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.

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.

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.