The metabolism of glycine into glutathione was monitored noninvasively in vivo in intact rat mammary adenocarcinomas (R3230Ac) by MRI and MRS. Metabolism was tracked by following the isotope label from intravenously infused [2‐13C]‐glycine into the glycinyl residue of glutathione. Signals from [2‐13C]‐glycine and γ‐glutamylcysteinyl‐[2‐13C]‐glycine (13C‐glutathione) were detected by nonlocalized 13C spectroscopy, as these resonances are distinct from background signals. In addition, using spectroscopic imaging methods, heterogeneity in the in vivo tumor distribution of glutathione was observed. In vivo spectroscopy also detected isotope incorporation from [2‐13C]‐glycine into both the 2‐ and 3‐carbons of serine. Analyses of tumor tissue extracts showed single‐ and multiple‐label incorporation from [2‐13C]‐glycine into serine from metabolism through the serine hydroxymethyltransferase and glycine cleavage system pathways. Mass spectrometric analysis of extracts also showed that isotope‐labeled serine is further metabolized via the trans‐sulfuration pathway, as 13C isotope labels appear in both the glycinyl and cysteinyl residues of glutathione. Our studies demonstrate the use of MRI and MRS for the monitoring of tumor metabolic processes central to oxidative stress defense. Copyright © 2011 John Wiley & Sons, Ltd.

Introduction Glutathione (GSH) is a tripeptide found in almost all tissues, where it acts as an antioxidant and forms part of the cellular defences against oxidative stress. Cancerous cells exhibit highly reduced intracellular environments, characterized by high levels of reduced GSH compared to its oxidized disulfide counterpart (1). Higher levels of GSH and its associated enzymes appear to play a significant role in therapy-resistance (2) and reduced overall survival (3). We’ve previously used C MRSI to image GSH distribution in rat fibrosarcoma tumours, using C-glycine infusion to introduce a C label into GSH and thus provide a biomarker synthesis rate and concentration. The fibrosarcoma studies demonstrated high GSH levels (>2 μmol/gram-tissue). We have extended our studies to the noninvasive detection of GSH in a rat mammary R3230Ac adenocarcinoma, allowing us to test our ability to detect and image GSH distribution in tumours with lower average levels of GSH, and also to probe the metabolic fate of C-labelled glycine via serine hydroxymethyltransferase and other pathways.

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5- 13 C]-thiazolidine-4-carboxylate, 13 C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by 13 C magnetic resonance spectroscopy. Although uptake and clearance of 13 C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of 13 C-OTZ over 20 h, however, resulted in 13 C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.