It has been demonstrated 2-oxoglutarate (2OG) a cerebral TCA cycle intermediate can be observed using hyperpolarized ¹³C labelled acetate (Ace) [1]. This observation provided a more direct analysis of glial TCA cycle activity than thermally polarized experiments reporting oxidative pathway activity trough glutamate measurements. The aim of this study is to examine saturation substrate dose conditions, calculate the production rate of 2OG from plasma acetate and measure it in a partially inhibited TCA cycle model. Beads of 3M [1-¹³C]Ace dissolved in H₂O and glycerol with 58mM TEMPOL radical were hyperpolarized using a custom-designed DNP polarizer operating at 7 T/1±0.05 K for 180 min [2]. Following solid-state polarization, samples were rapidly dissolved in 5-6 ml of super-heated D₂O (170°C). A variable substrate volume was injected into the femoral vein of male Sprague-Dawley rats (n=14, 250-275g, fasted 12h) 3 s after dissolution using an automated protocol [3], leading to several blood Ace concentrations. Animals were anesthetized using a gas containing 1.5% isoflurane and their physiology was monitored. Light TCA cycle inhibition was induced in 4 additional rats by administrating one hour before dissolution, by the aconitase inhibitor fluoroacete (9µg/kg, i.v) [4]. MR measurements were carried out on a 9.4 T/ 31 cm actively shielded animal scanner (Varian/Magnex) using a home-built single loop ¹³C/¹H quadrature surface coils. Field inhomogeneity was corrected using the FASTMAP protocol. Neurochemical profile was tracked before and after Ace injection, using SPECIAL [5] sequence (TR/TE = 4000/2.8ms in 20 blocks of 16 scans). ¹³C MRS spectra were acquired every 1.5 s using the SIRENE scheme [6] starting 5 s after the beginning of the [1-¹³C]Ace injection. Ace concentrations were confirmed by high resolution NMR.

After Ace injection, no significant changes were observed in the physiology and the neurochemical profile. For all substrate concentrations, hyperpolarized [1-¹³C]Ace (182.2ppm) and [5-¹³C]2-OG (182.05ppm) were detected. (Fig1) The conversion rate (K_OG) between Ace and 2OG were calculated as following $$$K_{OG}=\frac{1}{T_{1,OG}}\frac{\int AUC_{OG}}{\int AUC_{Ace}}$$$ [7] and were found dependent on the substrate dose. (Fig2) We estimate 2OG production rate (v_OG) from Ace plasma by multiplying K_OG by the dose. We observed an asymptotic behavior at high doses supporting the assignment to a saturation state. (Fig3) When partially inhibition TCA cycle, a reduction in the production rate of 2OG of a quarter was observed for Ace dose of 8.3±1.8mM. (Fig4) Time course of Ace and OG in both intact and inhibited TCA cycle shown a delay of 1.5s between their maximum signals. Ace and OG maximum signals were delayed of 4.5 seconds compare to wild animal group (Fig5).

The different kinetics of the resonance observed at 182.05 ppm as compared to Ace and its dependence on substrate concentration supports its assignment to the downstream metabolite 2OG. Short delay between maximal signal of Ace and 2OG reports a fast turnover of 2OG ¹³C enrichment explained by its small pool size. Below 4mM, conversion rate distribution is scattered. In contrast, K_OG values followed closely the decay trend for higher substrate doses reporting a probable saturation of the system. It may result from the normalization by Ace and its cerebral accumulation. We conclude then to a saturation state for plasma concentrations upper to 4mM that is consistent with v_OG asymptotic behavior and coherent with previous studies [8]. v_OG reduction due to oxidative pathway impair matches with percentage of inhibition that we expected [9]. Because of unknown cerebral Ace concentration, production rate of 2OG is calculated directly from plasma Ace and englobes acetate transport rate, its consumption metabolic rate (CMR_Ace) and TCA cycle activity. Therefore, v_OG sensitivity to a general reduction of oxidative pathway activity reflects cerebral TCA activity but also cardiac inhibition changing the maximal signals timing and the potential Ace flow of signal in and out of the volume of interest. Fitting metabolites kinetics to a more detailed mathematical model and measuring the arterial input functions could help to dissociate both phenomena. We conclude that 2-oxoglutarate production rate can be calculated as a function of varying substrate concentrations and is affected by TCA cycle activity modulations.