式肉桂酸乙酯的不对称二羟化(AD)反应,得到手性连二醇,产物再经两步反应可获得高光学活性的紫杉醇C13侧链。结果各步反应产品纯化方法简单,配体可重复使用,紫杉醇侧链产率48．4％。结论该方法显著降低了紫杉醇的生产成本,为工业化生产抗癌药物紫杉醇打下基础。     

Accurate flip-angle calibration for 13C MRI.

(13)C imaging and spectroscopy in the presence of injected labeled compounds can vastly extend the capability of MRI to perform metabolic imaging. The details of imaging in the presence of injected compounds, however, pose new technological challenges. Pulse sequences, in general, rely on precise flip-angle (FA) calibration to create high signal-to-noise ratio (SNR), artifact-free images. Signal quantification also requires precise knowledge of the excitation FA. In MRI scans that rely on signal acquisitions from injected compounds, however, such FA calibration is challenged by low natural-abundance (13)C signal levels before injection, and by time-varying signal following injection. A method to precisely set the FA at the (13)C frequency based on FA calibration at the (23)Na frequency is presented here. A practical implementation of a coil (a dual-tuned, (23)Na/(13)C low-pass birdcage coil) suitable for this calibration in vivo is also documented. Accurate FA calibration is demonstrated at the (13)C frequency for in vivo rat experiments using this approach.     

Hyperpolarized 13C MR angiography using trueFISP.

A (13)C-enriched water-soluble compound (bis-1,1-(hydroxymethyl)-1-(13)C-cyclopropane-D(8)), with a (13)C-concentration of approximately 200 mM, was hyperpolarized to approximately 15% using dynamic nuclear polarization, and then used as a contrast medium (CM) for contrast-enhanced magnetic resonance angiography (CE-MRA). The long relaxation times (in vitro: T(1) approximately 82 s, T(2) approximately 18 s; in vivo: T(1) approximately 38 s, T(2) approximately 1.3 s) are ideal for steady-state free precession (SSFP) imaging with a true fast imaging and steady precession (trueFISP) pulse sequence. It was shown both theoretically and experimentally that the optimal flip angle was 180 degrees. CE-MRA was performed in four anesthetized live rats after intravenous injection of 3 ml CM. The angiograms covered the thoracic/abdominal region in two of the animals, and the head-neck region in the other two. Fifteen consecutive images were acquired in each experiment, with a flip-back pulse at the end of each image acquisition. In the angiograms, the vena cava (SNR approximately 240), aorta, renal arteries, carotid arteries (SNR approximately 75), jugular veins, and several other vessels were visible. The SNR in the cardiac region was 500. Magnetization was preserved from one image acquisition to the next using the flip-back technique (SNR(cardiac) approximately 10 in the 15th image).     

NMR imaging of 13C in animal tissues

Natural-abundance 13C NMR imaging of animal tissues is demonstrated using high-field (4.7-T) and surface coil techniques. Undecoupled images of an unfertilized egg yolk and an oxtail are presented. Analysis indicates the images map mainly methylene carbon or lipid signal intensity.     

13C NMR study of the generation of C2- and C3,-deuterated lactic acid by tumoral pancreatic islet cells exposed to D-[1-13C]-, D-[2-13C]- and D-[6-13C]-Glucose in 2H2O

Tumoral pancreatic islet cells of the RIN5mF line were incubated for 120 min in media prepared in ²H₂O and containing D -[1-¹³C]glucose, and D -[2-¹³C]glucose, and D -[6-¹³C]glucose. The generation of C₂- and C₃- deuterated lactic acid was assessed by ¹³C NMR. The interpretation of experimental results suggests that a) the efficiency of deuteration on the C₁ of D-fructose 6-phosphate does not exceed about 47% and 4% in the phosphoglucoisomerase and phosphomannoisomerase reactions, respectively; b) approximately 38% of the molecules of D -glyceraldehyde 3-phosphate generated from D -glucose escape deuteration in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase; and c) about 41% of the molecules of pyruvate generated by glycolysis are immediately converted to lactate, the remaining 59% of pyruvate molecules undergoing first a single or double back-and-forth interconversion with L -alanine. It is proposed that this methodological approach, based on high resolution ¹³C NMR spectroscopy, may provide novel information on the regulation of back-and-forth interconversion of glycolytic intermediates in intact cells as modulated, for instance, by enzyme-to-enzyme tunneling.     

Detection of metabolites in rabbit brain by 13C NMR spectroscopy following administration of [1-13C]glucose

1H-decoupled 13C NMR spectra (20.2 MHz) of the living rabbit brain were collected with a surface coil following the intravenous infusion of [1-13C]glucose. Within 15 min of infusion, the alpha and beta anomers of glucose were detected and, shortly thereafter, the carbon atoms at positions C4, C3, and C2 of glutamate and(or) glutamine. After reductions of inspired oxygen from 30 to 5%, lactate C3 was detected. The intensity of the lactate resonance rose progressively during hypoxia and later fell during recovery with oxygen. The 13C fractional isotopic enrichment of arterial blood glucose was measured by 1H NMR providing information on the rate and extent of blood glucose labeling.     

Sensitivity enhancement in whole-body natural abundance 13C spectroscopy using 13C/1H double-resonance techniques at 4 tesla.

In vivo 13C spectroscopy experiments were performed using a whole-body MR system at a static field of 4 T. The main goal of the investigations was to evaluate the sensitivity increase achievable by means of 13C/1H double-resonance techniques at 4 T. Spectra from subcutaneous fat as well as muscle glycogen from the lower leg were acquired using frequency selective proton decoupling and the polarization transfer method SINEPT. With respect to measurements on subcutaneous fat, polarization transfer turned out to be more efficient than selective decoupling. About a fourfold enhancement in spectral peak intensity for the C = C line doublet of the unsaturated fatty acid chain was obtained. Combining polarization transfer with decoupling yielded a factor of 6 in signal amplitude. In contrast to that, the signal enhancement observed in measurements on the glycogen C-1 resonance was only around twofold. The lower efficiency is explained by fast T2 relaxation of the proton transition. A T2 value of about 3 ms was derived from the experimental data. Acquisition times as low as 3 min were realized for normal level glycogen in human calf muscle, enabling a time resolution adequate for dynamic studies on muscle glycogen depletion. Aspects of RF power absorption in tissue and the generally higher efficiency make polarization transfer methods preferable to selective decoupling in whole-body 13C spectroscopy at 4 T.     

Tumor imaging using hyperpolarized 13C magnetic resonance spectroscopy

Dynamic nuclear polarization is an emerging technique for increasing the sensitivity of magnetic resonance imaging and spectroscopy, particularly for low‐γ nuclei. The technique has been applied recently to a number of ¹³C‐labeled cell metabolites in biological systems: the increase in signal‐to‐noise allows the spatial distribution of an injected molecule to be imaged as well as its metabolic product or products. This review highlights the most significant molecules investigated to date in preclinical cancer models, either in terms of their demonstrated metabolism in vivo or the biological processes that they can probe. In particular, label exchange between hyperpolarized ¹³C‐labeled pyruvate and lactate, catalyzed by lactate dehydrogenase, has been shown to have a number of potential applications. Finally, techniques to image these molecules are also discussed as well as methods that may extend the lifetime of the hyperpolarized signal. Hyperpolarized magnetic resonance imaging and magnetic resonance spectroscopic imaging have shown great promise for the imaging of cancer in preclinical work, both for diagnosis and for monitoring therapy response. If the challenges in translating this technique to human imaging can be overcome, then it has the potential to significantly alter the management of cancer patients. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.