N‐acetyl resonances in in vivo and in vitro NMR spectroscopy of cystic ovarian tumors

An unassigned and prominent resonance in the region from δ 2.0–2.1 ppm has frequently been found in the in vivo MR spectra of cancer patients. We demonstrated the presence of this resonance with in vivo MRS in the cyst fluid of a patient with an ovarian tumor. ¹H‐NMRS on the aspirated cyst fluid of this patient confirmed the observation. A complex of resonances was observed between 2.0 and 2.1 ppm. It was also present in 11 additional ovarian cyst fluid samples randomly chosen from our biobank. The resonance complex was significantly more prominent in samples from mucinous tumors than in samples from other histological subtypes. A macromolecule (>10 kDa) was found responsible for this complex of resonances. A correlation spectroscopy (COSY) experiment revealed cross peaks of two different types of bound sialic acid suggesting that N‐glycans from glycoproteins and/or glycolipids cause this resonance complex. In the literature, plasma α‐1 acid glycoprotein (AGP), known for its high content of N‐linked glycans, has been suggested to contribute to the δ 2.0–2.1 spectral region. The AGP cyst fluid concentration did not correlate significantly with the peak height of the δ 2.0–2.1 resonance complex in our study. AGP may be partly responsible for the resonance complex but other N‐acetylated glycoproteins and/or glycolipids also contribute. After deproteinization of the cyst fluid, N‐acetyl‐L ‐aspartic acid (NAA) was found to contribute significantly to the signal in this spectral region in three of the 12 samples. GC‐MS independently confirmed the presence of NAA in high concentration in the three samples, which all derived from benign serous tumors. We conclude that both NAA and N‐acetyl groups from glycoproteins and/or glycolipids may contribute to the δ 2.0–2.1 ppm resonance complex in ovarian cyst fluid. This spectral region seems to contain resonances from biomarkers that provide relevant clinical information on the type of ovarian tumor. Copyright © 2009 John Wiley & Sons, Ltd.     

Rat strain-dependent variations in brain metabolites detected by in vivo (1) H NMR spectroscopy at 16.4T

Localized in vivo (1) H NMR spectroscopy is playing an increasing role in preclinical studies, because of its ability to quantify the concentrations of up to 20 metabolites in rat brain. To assess the differences between often-used rat strains, the neurochemical profiles of Sprague-Dawley, Wistar and Fischer rats were determined at ultrashort TE at 16.4 T. To ascertain high-qualitative quantification, a first experiment examined the dependence of the measuring time on the quantification results and precision by precisely the number of averages between 16 and 320. It was shown that most metabolites can be quantified accurately within a short scan time, yielding Cramér-Rao lower bounds below 20% and stable concentrations for 16 metabolites with as few as 32 or 64 averages in the thalamus and hippocampus, respectively. Interstrain differences in metabolite concentrations were shown to be moderate, with taurine varying significantly between Sprague-Dawley and Wistar rats, and slightly more distinct differences from Fischer rats, including variations in glutamate and myo-inositol. The high spectral quality and quantification precision of all data again demonstrated the potential of in vivo (1)H NMR spectroscopy at ultrahigh field.     

Dimethyl sulfone in human cerebrospinal fluid and blood plasma confirmed by one-dimensional (1)H and two-dimensional (1)H-(13)C NMR

(1)H-NMR spectroscopy at 500 MHz was used to confirm that a previously unidentified singlet resonance at 3.14 ppm in the spectra of cerebrospinal fluid and plasma samples corresponds to dimethyl sulfone (DMSO(2)). A triple resonance inverse cryogenic NMR probe, with pre-amplifier and the RF-coils cooled to low temperature, was used to obtain an (1)H-(13)C HSQC spectrum of CSF containing 8 microM (753 ng/ml) DMSO(2). The (1)H-(13)C correlation signal for DMSO(2) was assigned by comparison with the spectrum from an authentic reference sample. In plasma and CSF from healthy controls, the concentration of DMSO(2) ranged between 0 and 25 micromol/l. The concentration of DMSO(2) in plasma from three of four patients with severe methionine adenosyltransferase I/III (MAT I/III) deficiency was about twice the maximum observed for controls. Thus, DMSO(2) occurs as a regular metabolite at low micromolar concentrations in cerebrospinal fluid and plasma. It derives from dietary sources, from intestinal bacterial metabolism and from human endogenous methanethiol metabolism.     

Lipid profiling of cancerous and benign gallbladder tissues by (1) H NMR spectroscopy

Qualitative and quantitative ¹H NMR analysis of lipid extracts of gallbladder tissue in chronic cholecystitis (CC, benign) (n = 14), xanthogranulomatous cholecystitis (XGC, intermediate) (n = 9) and gallbladder cancer (GBC, malignant) (n = 8) was carried out to understand the mechanisms involved in the transformation of benign gallbladder tissue to intermediate and malignant tissue. The results revealed alterations in various tissue lipid components in gallbladder in CC, XGC and GBC. The difference in the nature of lipid components in benign and malignant disease may aid in the identification of the biological pathways involved in the etiopathogenesis of GBC. This is the first study on lipid profiling of gallbladder tissue by ¹H NMR spectroscopy, and has possible implications for the development of future diagnostic approaches. Copyright © 2010 John Wiley & Sons, Ltd.     

Novel corrective equations for complete estimation of human tissue lipids after their partial destruction by perchloric acid pre-treatment: high-resolution (1)H-NMR-based study

Owing to the small quantity of tissue available in human biopsy specimens, aqueous and lipid components often have to be determined in the same tissue sample. Perchloric acid (PCA) used for the extraction of aqueous metabolites has a deleterious effect on lipid components; the severity of the damage is not known. In this study, human muscle tissue was first treated with PCA to extract aqueous metabolites, and the residue was then used for lipid extraction by conventional methods, i.e. the methods of Folch and Bligh & Dyer and a standardised one using methanol/chloroform (1:3, v/v) used in our laboratory. A (1)H-NMR spectrum was obtained for each lipid extract. Lipid was quantified by measuring the integral area of N(+)-(CH(3))(3) signals of phospholipids (PLs). Triacylglycerol (TG) and cholesterol (CHOL) were quantified using the -CH(2)- signals of glycerol and the C18 methyl signal, respectively. This study shows that prior use of PCA caused marked attenuation of TG, PL, and CHOL. This was confirmed by recovery experiments and observation of the direct effect of PCA on the standard lipid components. On the basis of the quantity of lipid lost in each case, three novel equations (with respect to TG, PL, and CHOL) were derived. Application of these equations to lipid quantities estimated in different pathological tissues after PCA pre-treatment produced values equivalent to those estimated without PCA use. This study conclusively shows that PCA pre-treatment damages all three lipid moieties, TG, PL, and CHOL. When PCA is used in a fixed ratio to the tissue, the lipid damage is also proportional and correctable by statistically derived equations. These equations will be useful in human biopsy specimens where aqueous and lipid components have to be studied using the same tissue sample because of the small quantity available.     

1H and 13C NMR Study of the Intermediates Formed by (Cp‐R)2ZrCl2 Activation with MAO and AlMe3/[CPh3][B(C6F5)4]. Correlation of Spectroscopic and Ethene Polymerization Data

Using ¹H and ¹³C NMR spectroscopy, cationic intermediates formed by activation of (Cp‐R)₂ZrCl₂ (R = nBu, tBu and 1,2,3‐Me₃) with MAO in toluene were monitored at Al/Zr ratios from 50 to 1 000. The catalysts (Cp‐R)₂ZrCl₂/AlMe₃/CPh₃⁺B(C₆F₅)₄^? (nBu, tBu and 1,2,3‐Me₃) were also studied for comparison of spectroscopic and polymerization data with MAO based systems. Complexes of type (Cp‐R)₂ZrMe⁺ ← Me^?‐Al?MAO ( IV ) with different Me‐MAO^? counter anions have been identified in the (Cp‐R)₂ZrCl₂/MAO systems at low Al/Zr ratios. At Al/Zr ratios of 200–1 000, the complex [(Cp‐R)₂Zr(μ‐Me)₂AlMe₂]⁺ Me‐MAO^? ( III ) dominates in all MAO‐based reaction systems. Ethene polymerization activity strongly depends on the Al/Zr ratio (Al/Zr = 200–1 000) for the systems (Cp‐nBu)₂ZrCl₂/MAO and (Cp‐tBu)₂ZrCl₂/MAO, while it is virtually constant in the same range of Al/Zr ratios for the catalytic system (Cp‐1,2,3‐Me₃)₂ZrCl₂/MAO. The data obtained are interpreted on the assumption that complex III is the actual precursor of active centers of polymerization in MAO based systems.

Formation of cationic intermediates by activation with MAO.