Determination of Absolute Concentrations of Metabolites from NMR Spectra

Abstract

Although a great deal of useful information is obtained from relative concentrations, absolute concentrations can further enhance the usefulness of in vivo NMR spectroscopy (MRS). Because of the non-invasive nature of MRS, clinical measurements of absolute concentrations can be made (and repeated if required) on tissues such as neonatal brain that are normally inaccessible to biopsy. Furthermore, because observations can be made on tissue in situ, metabolite concentrations can be obtained without the risk of the freezing artefacts that can affect biopsy results. With the absence of reliable direct methods of absolute quantitation, early attempts to estimate metabolite concentrations in human tissue by MRS (mainly in muscle [1–5] and brain [6–10]) were indirect and depended on knowledge of either the total mobile phosphate (Ptot) concentration or the concentration of a particular, normally stable metabolite (e.g. adenosine triphosphate (ATP)). For instance, in studies of normal, resting, human muscle [1, 3] phosphocreatine (PCr) and inorganic orthophosphate (Pi) concentrations have been estimated from relative peak areas assuming either [ATP] = 5.5 mmol kg−1 wet wt or [Ptot] = 49.5 mmol kg−1 wet wt (see Table 1). In calculations based on [ATP], absolute concentrations were derived using the β-ATP resonance because, unlike the γ and α resonances, the β peak does not have contributions from other underlying resonances. (The γ and α peaks include contributions from adenosine diphosphate (ADP) and the α peak also includes contributions from nicotinamide dinucleotides (NAD + NADH).) In the calculations based on [Ptot] the following was assumed: $$\left[ {P_{tot} } \right] = \left[ {PCr} \right] + \left[ {Pi} \right] + 3 \times \left[ {ATP} \right] + 2 \times \left[ {NAD + NADH} \right]$$ (1) These two approaches gave very similar results and favourable comparison with biopsy data. In fact, due to the inadequate cessation of metabolic processes prior to and during the freezing of biopsy samples, it appears that MRS has the potential for providing better measurements of [Pi] and [PCr] than biopsy. However, in many important situations these simple approaches are inadequate because of the invalidity of the assumption of stability for the concentration reference and therefore alternative methods are required.