magnetic resonance imaging is proving to be a powerful tool for the evaluation of structural and functional changes [ 1 , 2 ] . additionally , magnetic resonance spectroscopy ( mrs ) can provide information on brain metabolites such as n - acetyl aspartate ( naa ) , choline , creatine , and glutamate . since metabolites are related to cellular function , it is understandable that brain metabolites have been studied for their associations with cognitive function . although brain metabolite levels have been measured in modest groups of young healthy subjects , there is relatively little literature concerning older adults . as early as 1993 , christiansen et al . measured metabolite levels and relaxation times in healthy young adults ( n  =  8 ; age 2030 ) compared with healthy elderly adults ( n  =  8 ; age 6080 ) . they found a 15  % decline in occipital naa in the older group compared with the younger group . in a recent meta - analysis of the available literature , haga et al .   reported reduced naa and raised choline and creatine in healthy older subjects ( > 60  years ) relative to healthy younger subjects ( < 60  years ) . previously , we used a single - voxel mrs technique and found an association between raised creatine and a decline in cognitive ability . reported a decreased naa / creatine ratio in people with mild cognitive impairment and alzheimer 's disease relative to cognitively normal , age - matched controls . magnetic resonance spectroscopic imaging ( mrsi ) enables measurement of multiple voxels in a slice across the brain and thereby opens up the possibility of producing maps of metabolite distribution . the objective of the current study was to establish brain metabolite levels in healthy older adults within a narrow age range . forty participants ( 25 female ) were recruited from healthy , community - living people comprising the lothian birth cohort 1936 ( lbc 1936 [ 7 , 8 ] ) . all participants were aged 73 or 74 at the time of mri scanning and formed a sub group of approximately 650 volunteers who were part of a longitudinal study on cognitive ageing   and for whom recent detailed structural imaging   and cognitive scores were available . an experienced observer determined brain atrophy , iron , and white matter ( wm ) lesion scores   from the structural images . the study was approved by the local research ethics committee and all participants gave informed written consent . in order to avoid any possible confounding effects from diurnal variation , all scans were performed at the same time of day ( between 11 a.m. and 1 p.m. ) . all imaging was performed on a 1.5 t clinical research scanner ( hdxt , ge healthcare , uk ) . the imaging protocol for this spectroscopy study used a standard quadrature head coil and included localiser , coronal t1-weighted ( t1w : 3d - ir - prep - fast - gre ) , and axial t2-weighted ( t2w : fse ) scans in addition to an mrsi scan for brain metabolite measurements . key acquisition parameters for the t1w scans were 3d inversion recovery - prepared , ti / tr / te  =  500/10/4  ms , flip angle 8 , matrix 192    192 , fov 256  mm slices of 1.3  mm thickness ( 156 slices total ) . parameters for the t2w scans were tr / te  =  4,000/102  ms , matrix 256    256 , fov 240  mm , slices of 5  mm thickness ( 28 slices total ) , and 2 signal averages . the t2w images were used to plan a 10  mm thick axial mrsi slice at a level that included the basal ganglia . press excitation was used with a volume of interest ( voi ) selected to cover the brain laterally and extend forward from the posterior edge of the brain , avoiding the frontal sinuses and orbits ( fig .  1 ) . 24    24 phase - encoding steps were applied over a field of view of 240  mm . for each phase encode , 512 complex data points were acquired with a sampling interval of 1  ms . manufacturer - provided ( first order ) automatic shimming was employed to reduce the b0 magnetic field inhomogeneity prior to the mrsi acquisitions . water suppression used the default chess technique with three selective rf pulses . outer volume suppression employed pre - saturation slabs parallel to the faces of the voi and an additional four slabs positioned around the edges of the brain to suppress lipid artifacts from the scalp ( fig .  1 ) . 1sagittal and axial localiser scan showing placement of press  mrsi slab and additional saturation bands ( hatched ) to minimise lipid contamination sagittal and axial localiser scan showing placement of press  mrsi slab and additional saturation bands ( hatched ) to minimise lipid contamination mrsi data were processed offline as described previously . briefly , a two - dimensional spatial fourier transform with cosine - weighted apodisation was applied to the raw spectroscopic data , followed by zero - order phase and eddy - current correction on a voxel - by - voxel basis using the residual water signals , bringing water to a fixed nominal chemical shift of 4.70  ppm . the residual water component was removed from each signal using the hankel lanczos singular value decomposition technique and the resulting signals quantified using the amares algorithm within the mrui package ( www.mrui.uab.es ) . a model consisting of three in - phase gaussian components ( corresponding to the major metabolite peaks of choline , creatine , and naa ) and two inverted components ( corresponding to lactate ) was used . spectra were automatically discarded if fitted line widths were < 1  hz or > 10  hz ; if the metabolite peaks were more than 0.1  ppm offset from their nominal values ; or if the voxels lay on the edges of the press excitation region . all spectra were also inspected visually and discarded if judged to be of poor quality , for example having a badly elevated baseline or containing spurious peaks . spectral peak areas were corrected for relaxation effects using literature values for t1 and t2 in basal ganglia in elderly subjects   and normalised according to the number of contributing protons . the resulting institutional units are directly related to molecular concentrations and enable comparison between studies . tissue segmentation maps for grey matter ( gm ) , wm , cerebrospinal fluid ( csf ) , and  background ( bgnd ) were computed from the t1-weighted images using the fsl - fast algorithm ( http://www.fmrib.ox.ac.uk ) . previously available t2 * ( gradient - echo ) images   were first registered with the t1w and t2w images and used to create brain masks . for each spectroscopic voxel , the percentage of gm , wm , csf , and bgnd content was calculated based on three contiguous slices to match the extent of the mrsi acquisition . spectroscopy voxels containing more than 20  % csf or 5  % bgnd were rejected . anova ( matlab ) and linear mixed effects ( pasw statistics ) analyses were carried out on metabolite levels , with ( % wm%gm ) as a variable and " subject " as a random factor . subject - by - subject associations between metabolite levels and structural and cognitive scores were explored . forty participants ( 25 female ) were recruited from healthy , community - living people comprising the lothian birth cohort 1936 ( lbc 1936 [ 7 , 8 ] ) . all participants were aged 73 or 74 at the time of mri scanning and formed a sub group of approximately 650 volunteers who were part of a longitudinal study on cognitive ageing   and for whom recent detailed structural imaging   and cognitive scores were available . an experienced observer determined brain atrophy , iron , and white matter ( wm ) lesion scores   from the structural images . the study was approved by the local research ethics committee and all participants gave informed written consent . in order to avoid any possible confounding effects from diurnal variation , all scans were performed at the same time of day ( between 11 a.m. and 1 p.m. ) . all imaging was performed on a 1.5 t clinical research scanner ( hdxt , ge healthcare , uk ) . the imaging protocol for this spectroscopy study used a standard quadrature head coil and included localiser , coronal t1-weighted ( t1w : 3d - ir - prep - fast - gre ) , and axial t2-weighted ( t2w : fse ) scans in addition to an mrsi scan for brain metabolite measurements . key acquisition parameters for the t1w scans were 3d inversion recovery - prepared , ti / tr / te  =  500/10/4  ms , flip angle 8 , matrix 192    192 , fov 256  mm slices of 1.3  mm thickness ( 156 slices total ) . parameters for the t2w scans were tr / te  =  4,000/102  ms , matrix 256    256 , fov 240  mm , slices of 5  mm thickness ( 28 slices total ) , and 2 signal averages . the t2w images were used to plan a 10  mm thick axial mrsi slice at a level that included the basal ganglia . press excitation was used with a volume of interest ( voi ) selected to cover the brain laterally and extend forward from the posterior edge of the brain , avoiding the frontal sinuses and orbits ( fig .  1 ) . 24    24 phase - encoding steps were applied over a field of view of 240  mm . for each phase encode , 512 complex data points were acquired with a sampling interval of 1  ms . manufacturer - provided ( first order ) automatic shimming was employed to reduce the b0 magnetic field inhomogeneity prior to the mrsi acquisitions . outer volume suppression employed pre - saturation slabs parallel to the faces of the voi and an additional four slabs positioned around the edges of the brain to suppress lipid artifacts from the scalp ( fig .  1 ) . 1sagittal and axial localiser scan showing placement of press  mrsi slab and additional saturation bands ( hatched ) to minimise lipid contamination sagittal and axial localiser scan showing placement of press  mrsi slab and additional saturation bands ( hatched ) to minimise lipid contamination briefly , a two - dimensional spatial fourier transform with cosine - weighted apodisation was applied to the raw spectroscopic data , followed by zero - order phase and eddy - current correction on a voxel - by - voxel basis using the residual water signals , bringing water to a fixed nominal chemical shift of 4.70  ppm . the residual water component was removed from each signal using the hankel lanczos singular value decomposition technique and the resulting signals quantified using the amares algorithm within the mrui package ( www.mrui.uab.es ) . a model consisting of three in - phase gaussian components ( corresponding to the major metabolite peaks of choline , creatine , and naa ) and two inverted components ( corresponding to lactate ) was used . spectra were automatically discarded if fitted line widths were < 1  hz or > 10  hz ; if the metabolite peaks were more than 0.1  ppm offset from their nominal values ; or if the voxels lay on the edges of the press excitation region . all spectra were also inspected visually and discarded if judged to be of poor quality , for example having a badly elevated baseline or containing spurious peaks . spectral peak areas were corrected for relaxation effects using literature values for t1 and t2 in basal ganglia in elderly subjects   and normalised according to the number of contributing protons . the resulting institutional units are directly related to molecular concentrations and enable comparison between studies . tissue segmentation maps for grey matter ( gm ) , wm , cerebrospinal fluid ( csf ) , and  background ( bgnd ) were computed from the t1-weighted images using the fsl - fast algorithm ( http://www.fmrib.ox.ac.uk ) . previously available t2 * ( gradient - echo ) images   were first registered with the t1w and t2w images and used to create brain masks . for each spectroscopic voxel , the percentage of gm , wm , csf , and bgnd content was calculated based on three contiguous slices to match the extent of the mrsi acquisition . spectroscopy voxels containing more than 20  % csf or 5  % bgnd were rejected . anova ( matlab ) and linear mixed effects ( pasw statistics ) analyses were carried out on metabolite levels , with ( % wm%gm ) as a variable and " subject " as a random factor . subject - by - subject associations between metabolite levels and structural and cognitive scores were explored . three participants who had fewer than five spectroscopic voxels valid for naa , choline , or creatine were excluded from the subject - by - subject analysis . of the combined total of 6,476 voxels contained within the mrsi press excitation regions ( fig .  1 ) , 1,432 survived the spectral tests for naa , and only 703 survived when including choline and creatine . these 703 voxels were included in the full anova analyses reported in table  1 . there was a significant negative correlation between the percentage of valid voxels and the mean naa line width ( of retained voxels ) for each subject ( r  =  0.44 , p  =  0.005 ) . the main metabolite findings are that creatine levels were significantly higher in gm than wm , whereas choline and naa levels were significantly higher in wm than gm . choline and creatine levels were highly correlated ( r  =  0.55 , p  <  0.001 ) , whilst creatine and naa showed a weak negative correlation ( r  =  0.32 , p  =  0.05 ) . naa / cre and cho / cre metabolite ratios were significantly greater in wm than gm . the naa / cho ratio was greater in gm than wm , although this difference did not quite reach significance ( p  =  0.06).table  1metabolite levels [ in institutional units ( iu ) : see text ] and metabolite ratios from anova analysis of spectroscopic voxels in 40 subjectswmgm 
 p ( gm vs wm)metabolites ( iu )  naa76700.03  choline1310<0.001  creatine4557<0.001metabolite ratios  naa / cho8.29.80.06  naa / cre2.11.4<0.001  cho / cre0.320.16<0.001spectral peak area ratios  naa / cho2.42.80.06  naa / cre2.41.6<0.001 cho / cre1.30.7<0.001ratios of spectral peak areas are also given for comparison with other studies . only voxels passing all spectral quality tests ( see figs .  2 , 3 and text ) are included 
 wm white matter , gm grey matterfig . below is shown the grid of spectra contained completely within the press box , colour coded according to : pale grey , background ( bgnd ) , where brain comprises < 95  % of the voxel volume ; grey , csf more than 20  % ; red , fitting of naa peak failed ; blue , failed visual quality test ; green , failed choline and/or creatine fitting but valid for naa ; black , passed all tests . tests were applied in the order described . for further details , refer to text . 40 subjects had a potential 6,476 voxels , of which 1,432 were valid for naa after spectral quality tests had been applied . reasons for exclusion were : background more than 5  % of voxel volume ( bgnd ) ; csf more than 20  % ( csf ) ; fitting of naa peak failed ( naa ) ; failed visual quality test ( vis ) ; failed choline and/or creatine fitting ( chocre ) , but valid for naa . tests were applied in the order described , and the number of additional voxels failing each test is given in the figure metabolite levels [ in institutional units ( iu ) : see text ] and metabolite ratios from anova analysis of spectroscopic voxels in 40 subjects ratios of spectral peak areas are also given for comparison with other studies . only voxels passing all spectral quality tests ( see figs .  2 , 3 and text ) are included 
 wm white matter , gm grey matter mrsi grid superimposed on corresponding t2-weighted image . the press excitation box is outlined in red . below is shown the grid of spectra contained completely within the press box , colour coded according to : pale grey , background ( bgnd ) , where brain comprises < 95  % of the voxel volume ; grey , csf more than 20  % ; red , fitting of naa peak failed ; blue , failed visual quality test ; green , failed choline and/or creatine fitting but valid for naa ; black , passed all tests . the spectral range displayed is from 4 to 1  ppm exclusion of spectroscopic voxels for the cohort . 40 subjects had a potential 6,476 voxels , of which 1,432 were valid for naa after spectral quality tests had been applied . reasons for exclusion were : background more than 5  % of voxel volume ( bgnd ) ; csf more than 20  % ( csf ) ; fitting of naa peak failed ( naa ) ; failed visual quality test ( vis ) ; failed choline and/or creatine fitting ( chocre ) , but valid for naa . tests were applied in the order described , and the number of additional voxels failing each test is given in the figure there was a trend for choline levels to be associated with wm lesion scores ( r  =  0.24 , p  =  0.1 ) . creatine levels were correlated with fluid intelligence ( r  =  0.33 , p  =  0.05 ) , and naa showed a trend towards association with memory scores ( r  =  0.27 , p  =  0.1 ) . of the combined total of 6,476 voxels contained within the mrsi press excitation regions ( fig .  1 ) , 1,432 survived the spectral tests for naa , and only 703 survived when including choline and creatine . these 703 voxels were included in the full anova analyses reported in table  1 . there was a significant negative correlation between the percentage of valid voxels and the mean naa line width ( of retained voxels ) for each subject ( r  =  0.44 , p  =  0.005 ) . the main metabolite findings are that creatine levels were significantly higher in gm than wm , whereas choline and naa levels were significantly higher in wm than gm . choline and creatine levels were highly correlated ( r  =  0.55 , p  <  0.001 ) , whilst creatine and naa showed a weak negative correlation ( r  =  0.32 , p  =  0.05 ) . naa / cre and cho / cre metabolite ratios were significantly greater in wm than gm . the naa / cho ratio was greater in gm than wm , although this difference did not quite reach significance ( p  =  0.06).table  1metabolite levels [ in institutional units ( iu ) : see text ] and metabolite ratios from anova analysis of spectroscopic voxels in 40 subjectswmgm 
 p ( gm vs wm)metabolites ( iu )  naa76700.03  choline1310<0.001  creatine4557<0.001metabolite ratios  naa / cho8.29.80.06  naa / cre2.11.4<0.001  cho / cre0.320.16<0.001spectral peak area ratios  naa / cho2.42.80.06  naa / cre2.41.6<0.001  cho / cre1.30.7<0.001ratios of spectral peak areas are also given for comparison with other studies . only voxels passing all spectral quality tests ( see figs .  2 , 3 and text ) are included 
 wm white matter , gm grey matterfig . below is shown the grid of spectra contained completely within the press box , colour coded according to : pale grey , background ( bgnd ) , where brain comprises < 95  % of the voxel volume ; grey , csf more than 20  % ; red , fitting of naa peak failed ; blue , failed visual quality test ; green , failed choline and/or creatine fitting but valid for naa ; black , passed all tests . 40 subjects had a potential 6,476 voxels , of which 1,432 were valid for naa after spectral quality tests had been applied . reasons for exclusion were : background more than 5  % of voxel volume ( bgnd ) ; csf more than 20  % ( csf ) ; fitting of naa peak failed ( naa ) ; failed visual quality test ( vis ) ; failed choline and/or creatine fitting ( chocre ) , but valid for naa . tests were applied in the order described , and the number of additional voxels failing each test is given in the figure metabolite levels [ in institutional units ( iu ) : see text ] and metabolite ratios from anova analysis of spectroscopic voxels in 40 subjects ratios of spectral peak areas are also given for comparison with other studies . only voxels passing all spectral quality tests ( see figs .  2 , 3 and text ) are included 
 wm white matter , gm grey matter mrsi grid superimposed on corresponding t2-weighted image . the press excitation box is outlined in red . below is shown the grid of spectra contained completely within the press box , colour coded according to : pale grey , background ( bgnd ) , where brain comprises < 95  % of the voxel volume ; grey , csf more than 20  % ; red , fitting of naa peak failed ; blue , failed visual quality test ; green , failed choline and/or creatine fitting but valid for naa ; black , passed all tests . the spectral range displayed is from 4 to 1  ppm exclusion of spectroscopic voxels for the cohort . 40 subjects had a potential 6,476 voxels , of which 1,432 were valid for naa after spectral quality tests had been applied . reasons for exclusion were : background more than 5  % of voxel volume ( bgnd ) ; csf more than 20  % ( csf ) ; fitting of naa peak failed ( naa ) ; failed visual quality test ( vis ) ; failed choline and/or creatine fitting ( chocre ) , but valid for naa . tests were applied in the order described , and the number of additional voxels failing each test is given in the figure there was a trend for choline levels to be associated with wm lesion scores ( r  =  0.24 , p  =  0.1 ) . creatine levels were correlated with fluid intelligence ( r  =  0.33 , p  =  0.05 ) , and naa showed a trend towards association with memory scores ( r  =  0.27 , p  =  0.1 ) . in this study we used mrsi to measure brain metabolite levels in healthy elderly subjects . we looked for evidence of tissue type differences and for associations with structural and cognitive scores . the narrow age range of the lothian birth cohort 1936 removes age as a potential confounding factor , although it does preclude looking for age - related effects in a single study such as this . our finding of higher creatine levels in gm than wm is in agreement with previous literature reports , although the magnitude of the difference varies greatly , being 27  % in the present study , 11  % in schuff et al . , 30  % in hetherington et al . , 40  % in tal et al . , and as much as 70  % in mclean et al . . our finding of lower naa levels in gm than in wm ( 70 vs. 76 iu ) is consistent with some previous reports [ 15 , 16 ] but different from others . for example , tal et al .   averaged metabolite values over a large centrally placed volume of brain and found that naa was 11  % higher in gm than wm . we found choline levels lower in gm than wm , consistent with previous reports [ 16 , 18 ] , although others have reported no significant difference [ 15 , 17 ] . christiansen et al .   found no difference in metabolite levels between basal ganglia and the occipital , temporal , and frontal regions in healthy elderly subjects . however , they used large ( 8  ml ) volumes of interest containing unspecified proportions of gm and wm . the reasons for the discrepancies in metabolite findings between various studies are unclear but will include exact details of the positioning , acquisition method and quantification , and possible ( unknown ) differences in metabolite t1 and t2 values between gm and wm . we used literature values for t1 and t2 correction and also normalised the results by the number of protons contributing to the spectral peaks ( 3 for naa and creatine , 9 for choline ) . the final results in institutional units are therefore closely related to molecular concentrations . other researchers , including   for example , have reported ratios of spectral peak areas under experimental conditions , and we have included these ratios in table  1 to enable comparisons with other studies . we did not explore regional variations because of the relatively low number of valid voxels in particular regions , especially the frontal and cortical regions . the chemical shift displacement between choline and naa amounted to approximately 0.5  mm through - plane ( i.e. 5  % of the slab thickness ) , 4 mm anterior - posterior , and 6  mm left  right . since we excluded voxels around the edge of the excitation volume we found a positive correlation between creatine and fluid intelligence score , whereas previously   we found a negative correlation between parietal cortex creatine and memory scores but no association with other cognitive measures . however , the two studies investigated different brain regions . as in recent work by bainbridge et al . , we employed strict criteria to discard spectroscopic voxels that failed various quality tests . although rather conservative , such criteria can be applied with minimal human intervention , will become important for future clinical studies , and will be essential when large data sets from whole - brain spectroscopy   become routinely available . the large loss of voxels ( fig . 3 ) was caused by a high proportion of csf and failures of spectral quantification . in older people with brain atrophy , it is inevitable that voxels will be lost due to csf and enlarged ventricles . failures of quantification were due in part to the problems of shimming over an extended region of the brain , which is especially difficult at the level of the basal ganglia . in some subjects , an unavoidable tilt of the head relative to the scanner led to portions of the frontal sinuses , ethmoid sinus , or eyes being included in the mrsi plane . this is a particular problem in older people , as they are less able to tilt their heads into the optimum posture . only first - order ( linear ) shimming was available on the scanner used . in other studies ( data not shown ) we have found greater success rates with mrsi slices at the level of the corpus callosum . the use of modern scanners with higher - order shims is likely to help , as are higher field strengths and more sensitive multi - channel coils enabling higher snr and hence smaller voxels with improved line widths . however , some brain areas , for example the frontal and temporal regions , will remain challenging in this regard . a limitation of this study is that time did not permit the collection of non - suppressed spectroscopic data for water referencing purposes . ideally , a rapid interleaved ( suppressed / reference ) technique would be used , as demonstrated by the echo - planar acquisition scheme of ebel and maudsley . the measurement of brain metabolites is potentially useful in studies of healthy ageing and acute and neurodegenerative diseases . the current study of a moderately sized cohort with a tight age range adds to the available literature . however , technical challenges such as shimming across the whole brain and water referencing need to be resolved before the technique can be more widely applied in large trials . future directions include rapid , three - dimensional , whole - brain coverage and longitudinal studies . this study was approved by the local research ethics committee and all participants gave informed written consent .