Preinvasive and invasive cervical cancer: an ex vivo proton magic angle spinning magnetic resonance spectroscopy study

The aim of this study was to obtain (1)H MR spectra using magic angle spinning (MAS) techniques from punch biopsies (<20 mg) of preinvasive and invasive cervical disease and to correlate the spectral profiles with sample classification on the basis of histopathology. Tissue samples were obtained at colposcopic examination, during local treatment of cervical intraepithelial neoplasia (CIN) or at hysterectomy. (1)H MAS MRS was performed at 25 degrees C while spinning the sample at 4.5 kHz. After measurement, the tissue was immersed in formalin and the pathology determined. Histological examination after (1)H MAS MRS defined 27 samples with squamous cell carcinoma (SCC), 12 with CIN and 39 with only normal tissue. The standardized integrals of the lipid, choline and creatine regions of the spectra were significantly higher in SCC than in normal or CIN tissue. There was no obvious difference in the standardized integral of the region 4.15-3.5 ppm. The acyl fatty acid side-chain length was longer or less unsaturated in SCC than in normal tissue. Normal tissue from patients with SCC showed significantly higher triglycerides than normal tissue from patients with benign uterine disease but significantly lower triglycerides than SCC tissue. (1)H MAS MRS of the uterine cervix ex vivo may be used to differentiate non-invasive from invasive cervical lesions, increase interpretation of in vivo MRS and provide insights into tumor biology.     

Comparisons of brain metabolites observed by HRMAS 1H NMR of intact tissue and solution 1H NMR of tissue extracts in SIV-infected macaques

The objective of this study was to compare ex vivo proton high-resolution magic angle spinning magnetic resonance spectra of intact tissue with those spectra obtained by solution (1)H NMR of brain extracts of the same sample. Sixteen brain tissue samples from simian immunodeficiency virus-infected rhesus macaques from both frontal cortex and putamen were evaluated by comparing brain metabolite quantities of N-acetylaspartate (NAA), choline-containing compounds (Cho), myo-inositol (MI), creatine (Cr), lactate (Lac), glutamate (Glu) and acetate (Ace). The ratios of the individual NMR peak areas of all metabolites relative to the creatine peak area were calculated. Linear regression analysis revealed significant correlations between measurements using the two methods. The strength of the correlations varied depending on the metabolite studied. We found highly significant correlations for NAA/Cr (r2 = 0.77; p < 0.0001), NAA + Ace/Cr (r2 = 0.73; p < 0.0001) and MI/Cr (r2 = 0.75; p < 0.0001). We observed somewhat less strong correlations for Glu/Cr (r2 = 0.54; p < 0.002) and Lac/Cr (r2 = 0.54; p < 0.002). There was a substantially weaker correlation for Cho/Cr (r2 = 0.32; p = 0.02). When plotting the metabolite ratios obtained by 1H HRMAS NMR of the intact tissue sample on the ordinate vs 1H NMR of the tissue extract on the abscissa, most metabolites exhibited a slope close to unity, and a positive intercept probably due to macromolecular contributions to the MAS spectra. The slope for Cho/Cr was substantially less than unity. Generally, samples from the frontal cortex showed a better correlation between intact and extracted tissue samples than putamen. This is most prominent in the cases of NAA/Cr and Cho/Cr. We conclude that both methods provide substantially the same information for most major brain metabolites, with the exception of the Cho resonance.     

Evaluation of 31P high-resolution magic angle spinning of intact tissue samples

The first detailed evaluation is presented of high-resolution (31)P MRS using magic angle spinning (MAS) of intact tissue samples and comparison with the conventional method of studying tissue extracts. The main motivation is that MAS leaves the sample intact at the end of the study for histopathological evaluation. While MAS of tissue samples has previously been demonstrated for (1)H MRS, (31)P MRS is better suited to study of the phospholipid metabolites of importance in cancer. Samples of rhabdomyosarcoma and RIF-1 experimental tumours were maintained at 4 degrees C, spun at 3 kHz and measured in 28-min acquisitions at 11.7 and 14 T. Metabolite stability was evaluated using four sequential 28-min acquisitions. High-resolution MRS was performed on extracts of the same tissue samples. (31)P HR-MAS yielded well-resolved high-resolution spectra, showing peaks from phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate, glycerophosphoethanolamine and glycerophosphocholine, with linewidths in the range 3-20 Hz. In tumour samples there was no significant change in peak areas over a 2-h period, while peaks sensitive to pH (inorganic phosphate, PE and PC) showed a small change in chemical shift, corresponding to a change of 0.13 +/- 0.06 pH units. Tissue metabolite concentrations showed good agreement with concentrations measured from extracts of the same pieces of tissue. For calculation of metabolite concentrations, the measurement of a reference compound in a separate measurement is more robust than using the signal from a reference compound in the rotor with the sample. Compared with performing tissue extracts, use of MAS of intact tissue samples requires less preparation, is quicker and permits the same sample to be used for subsequent histopathology. The methodology has particular application in studying phospholipid metabolism in cancer and in monitoring tumour response to treatment, where concentrations of phospholipid-related metabolites are found to alter following response to a wide range of anti-cancer therapies.     

Magnetic resonance spectroscopy of the occipital cortex and the cerebellar vermis distinguishes individual cats affected with alpha‐mannosidosis from normal cats

A genetic deficiency of lysosomal alpha‐mannosidase causes the lysosomal storage disease alpha‐mannosidosis (AMD), in which oligosaccharide accumulation occurs in neurons and glia. The purpose of this study was to evaluate the role of magnetic resonance spectroscopy (MRS) in detecting the oligosaccharide accumulation in AMD. Five cats with AMD and eight age‐matched normal cats underwent in vivo MRS studies with a single voxel short echo time (20 ms) STEAM spectroscopy sequence on a 4.7T magnet. Two voxels were studied in each cat, from the cerebellar vermis and the occipital cortex. Metabolites of brain samples from these regions were extracted with perchloric acid and analyzed by high resolution NMR spectroscopy. A significantly elevated unresolved resonance signal between 3.4 and 4. ppm was observed in the cerebellar vermis and occipital cortex of all AMD cats, which was absent in normal cats. This resonance was shown to be from carbohydrate moieties by high resolution NMR of tissue extracts. Resonances from the Glc‐NAc group (1.8–2.2 ppm) along with anomeric proton signals (4.6–5.4 ppm) from undigested oligosaccharides were also observed in the extract spectra from AMD cats. This MRS spectral pattern may be a useful biomarker for AMD diagnosis as well as for assessing responses to therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Classification of tumour 1H NMR spectra by pattern recognition.

1H spectra of tumours or normal tissues, which include signals from all hydrogen-containing metabolites, are too complex for the human eye to interpret. We have studied 58 1H spectra from perchloric acid extracts of three normal tissues (liver, kidney and spleen) and five rat tumours (GH3 pituitary, fibrosarcoma, Morris Hepatomas 7777 and 9618a and Walker carcinosarcoma). Instead of editing them or quantifying individual metabolites, we have used statistical pattern recognition techniques to classify them into groups. This automatic, objective method differentiated spectra from normal and malignant rat tissue biopsies, and from different types of cancer. It seems likely that this technique can be applied to human tissues and thus used for cancer diagnosis.     

A quantitative comparison of metabolite signals as detected by in vivo MRS with ex vivo 1H HR‐MAS for childhood brain tumours

¹H MRS provides a powerful method for investigating tumour metabolism by allowing the measurement of metabolites in vivo. Recently, the technique of ¹H high‐resolution magic angle spinning (HR‐MAS) has been shown to produce high‐quality data, allowing the accurate measurement of many metabolites present in unprocessed biopsy tissue. The purpose of this study was to evaluate the agreement between the techniques of in vivo MRS and ex vivo HR‐MAS for investigating childhood brain tumours. Short‐TE (30 ms), single‐voxel, in vivo MRS was performed on 16 paediatric patients with brain tumours at 1.5 T. A frozen biopsy sample was available for each patient. HR‐MAS was performed on the biopsy samples, and metabolite quantities were determined from the MRS and HR‐MAS data using the LCModel? and TARQUIN algorithms, respectively. Linear regression was performed on the metabolite quantities to asses the agreement between MRS and HR‐MAS. Eight of the 12 metabolite quantities were found to correlate significantly (P < 0.05). The four worst correlating metabolites were aspartate, scyllo‐inositol, glycerophosphocholine and N‐acetylaspartate, and, except for glycerophosphocholine, this error was reflected in their higher Cramer–Rao lower bounds (CRLBs), suggesting that low signal‐to‐noise was the greatest source of error for these metabolites. Glycerophosphocholine had a lower CRLB implying that interference with phosphocholine and choline was the most significant source of error. The generally good agreement observed between the two techniques suggests that both MRS and HR‐MAS can be used to reliably estimate metabolite quantities in brain tumour tissue and that tumour heterogeneity and metabolite degradation do not have an important effect on the HR‐MAS metabolite profile for the tumours investigated. HR‐MAS can be used to improve the analysis and understanding of MRS data. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolomic studies of human lung carcinoma cell lines using in vitro (1)H NMR of whole cells and cellular extracts

We report principal component analysis (PCA) of (1)H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and (1)H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumour origin with different chemotherapy drug resistance patterns. For whole cells, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene mobile lipid (1)H resonance intensities, which correlate with our knowledge of the level of resistance displayed by the different cells. Similarly, in the (1)H NMR spectra of the aqueous and lipophilic extracts, significant quantitative differences in the metabolite distributions were apparent, which are consistent with the PCA results. Copyright (c) 2008 John Wiley & Sons, Ltd.     

1H NMR spectroscopic method for diagnosis of malabsorption syndrome: a pilot study

Despite its well-documented limitations, colorimetry has been commonly used for the d-xylose test in the diagnosis of malabsorption syndrome (MAS). With a possibility of overcoming its limitations, the use of (1)H NMR spectroscopy for D-xylose test is explored herein. Urine samples from 35 adults with suspected MAS were obtained before and after oral ingestion of D-xylose. The diagnosis of MAS was based on fecal fat (72 h excretion using Van de Kamer's technique, normal < 7 g/24 h and/or Sudan III stain of spot stool specimen, normalor=1 g/5 g/5 h). In vitro experiments on the standard specimens of D-xylose were also performed independently using both methods. Colorimetry showed a lower value for the quantity of D-xylose excreted in urine than NMR [median 0.73 (0.17-1.89 g) vs 1.37 (0.17-3.23 g), respectively; p<0.0001, Wilcoxon's signed ranks test]. Colorimetry and NMR correctly diagnosed 11/12 and 10/12 (p=N.S.) patients with MAS and 14/23 and 20/23 (p<0.05) without MAS, respectively. Sensitivity and specificity of colorimetry and NMR were 91.6 and 60.7% vs 83.3 and 86.9%, respectively. In in vitro experiments, the values obtained for standard xylose using NMR showed a maximum error of 7%, whereas the colorimetric method showed 20%. The NMR method is simple and may be more accurate for the D-xylose absorption test. Colorimetry was found to be inferior as compared with NMR due to its low specificity.     

1H and 13C HR-MAS spectroscopy of intact biopsy samples ex vivo and in vivo 1H MRS study of human high grade gliomas

High-resolution magic angle spinning (HR-MAS) one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy has been used to study intact glioblastoma (GBM) brain tumour tissue. The results were compared with in vitro chemical extract and in vivo spectra. The resolution of 1H one-dimensional, 1H TOCSY and 13C HSQC HR-MAS spectra is comparable to that obtained on perchloric extracts. 13C HSQC HR-MAS spectra have been particularly useful for the identification of 37 different metabolites in intact biopsy tumours, excluding water and DSS components. To our knowledge, this is the most detailed assignment of biochemical compounds obtained in intact human tissue, in particular in brain tumour tissue. Tissue degradation during the recording of the NMR experiment was avoided by keeping the sample at a temperature of 4 degrees C. Detailed metabolical compositions of 10 GBM (six primary, two secondary and two unclassified) were obtained. A good correlation between ex vivo and in vivo MRS has been found.     

Comparison of HR MAS MR spectroscopic profiles of breast cancer tissue with clinical parameters

Breast cancer is the most frequent form of cancer in women and improved diagnostic methods are desirable. Malignant cells have altered metabolism and metabolic mapping might become a tool in cancer diagnostics. High-resolution magic angle spinning (HR MAS) MR spectroscopy of tissue biopsies provides detailed information on metabolic composition. The 600 MHz 1H HR MAS spectra were acquired of breast cancer tissue from 85 patients and adjacent non-involved tissue from 18 of these patients. Tissue specimens were investigated by microscopy after MR analysis. The resulting spectra were examined by three different approaches. Relative intensities of glycerophosphocholine (GPC), phosphocholine (PC) and choline were compared for cancerous and non-involved specimens. Eight metabolites, choline, creatine, beta-glucose, GPC, glycine, myo-inositol, PC and taurine, were quantified from the recorded spectra and compared with tumor histological type and size, patient's lymph node status and tissue composition of sample. The spectra were also compared with tumor histological type and size, lymph node status and tissue composition of samples using principal component analysis (PCA). Tumor samples could be distinguished from non-involved samples (82% sensitivity, 100% specificity) based on relative intensities of signals from GPC, PC and choline in 1H HR MAS spectra. Tissue concentrations of metabolites showed few differences between groups of samples, which can be caused by limitations in the quantification procedure. Choline and glycine concentrations were found to be significantly higher in tumors larger than 2 cm compared with smaller tumors. PCA of MAS spectra from patients with invasive ductal carcinomas indicated a possible prediction of spread to axillary lymph nodes. Metabolite estimates and PCA of MAS spectra were influenced by the percentage of tumor cells in the investigated specimens.     

Investigation of metabolite changes in the transition from pre-invasive to invasive cervical cancer measured using (1)H and (31)P magic angle spinning MRS of intact tissue

The aim of this study was to determine the metabolic changes in the transition from pre-invasive to invasive cervical cancer using high-resolution magic angle spinning (HR-MAS) MRS. Biopsy specimens were obtained from women with histologically normal cervix (n = 5), cervical intraepithelial neoplasia (CIN; mild, n = 5; moderate/severe, n = 40), and invasive cancer (n = 23). (1)H HR-MAS MRS data were acquired using a Bruker Avance 11.74 T spectrometer (Carr-Purcell-Meiboom-Gill sequence; TR = 4.8 s; TE = 135 ms; 512 scans; 41 min acquisition). (31)P HR-MAS spectra were obtained from the normal subjects and cancer patients only (as acetic acid applied before tissue sampling in patients with CIN impaired spectral quality) using a (1)H-decoupled pulse-acquire sequence (TR = 2.82 s; 2048 scans; 96 min acquisition). Peak assignments were based on values reported in the literature. Peak areas were measured using the AMARES algorithm. Estimated metabolite concentrations were compared between patient diagnostic categories and tissue histology using independent samples t tests. Comparisons based on patient category at diagnosis showed significantly higher estimated concentrations of choline (P = 0.0001) and phosphocholine (P = 0.002) in tissue from patients with cancer than from patients with high-grade dyskaryosis, but no differences between non-cancer groups. Division by histology of the sample also showed increases in choline (P = 0.002) and phosphocholine (P = 0.002) in cancer compared with high-grade CIN tissue. Phosphoethanolamine was increased in cancer compared with normal tissue (P = 0.0001). Estimated concentrations of alanine (P = 0.01) and creatine (P = 0.008) were significantly reduced in normal tissue from cancer patients compared with normal tissue from non-cancer patients. The estimated concentration of choline was significantly increased in CIN tissue from cancer patients compared with CIN tissue from non-cancer patients (P = 0.0001). Estimated concentrations of choline-containing metabolites increased from pre-invasive to invasive cervical cancer. Concurrent metabolite depletion occurs in normal tissue adjacent to cancer tissue.     

Phase transition from a C-centered to a B-centered orthorhombic crystalline form of syndiotactic poly(propylene)

Samples of highly syndiotactic poly(propylene) (s-PP) crystallized in a C-pseudo-centered orthorhombic form are here characterized through high resolution solid state ¹³C NMR sepctroscopy and wide angle X-ray diffraction. The ¹³C NMR CP MAS (cross-polarization, magic angle spinning) spectra of highly disordered quench-precipitated s-PP samples (with a structure very close to a C-pseudo-centered orthrohombic form) yield additional resonances beside those already reported in the literature for s-PP samples crystallized with the chains in a fully helical $ \rlap{--} (TTGG\rlap{--} )_n $ conformation (T: trans; G: gauche). From the correlation of ¹³C NMR CP MAS spectra with the X-ray diffraction profiles recorded at the same temperatures, it is possible to establish that at a given temperature (approximately 100°C, for our samples) a phase transition from the C- to the B-pseudo-centered orthorhombic form starts to occur. Correspondingly, the aforementioned additional resonances in the ¹³C NMR CP MAS spectra progressively disappear with the onset of the phase transition. These extra resonances are indeed completely absent in the ¹³C NMR CP MAS spectrum of samles crystallized in the B-pseudo-centered structure. NMR and X-ray diffraction data, according to differential scanning calorimetry, were interpreted with a recrystallization phenomenon; at temperatures below 140°C for our samples, the variously sized C-pseudo-centered orthorhombic crystallites melt and readily re-crystallize in the B-pseudocentered orthorhombic form.     

Localized NMR spectroscopy in vivo. Progress and problems

Metabolites in brain and muscle of normal human volunteers have been studied by localized 1H and 31P NMR spectroscopy in vivo. Localization was achieved by means of stimulated echo (STEAM) sequences for both water-suppressed 1H NMR (TE = 20 ms) and 31P NMR (TE = 3 ms). Volumes-of-interest and measuring times selected for brain spectroscopy were 8 mL and 6.5 min for 1H NMR and 125 mL and 13 min for 31P NMR, respectively. General problems relating to spatial localization, spectral resolution, and quantitation of in vivo NMR data are discussed with respect to the nucleus and organs under investigation. They are correlated to studies of tissue extracts obtained at field strengths of 2.35 T (Bruker Biospec) and 7.0 T (Bruker MSL 300). Human studies were performed at 2.0 T on a whole-body research system (Siemens Magnetom).     

Molecular characterization of human healthy and neoplastic cerebral and renal tissues by in vitro (1)H NMR spectroscopy (review)

The clinical impact of (1)H NMR spectroscopy in the study of human organs, brain and kidney in particular, is well demonstrated. The in vitro (1)H NMR technique is a powerful tool for monitoring changes in intracellular metabolites of human normal and neoplastic cerebral and renal tissues. Healthy and tumoral tissues of different histologic types have been fully characterized from a biochemical standpoint. Molecular characterization is performed on both the aqueous and lipid extracts of surgically removed tissue biopsies yielding a full picture of tissue biochemistry. These analyses have disclosed markers of healthy brain and kidney and of their respective neoplastic lesions. Moreover, some biochemical features can differentiate neoplasms within the same histological type. In particular, lipidic components, like cholesteryl esters (namely oleate), detected in highest grade tumors, warrant further investigation. A better understanding of the biochemistry of diseased human tissues could open the way to new diagnostic and treatment strategies.     

Quantification of amounts and 13C content of metabolites in brain tissue using high‐ resolution magic angle spinning 13C NMR spectroscopy

Metabolic pathway mapping using ¹³C NMR spectroscopy has been used extensively to study interactions between neurons and glia in the brain. Established extraction procedures of brain tissue are time consuming and may result in degradation of labile substances. We examined the potential of mapping ¹³C‐enriched compounds in intact brain tissue using high‐resolution magic angle spinning (HR‐MAS) NMR spectroscopy. Sprague–Dawley rats received an intraperitoneal injection of [1,6‐¹³C]glucose, and 15 min later the animals were subjected to microwave fixation of the brain. Quantification of concentration and ¹³C labelling of metabolites in intact rat thalamus were carried out based on exogenous ethylene glycol concentrations measured from ¹H NMR spectra using an ERETIC (Electronic REference To access In vivo Concentrations) signal. The results from intact tissue were compared with those from perchloric acid‐extracted brain tissue. Amounts of ¹³C labelling at different positions (C2, C3 and C4) in glutamate, glutamine, γ‐aminobutyric acid and aspartate measured in either intact tissue or perchloric acid extracts were not significantly different. Proton NMR spectra were used for quantification of six different amino acids plus lactate, inositol, N‐acetylaspartate, creatine and phosphocreatine. Again, results were very similar when comparing the methods. To our knowledge, this is the first time quantitative ¹³C NMR spectroscopy measurements have been carried out on intact brain tissue ex vivo using the HR‐MAS technique. The results show that HR‐MAS ¹³C NMR spectroscopy in combination with ¹H NMR spectroscopy and the ERETIC method is useful for metabolic studies of intact brain tissue ex vivo. Copyright © 2008 John Wiley & Sons, Ltd.     

Can nuclear magnetic resonance (NMR) spectroscopy reveal different metabolic signatures for lung tumours?

This study aims to evaluate the potential of ¹H NMR spectroscopy, combined with multivariate statistics, for discriminating between tumour and non-involved (control) pulmonary parenchyma and for providing biochemical information on different histological types. Paired tissue samples from 24 primary lung tumours were directly analysed by high-resolution magic angle spinning (HRMAS) ¹H NMR spectroscopy (500 MHz), and their spectral profiles subjected to principal component analysis (PCA) and partial least squares regression discriminant analysis (PLS-DA). Tumour and adjacent control parenchyma were clearly discriminated in the PLS-DA model with a high level of sensitivity (95% of tumour samples correctly classified) and 100% specificity (no false positives). The metabolites giving rise to this separation were mainly lactate, glycerophosphocholine, phosphocholine, taurine, reduced glutathione and uridine di-phosphate (elevated in tumours) and glucose, phosphoethanolamine, acetate, lysine, methionine, glycine, myo- and scyllo-inositol (reduced in tumours compared to control tissues). Furthermore, PLS-DA of a sub-set of tumour samples allowed adenocarcinomas to be discriminated from carcinoid tumours and epidermoid carcinomas, highlighting differences in metabolite levels between these histological types, and therefore revealing valuable knowledge on the biochemistry of different types of bronchial-pulmonary carcinomas.     

Metabolite identification in human liver needle biopsies by high-resolution magic angle spinning 1H NMR spectroscopy

High-resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy of intact human liver needle biopsies has not been previously reported. HR-MAS NMR spectra collected on 17 specimens with tissue amounts between approximately 0.5 and 12 mg showed very good spectral resolution and signal-to-noise ratios. One-dimensional 1H spectra revealed many intense signals corresponding to cellular metabolites. In addition, some high molecular weight metabolites, such as glycogen and mobile fatty acids, could be observed in some spectra. Resonance assignments for 22 metabolites were obtained by combining the analysis of three different types of 1D 1H spectral editing, such as T2 filtering or the nuclear Overhauser effect and 2D TOCSY and 13C-HSQC spectra. Biochemical stability of the liver tissue during up to 16 h of magic angle spinning at 277 K was studied. Biochemical trends corresponding to the different pathologies were observed, involving free fragments of lipids among other metabolites. NMR signal intensity ratios can be useful for discrimination among non-pathological, hepatitis C affected and cirrhotic liver tissues. Overall, this work demonstrates the applicability of HR-MAS NMR spectroscopy to the biochemical characterization of needle biopsies of the human liver.     

An assessment of the effects of sample ischaemia and spinning time on the metabolic profile of brain tumour biopsy specimens as determined by high-resolution magic angle spinning (1)H NMR

High-resolution magic angle spinning (HRMAS) (1)H NMR of biopsy tissue provides a biochemical profile that has potential diagnostic and prognostic value, and can aid interpretation of the lower-resolution (1)H-NMR spectra obtained in vivo. However, the biochemical profile obtained may be affected by experimental factors such as a period of ischaemia before snap-freezing of the biopsy tissue for subsequent analysis and the mechanical stress of the spinning procedure of HRMAS itself. We have used normal rat brain cortex as a 'gold standard', either funnel-frozen or deliberately allowed to become ischaemic for set periods of time before snap-freezing, to quantitatively investigate these two effects. In addition, we have compared biochemical changes that occur in normal rat brain during HRMAS (spun continuously at 5 kHz for 4 h at 4 degrees C as could be required for a two-dimensional acquisition) with those that occur in biopsy samples from low-grade and high-grade adult human astrocytomas, during the same HRMAS procedure. Significant changes due to delayed initial sample freezing were noted in metabolites associated with glycolysis (alanine, glucose and lactate), as expected. However, for the funnel-frozen rat tissue at 4 degrees C, there were even more significant changes, which appear to be the result of extended spinning at 5 kHz. In particular, the 18% total creatine increase observed is unlikely to be de novo synthesis of creatine. More likely, the asymptotic exponential increase in creatine suggests an exponential release of an NMR-invisible bound creatine store as a result of tissue damage from mechanical stress of sample spinning. Overall, it appears that tissue ischaemia during biopsy excision and delays in snap-freezing may have less significant effects on metabolite profile than the prolonged spinning times required for two-dimensional HRMAS, and this must be accounted for when results are being interpreted.     

Metabolic characterisation of plasma in juveniles with glycogen storage disease type 1a (GSD1a) by high-resolution (1)H NMR spectroscopy

This paper reports the first application of high-resolution (1)H NMR spectroscopy to the plasma of five juveniles with glycogen storage disease type 1a (GSD1a), permitting the characterisation of the plasma metabolic profile and the identification of alterations relative to a set of control samples. The relaxation-weighted spectra allowed changes in low molecular weight compounds to be detected more clearly, whereas diffusion-edited spectra were used to characterise the plasma lipoprotein profile. Low molecular weight metabolites with altered levels in most patients were lactate, ketone bodies, acetate, creatine/creatinine and glucose. One of the patients showed distinctively lower glucose levels and higher lactate and ketone body contents, suggesting poorer metabolic control of the disease compared with other patients. In addition, a metabolite tentatively identified as alpha-hydroxyisobutyrate was only detected in the spectra of GSD1a plasmas, representing, therefore, a possible novel GSD1a biomarker. Total lipoprotein contents were higher in the plasma from GSD1a patients. Furthermore, lower HDL and higher VLDL + LDL levels also characterised the plasma of these patients. Preliminary results on principal component analysis of (1)H NMR spectra allowed a clear separation between GSD1a and control plasmas. The specificity of the changes observed to GSD1a is discussed, together with the recognised potential of NMR and pattern recognition methods for aiding the diagnosis of GSD1a.     

Renal distribution and metabolism of [2H9]choline. A 2H NMR and MRI study

Trimethylamines are required as substrates in the biosynthesis of a number of important molecules in the cell. Herein, we describe the use of choline, deuterated in its 9 methyl positions, as an NMR label for following the distribution and metabolism of methyl groups after intravenous choline infusion. Deuterium (2H) NMR spectroscopy of the rabbit kidney in vivo revealed a linear uptake of infused choline that was directly proportional to the rate of infusion. The sensitivity limit for the spectroscopic studies in vivo was in the order of 100 microM for a 2 min data collection. After the infusion, 2H NMR imaging of the kidney in vivo demonstrated high trimethylamine concentrations in both the cortex and inner medulla but not in the outer medulla. The inner medullary fraction, however, was more labile to diuresis induced by furosemide. Companion high resolution 2H NMR studies of extracts revealed a cortex betaine/choline concentration ratio of 0.69 +/- 0.05 (mean +/- SEM, n = 3) before furosemide administration. Following furosemide infusion, the cortex betaine/choline concentration ratio was 3 +/- 1 (n = 6). Thus, 2H renal images following furosemide treatment can be interpreted as metabolic maps of betaine distribution. In addition, extraction studies revealed high concentrations of labelled choline and betaine in the liver. These data demonstrate that 2H-labelled choline is an effective marker of choline methyl metabolism in vivo and should provide a unique tool for the investigation of this important substrate.