Acetate (Ace), an astrocyte-specific substrate, is proposed as an attractive alternative to glucose for infusion studies of glial metabolism ¹. Metabolic modeling of metabolite ¹³C turnover curves in brain with ¹³C-labeled acetate infused as tracer substrate requires prior knowledge of the transport and uptake kinetics of Ace. The aim of this study was to determine the kinetics of transport and utilization for acetate uptake in the rat brain using specific distribution volume (V_d) of Ace in the rat brain. The effect of distribution volume considered for Ace in the rat brain on estimated cerebral metabolic rate of acetate (CMR_ace) were investigated in details.The experimental procedures involved four male adult Sprague–Dawley rats(200-225g). Briefly, two femoral arteries were cannulated for animal physiology monitoring and periodic plasma sampling (every 10min) and two femoral veins for intravenous infusion of α-chloralose for anesthesia and Ace. The Ace infusion protocol was optimized to reach a steady-state concentration of Ace in the brain in less than 20 min (Fig 1). All experiments were performed on a 14.1T/26cm horizontal magnet with a 12-cm gradient coil insert (400mT/m,120µs). A home-built quadrature surface coil with two physically decoupled 12mm-diameter loops was used. Brain ¹H-MRS data were acquired using STEAM sequence with optimized parameters(TM=49ms,TR=4s and TE=50ms) to minimize GABA resonance overlapping with Ace (@1.89 ppm)². Spectral analysis was done using LCModel(Stephen Provencher Inc., Oakville, ON, Canada) for estimating brain acetate concentrations. Plasma acetate concentration was determined using high-resolution ¹H NMR (9.4 Tesla, Avance-400 spectrometer) based on diffusion NMR method to suppress macromolecules background signals³. The diffusion characteristic of Ace in rat brain has been determined in the same group of Ace infused rats in recent diffusion spectroscopy study⁴, where the distribution volume of Ace in rat brain has been experimentally estimated to 0.48 ml/g. Kinetic parameters of the reversible non-steady-state Michaelis-Menten model¹: Michaelis-Menten constant transport rate of acetate through the blood brain barrier (K^t_M); Maximal transport rate of acetate (V^t_max); the apparent Michaelis-Menten constant and the maximal rate (K^u_M and V^u_max) for acetate transport through the mitochondrial inner membrane and acetyl-CoA synthetase from Ace were estimated simultaneously by fitting the apparent concentration time course of brain Ace [S_brain] and plasma Ace [S_plasma] (Fig 2). To investigate the effect of V_d allocated for Ace in the rat brain, the estimated kinetic parameters were first evaluated with V_d = 0.48⁴ and in a second step by assuming V_d as 0.77ml/g¹.Plasma and brain Ace concentrations increased rapidly after Ace infusion, however brain Ace concentration reached steady state with some delay compared to plasma Ace (Fig 1). V^t_max was found higher than V^u_max (Table1) which is in line with previously reported values ¹. Increasing CMR_ace following Ace infusion illustrates the fact that acetate utilization is not saturated under normal physiological conditions. Considering specific V_d for Ace as 0.48 ml/g, the estimated CMR_ace is in better agreement with reported value for glial TCA cycle rate in case of glucose and Ace infusions^{5, 6}. Increasing the V_d to the value of water and glucose in the rat brain as previously applied¹(0.77ml/g) resulted in a doubled CMR_ace value, while the same saturation behavior as in case of 0.48 ml/g (Fig 3) was observed.

The localized ¹H NMR spectroscopy acquired with STEAM sequence at ultra-high magnetic field (14.1T) allowed to investigate non-invasively the kinetic parameters of Ace transport and utilization in the rat brain. The CMR_ace with Ace V_d of 0.48ml/g estimated from specific diffusion characteristics of Ace in rat brain⁴ was in better agreement with reported glial TCA cycle rates ^{5, 6}. In addition it is in consistency with the fact that most acetyl-CoA entering the glial TCA cycle is synthesized from Ace transported to the rat brain. The dependency of estimated CMRace to distribution volume of Ace in the rat brain in the present study highlights the importance about a refined determination of V_d for Ace in brain metabolic studies.