In vivo studies in Alzheimer’s Disease (AD) have shown reduced glucose uptake in the retrosplenial cortex (RSC), as measured by FDG-PET¹, and altered resting state activity in the default mode network of the RSC, mediotemporal and frontal brain regions, as measured by fMRI². Additionally, post-mortem and in vitro studies indicate alterations in membrane phospholipid metabolism³ as well as in basic energy mechanisms (e.g. creatine kinase (CK) activity⁴) in AD patients. Both phospholipid and energy metabolism can be investigated in vivo by phosphorus MR Spectrocopy (³¹P MRS). Previous ³¹P MRS studies^5,6,7 have so far provided inconclusive results, due to variation in disease stage and medication status of AD patients, and differences in investigated brain regions and control groups. In addition, these studies were often hampered by low spectral resolution and small sample sizes. To assess phospholipid and energy metabolism in multiple brain regions in mild AD patients and healthy age-matched control subjects by 3D ³¹P MRS imaging.³¹P MRSI was performed in 31 drug-naïve mild AD patients (MMSE ≥20; 73.5±6.9 [mean±SD] yrs) and 31 age- and gender-matched controls (73.5±6.3 yrs). Whole brain ³¹P-MRSI data was obtained on a Siemens Magnetom Trio 3T system with a dual-tuned ¹H/³¹P volume head coil (Rapid) and a 3D pulse-acquire sequence with the following parameter values: TR=2000 ms, TE=0.10 ms, 40° flip-angle, NA=4, WALTZ4 proton decoupling, nominal voxel size=16.25x16.25x16.25mm, TA=13:08 min. Resonances in MR spectra from four regions of interest (ROI; left and right hippocampus, HL and HR; anterior cingulate cortex, ACC; and RSC) were fitted using Metabolite Report (Siemens) (Figure 1), resulting in metabolite levels of phosphocholine (PCho),phosphoethanolamine (PEtn), glycerophosphocholine (GPCho), glycerophosphoethanolamine (GPEtn), inorganic phosphate (Pi), phosphocreatine (PCr), nicotinamide adenine dinucleotide (NAD(H)) and alpha, beta and gamma adenosine triphosphate (a/b/yATP). For all metabolites the signal percentage with respect to the total phosphorus signal was calculated. Both a quantitative evaluation of the fitting results (Cramer–Rao lower bound ≤30%) and a visual quality control (independently by two spectroscopists) were performed. Only metabolites that passed both quality checks were included in the statistical analyses. Level of phosphomonoesters (PME) was calculated as the sum of PCh and PEth, phosphodiesters (PDE) as the sum of GPCh and GPEth, and total ATP (tATP) as the sum of aATP, bATP and yATP. In addition, we calculated pH and ratios for phosphomonoesters to phosphodiesters (PME/PDE as well as PEth/GPEth and PCh/GPCh), and PCr to Pi. Group differences were analyzed using a mixed model with group (AD or control), sex, and brain region as fixed factors, considering brain region as a within subject factor, and adjusting for age. Quality control led to the rejection of 3-20% of metabolite fits for phospholipid metabolites and NAD(H). Excellent fits (<2% rejected) for all other metabolites was achieved. An interaction between group and brain region was found for PCr, showing that levels of PCr were higher in mild AD patients compared with healthy control (HC) subjects in the RSC (LS mean difference=0.86, SEM=0.29 [p=0.004]) and left (LS mean difference=1.52, SEM=0.52 [p=0.005]) and right hippocampus (LS mean difference=1.47, SEM=0.38 [p<0.001]), but not in the ACC (Figure 2A). A main effect of group was found for PCr/Pi (AD>HC; LS mean difference=0.22, SEM=0.11 [p=0.046]; Figure 2B) and pH (AD>HC; LS mean difference=0.008, SEM=0.004 [p=0.032]; Figure 2C). No effect of group was found for Pi, NAD(H), t/a/b/yATP or any of the phospholipid compounds measured. A main effect of brain region was found for PEth, PDE, GPEtn, GPCh, PME/PDE, PEth/GPEth, PCh/GPCh, PCr/Pi, Pi, t/a/b/yATP, NAD(H), and pH.To the best of our knowledge, this is the largest ³¹P MRS study performed in AD patients. By using 3D MRSI we were able to measure multiple brain regions simultaneously and provide both disease specific as well as regional information. Most phospholipid compounds and all high energy phosphates displayed regional variation. This may be caused by true intra-cellular metabolic differences between brain regions, or arise from differences in tissue composition. The differences in PCr and pH found between AD patients and healthy aged controls indicate a redistribution of metabolites involved in the CK reaction equilibrium [K = PCr∙ADP∙H+ / Cr∙ATP]. Taken together with changes in PCr/Pi, this could reflect altered energy metabolism in AD, or alternatively, be a consequence of morphological differences (altered ratio of neurons to glia cells, e.g. gliosis).