A method was developed to quantify prostate metabolite concentrations using (1)H high-resolution magic angle spinning (HR-MAS) spectroscopy. T(1) and T(2) relaxation times (in milliseconds) were determined for the major prostate metabolites and an internal TSP standard, and used to optimize the acquisition and repetition times (TRs) at 11.7 T. At 1 degrees C, polyamines (PAs; T(1mean) = 100 +/- 13, T(2mean) = 30.8 +/- 7.4) and citrate (Cit; T(1mean) = 237 +/- 39, T(2mean) = 68.1 +/- 8.2) demonstrated the shortest relaxation times, while taurine (Tau; T(1mean) = 636 +/- 78, T(2mean) = 331 +/- 71) and choline (Cho; T(1mean) = 608 +/- 60, T(2mean) = 393 +/- 81) demonstrated the longest relaxation times. Millimolal metabolite concentrations were calculated for 60 postsurgical tissues using metabolite and TSP peak areas, and the mass of tissue and TSP. Phosphocholine plus glycerophosphocholine (PC+GPC), total choline (tCho), lactate (Lac), and alanine (Ala) concentrations were higher in prostate cancer ([PC+GPC](mean) = 9.34 +/- 6.43, [tCho](mean) = 13.8 +/- 7.4, [Lac](mean) = 69.8 +/- 27.1, [Ala](mean) = 12.6 +/- 6.8) than in healthy glandular ([PC+GPC](mean) = 3.55 +/- 1.53, P < 0.01; [tCho](mean) = 7.06 +/- 2.36, P < 0.01; [Lac](mean) = 46.5 +/- 17.4, P < 0.01; [Ala](mean) = 8.63 +/- 4.91, P = 0.051) and healthy stromal tissues ([PC+GPC](mean) = 4.34 +/- 2.46, P < 0.01; [tCho](mean) = 7.04 +/- 3.10, P < 0.01; [Lac](mean) = 45.1 +/- 18.6, P < 0.01; [Ala](mean) = 6.80 +/- 2.95, P < 0.01), while Cit and PA concentrations were significantly higher in healthy glandular tissues ([Cit](mean) = 43.1 +/- 21.2, [PAs](mean) = 18.5 +/- 15.6) than in healthy stromal ([Cit](mean) = 16.1 +/- 5.6, P < 0.01; [PAs](mean) = 3.15 +/- 1.81, P < 0.01) and prostate cancer tissues ([Cit](mean) = 19.6 +/- 12.7, P < 0.01; [PAs](mean) = 5.28 +/- 5.44, P < 0.01). Serial spectra acquired over 12 hr indicated that the degradation of Cho-containing metabolites was minimized by acquiring HR-MAS data at 1 degree C compared to 20 degrees C.

A fast and quantitative 2D high-resolution magic angle spinning (HR-MAS) total correlation spectroscopy (TOCSY) experiment was developed to resolve and quantify the choline-and ethanolamine-containing metabolites in human prostate tissues in Ϸ1 hr prior to pathologic analysis. At a 40-ms mixing time, magnetization transfer efficiency constants were empirically determined in solution and used to calculate metabolite concentrations in tissue. Phosphocholine (PC) was observed in 11/15 (73%) cancer tissues but only 6/32 (19%) benign tissues. The choline-(Cho) and ethanolamine (Eth)-containing metabolites are major cytosolic precursors and degradation products of phospholipid membrane assembly and catabolism (1). There is currently much interest in correlating the individual Cho-and Eth-containing metabolites with cancer presence, aggressiveness, and therapeutic response because of the information they contain about cellular proliferation, apoptosis, and the activity of key enzymes (2-7). Historically, 31 P spectroscopy has been used both in vitro (8 -12) and in vivo (13-15) to resolve the phosphomonoesters (phosphocholine [PC] and phosphoethanolamine [PE]) and phosphodiesters (glycerophosphocholine [GPC] and glycerophosphoethanolamine [GPE]). 1 H spectroscopy has mainly focused on detection of the choline head group singlets of free Cho, PC, and GPC (16 -18), which give rise to a composite choline peak at Ϸ3.2 ppm in vivo (19). Although Cho, PC, and GPC can be resolved from each other in extracts, they cannot typically be resolved from each other in intact prostate tissue at 11.7T, and free ethanolamine (Eth), PE, GPE, glucose, taurine, and myo-inositol (mI) also co-resonate in this highly overlapping region (17).Total correlation spectroscopy (TOCSY) (20) is a powerful 2D NMR experiment which has previously been used in HR-MAS studies of cultured melanoma cells (21) and intact prostate (22), liver (23,24), brain (25), and kidney (26) tissues. In TOCSY, an isotropic mixing pulse is used to transfer magnetization between protons within a spin system such that, depending on the duration of the mixing time, correlations can be observed between protons that are several bonds apart. One advantage of TOCSY over conventional correlation spectroscopy (COSY) is that the cross-peak multiplets have an absorption phase rather than dispersive phase character. Consequently, TOCSY can be used quantitatively provided the magnetization transfer (MT) efficiency is known for the specific metabolites under investigation at the mixing time being used (27). Recent studies have demonstrated that TOCSY can be used to resolve and quantify the sidechain CH 2 -CH 2 crosspeaks of several including PC, GPC, Eth, PE, and GPE (21,22). For the Cho and Eth metabolites, the sidechain methylene protons give rise to a single cross-peak on each side of the diagonal, which simplifies their quantification compared to more complicated spin systems (e.g., myo-inositol).Previous studies have also demonstrated that rotor synchronization, in which the dur...

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The goal of this study was to determine the ability of highresolution magic angle spinning (HR-MAS) NMR spectroscopy to distinguish different stages of intervertebral disc degeneration (IVDD). 17 discs were removed from human cadavers and analyzed them using 1D and 2D (total correlation spectroscopy (TOCSY)) 1 H HR-MAS spectroscopy, and T 1 and T 2 relaxation time measurements to determine the chemical composition and changes in chemical environment of discs with increasing levels of degeneration (Thompson grade). Among the significant findings were that spectra were very similar for samples taken from annular and nuclear regions of discs, and that visually apparent changes were observed in the spectra of the annular and nuclear samples from discs with increasing Intervertebral disc degeneration (IVDD) is a leading cause of lumbar spine-related lower-back pain, a common medical problem that affects 60 -80% of aging Americans (1). The intervertebral disc is a flexible fibrocartilaginous structure that supports forces and facilitates spinal movement. Healthy discs consist of three specific tissue components: 1) the annulus fibrosus, a collagenous region tightly packed circumferentially around the periphery of the disc, which allows for pliability; 2) the nucleus pulposus, a hydrated proteoglycan gel located at the center of the disc, which when compressed expands radially and braces the annulus fibrosus to maintain stiffness and prevents the annulus from buckling under compression; and 3) a cartilaginous end-plate that separates the nucleus from the adjacent vertebral bone (2).Disc degeneration is characterized by a complex series of physical and chemical degradative processes. The extent or severity of IVDD is most commonly described clinically by means of the Thompson grading scale (3): following a set of parameters, a x-ray radiographic inspection of the disc is conducted and the gross morphology is used to determine the extent of degeneration. Iatridis and coworkers (4) concluded that changes in the mechanical properties of the intervertebral disc suggest a shift from a "fluidlike" behavior to a more "solid-like" behavior with degeneration. Fixed charge density (FCD) and the biochemical environment of the surrounding water have also been shown to greatly influence degeneration. As highly charged proteoglycans attract water and cause the tissue to swell, disc pressurization and spinal load support are directly affected (5-7). Differences in the Thompson grade are reflected by changes in the concentrations of constituents such as collagen and proteoglycans in both the annulus and nucleus (8 -11). It has been proposed that biochemical degradation, upregulation of genes associated with collagen matrix degradation, and the cumulative effect of mechanical loading all stimulate the degenerative disc process.Several in vivo MRI studies have been performed in an attempt to better characterize IVDD. T 1 -and T 2 -weighted MRI has been used to analyze the structure of intervertebral discs (11-18). Pfirrmann and coworkers...

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