Quantification of choline‐ and ethanolamine‐containing metabolites in human prostate tissues using ¹H HR‐MAS total correlation spectroscopy

Abstract

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...

31P MR spectroscopic imaging of the human prostate at 7 T: T1relaxation times, Nuclear Overhauser Effect, and spectral characterization

“…This contrasts with the 1 H HR-MAS spectra, which showed clear signal of the phosphodiesters, whereas PC was nearly undetectable. The ratio between PE and PC observed in vivo differed from in vitro observations, in which PE was more abundant than PC, especially in the 1 H HR-MAS spectra (%6Â in 31 P highresolution NMR spectra; 30-250Â in 1 H HR-MAS spectra) (7,(41)(42)(43). The differences between in vivo and in vitro results suggest metabolite content changes during extraction and in vitro measurements.…”

“…Earlier 31 P work of the human prostate with an endorectal coil at 2 T, without any localization apart from the coil reception profile, showed separate resonances for phosphocreatine (PCr), adenosine triphosphate (ATP), and inorganic phosphate (Pi), but could not separate resonances related to phospholipid metabolism: phosphoethanolamine (PE), phosphocholine (PC), glycerophosphoethanolamine (GPE), and glycerophosphocholine (GPC) (4,5). These individual metabolites are of particular interest as their mutual ratios and levels have repeatedly been linked to malignancy in cell studies and tissue samples (6)(7)(8) and also recently in vivo in the human brain (9). 1 H-decoupling can improve the sensitivity of in vivo 31 P MR spectroscopy at clinical field strengths (10,11).…”

Preclinical Evaluation of 11C-Sarcosine as a Substrate of Proton-Coupled Amino Acid Transporters and First Human Application in Prostate Cancer

“…The prostate cancer metabolome is characterized by an increased amino acid metabolism and a perturbation of nitrogen breakdown pathways (1), along with high total choline-containing compounds and phosphocholine levels (2). With metabolomic profiling, tissue levels of sarcosine-an N-methyl derivative of the amino acid glycine and a metabolic product of choline ( Fig.…”

GDPD5, a choline-generating enzyme and its novel role in tumor cell migration

“…Importantly, the authors describe for 1 3 2015; Petry et al 2010), activation of inflammatory pathways (Mattsson et al 2015), mutations (Suda and Mitsudomi 2015), altered miRNAs (Yanokura et al 2015), as well as loss of control in signaling and cell proliferation (Kim and Choi 2015;Cadenas et al 2014;Marchan 2014a, b). Although evidence for an association between choline metabolism and tumor progression has been published (e.g., Ackerstaff et al 2001;Franks et al 2002;Swanson et al 2008), the situation is far from being understood. Studying only individual metabolites or enzymes in relation to clinical parameters may also lead to conflicting results, because the activity of an entire pathway may be more relevant and should be taken into account.…”