Applications of PET imaging with the proliferation marker [18F]-FLT.

[18F]-3'-fluoro-3'-deoxythymidine (FLT) is a nucleoside-analog imaging agent for quantifying cellular proliferation that was first reported in 1998. It accumulates during the S-phase of the cell cycle through the action of cytosolic thymidine kinase, TK1. Since TK1 is primarily expressed in dividing cells, FLT uptake is essentially limited to dividing cells. Thus FLT is an effective measure of cell proliferation. FLT uptake has been shown to correlate with the more classic proliferation marker, the monoclonal antibody to Ki-67. Increased cellular proliferation is known to correlate with worse outcome in many cancers. However, the Ki-67 binding assay is performed on a sampled preparation, ex vivo, whereas FLT can be quantitatively measured in vivo using positron emission tomography (PET). FLT is an effective and quantitative marker of cell proliferation, and therefore a useful prognostic predictor in the setting of neoplastic disease. This review summarizes clinical studies from 2011 forward that used FLT-PET to assess tumor response to therapy. The paper focuses on our recommendations for a standardized clinical trial protocol and components of a report so multi center studies can be effectively conducted, and different studies can be compared. For example, since FLT is glucuronidated by the liver, and the metabolite is not transported into the cell, the plasma fraction of FLT can be significantly changed by treatment with particular drugs that deplete this enzyme, including some chemotherapy agents and pain medications. Therefore, the plasma level of metabolites should be measured to assure FLT uptake kinetics can be accurately calculated. This is important because the flux constant (KFLT) is a more accurate measure of proliferation and, by inference, a better discriminator of tumor recurrence than standardized uptake value (SUVFLT). This will allow FLT imaging to be a specific and clinically relevant prognostic predictor in the treatment of neoplastic disease.

Effects of pH and molecular charge on dipolar coupling interactions of solutes in skeletal muscle observed by DQF, 1H NMR spectroscopy.

In this study we tested the effect of molecular charge and chirality as well as tissue pH on dipolar coupling interaction in skeletal muscle. These results were demonstrated by double quantum filtered, DQF, 1H NMR spectra acquired on permeable skeletal muscle samples dialyzed against buffered solutions containing three classes of solutes-electrolytes (lactate and Tris), zwitterions (alanine and glycine), and non-electrolytes (dioxane and ethanol)-as a function of pH ranging from 5.0 to 8.5. The results show that charge density on the protein filaments strongly influences dipolar coupling of solutes in muscle whereas charge on the solutes themselves has only a small effect. The frequency splitting of the dipolar coupled peaks for all the molecules tested was strongly affected by muscle pH. Higher pH increased negative charge density on the filaments and resulted in weaker dipolar coupling for anions and zwitterions but stronger coupling for the cation TRIS. Molecular charge per se or chirality did not affect the frequency splitting of the dipolar coupled peaks. The molecules, lactate, ethanol, and alanine, have scalar coupled spins and consequently a double quantum signal in solution. However, spectra acquired from these molecules in muscle showed an additional frequency splitting due to additional dipolar coupling interactions. Due to lack of scalar coupling, spectra from Tris, glycine, and dioxane showed no double quantum signal in solution but did when in muscle. All these observations can be explained by the fact that the net charge on protein filaments dominates the mechanism of dipolar coupling interactions in the highly anisotropic structures in muscle.

Multinuclear NMR studies of an actively dividing artificial tumor.

Growth of the A549 cell line in a perfusion system suitable for use in a magnetic resonance study has been characterized and shown to be stable physiologically and hence appropriate for serial observations. Several methods of monitoring cell growth were compared to assess the behavior of the cells in this system. Comparison between NMR metabolite data and cell growth via cell counting showed that 31P NMR signals accurately reported cell doubling time. In contrast to most NMR cell culture systems, viable cells can be recovered from the perfusion system after the NMR measurements for further biochemical studies. These data further suggest that this system will be useful for studying the physiology and biochemistry of exponentially growing cells for at least two days in NMR tube culture.

Double quantum filtered (1)H NMR spectroscopy enables quantification of lactate in muscle.

In this study we address the question of quantification of muscle lactate using double quantum filtered (DQF) (1)H NMR spectroscopy where dipolar and scalar coupled spectra are acquired. For this, lactate content in muscle samples was independently determined using a conventional enzymatic assay and DQF, (1)H NMR spectroscopy. NMR quantification of lactate relied on comparison of muscle spectra with similarly acquired spectra of standard lactate solutions. Transverse relaxation, T(2), and dipolar coupling effects were investigated at two different orientations of muscle fibers relative to B(o) and at various lactate concentrations. In all cases, we found a biexponential T(2) decay of the lactate methyl signal with a long T(2) of 142 ms (+/-8 ms, n=24) and a short T(2) of 37 ms (+/-6 ms, n=24). Lactate content of muscle determined by NMR spectroscopy agreed with the results obtained from enzymatic assays of the same samples provided that T(2) effects as well as the presence of both scalar and dipolar coupling interactions of lactate in muscle were taken into account.

Validation of whole-body magnetic resonance spectroscopy as a tool to assess murine body composition.

OBJECTIVE: To evaluate proton magnetic resonance spectroscopy (MRS) as a tool for the non-invasive assessment of murine body composition. DESIGN: Twenty C57/BL6 male mice with a wide range of body adiposities underwent both pre- and post-mortem whole-body MRS to assess body composition. MRS measures were compared to the results obtained by chemical carcass analysis, the current 'gold standard' for determination of body composition. MEASUREMENTS: Areas under the curve (AUC) for lipid and water peaks of whole body MRS spectra (AUClipid and AUCH2O, respectively) were used to determine percentages of body fat (%FATMRS) and fat free mass by MRS (%FFMMRS). Total body fat, total body water, fat free mass, and total lean mass were determined by chloroform/methanol extraction of lipid from dessicated whole carcass and compared to MRS measures (%FATMRS, %FFMMRS, AUClipid, and AUCH2O). The variability of the MRS technique was assessed by determining the coefficients of variation (COV) associated with %FATMRS, AUClipid, and AUCH2O for mice of three different adiposities. RESULTS: %FATMRS in live mice was highly correlated with body fat percentage (r=0.994, P<0.001) and total body fat (r=0.980, P<0.001) derived from chemical carcass analysis over a broad range of adiposities (7-48% body fat content by carcass analysis). There was no difference in %FATMRS measured pre- vs post-mortem (r=1.00, P<0.001). AUClipid was highly correlated with chemically derived total fat mass (r=0.996, P<0.001) and body fat percentage (r=0.981, P<0.001), while %FFMMRS was strongly correlated to chemical determinations of percentage body water (r=0.994, P<0. 001), percentage fat free mass (r=0.993, P<0.001), and percentage lean mass (r=0.792, P<0.001). AUCH2O was strongly associated with carcass analysis determinations of total body water (r=0.964, P<0. 001), total fat free mass (r=0.953, P<0.001), and total lean mass (r=0.89, P<0.001). In mice of 6%, 12%, and 43% body fat, COVs determined for %FATMRS and AUClipid were less than 10%. The COVs for AUCH2O were less than 2%. CONCLUSIONS: MRS provides precise, accurate, rapid, and non-invasive measures of body fat, body water, fat free mass, and lean mass in living mice with a broad range of adiposities.

Anisotropic orientation of lactate in skeletal muscle observed by dipolar coupling in (1)H NMR spectroscopy.

Double quantum (DQ), J-resolved (1)H NMR spectra from rat and bovine skeletal muscle showed a splitting frequency ( approximately 24 Hz) for the lactate methyl protons that varied with the orientation of the muscle fibers relative to the magnetic field. In contrast, spectra of lactate in solution consist of a J-coupled methyl doublet and a J-coupled methine quartet (J(HH) = 7 Hz) with no sensitivity to sample orientation. Spectra acquired in magnetic fields of 4.7, 7, and 11 T showed that the splitting was not due to inhomogeneities in magnetic susceptibility within the muscle, because the magnitude of the splitting did not scale with the strength of B(0) fields. Triple quantum coherence (TQC) spectra revealed two distinct transition frequencies on the methyl resonance. These frequencies resulted from intra-methyl and methine-methyl couplings in this four spin system (A(3)X). Decoupling experiments on the triple quantum coherence showed that the observed frequency splitting was due mainly to the dipolar interactions between the methine and methyl protons of the lactate molecule. Thus, all the proton resonances of the lactate molecules in muscle behave anisotropically in the magnetic field. Adequate design and interpretation of spectroscopic experiments to measure lactate in muscle, and possibly in any cell and organ which contain asymmetric structures, require that both the dipolar coupling described here and the well-known scalar coupling be taken into account.

Incorporation of a phosphonium analogue of choline into the rat brain as measured by magnetic resonance spectroscopy.

A clear understanding of choline metabolism is important in our goal to modify demyelination and remyelination in multiple sclerosis. To develop a technique capable of measuring metabolic changes in the brain, we have studied the incorporation of a phosphonium analogue of choline (P-choline) in tissue extracts of rats. After feeding adult rats a choline-deficient diet supplemented with P-choline, the analogue was not detectable by in vivo volume-localized 1H spectroscopy. However, in vitro 31P measurements of brain extracts revealed an 11% incorporation of P-choline into phosphatidylcholine. We report that P-choline incorporates preferentially into the lipid pool over the lipid precursor pool and we provide evidence that the choline peak resolved by in vivo 1H spectroscopy is only composed of small molecular weight choline-containing compounds.

Magnetic resonance spectroscopic measurement of cellular thiol reduction-oxidation state.

Chromatographic and magnetic resonance spectroscopic measurements of thiol reduction-oxidation state in chemically constructed samples show close analytical agreement. This result, coupled with the synthesis of new probe molecules allowing greater sensitivity and lower toxicity, supports the development of an NMR method for non-invasive thiol redox measurement, an important variable in the response of tumors to radiation and chemotherapy.

Measurement of tissue oxidation-reduction state with carbon-13 nuclear magnetic resonance spectroscopy.

The oxidation state of tissues influences their response to cancer therapy. We have devised a novel approach to the measurement of thiol redox which is based on the relative nuclear magnetic resonance signal intensity from carbon-13 adjacent to sulfur in metabolites of the redox-sensitive phosphorothioate drug, S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR3689). Incubation of WR3689 metabolites under oxidizing conditions results in quantifiable changes in the 13C nuclear magnetic resonance spectrum stoichiometrically related to the degree of oxidation in mouse liver homogenate in vitro. Drug oxidation is competitive with the oxidation of tissue-derived thiol groups under these conditions. Noninvasive measurement of redox state may assist in designing more effective strategies for altering normal and malignant tissue response to cancer therapy.

Nuclear medicine technology.

Combined liver-lung scanning has proven to be an extremely rapid and useful tool in the diagnosis and localisation of sub-phrenic abscess and differentiation of basal defects of the right lung. There is little discomfort to the patient who may be experiencing considerable chest pain. The patient spends less than 20 minutes in the nuclear medicine department. The results are quickly obtained, no complex mathematical analyses are needed, and the images are easily interpreted.