Metabolites in Proton Spectroscopy of the Brain: Neurochemistry and Physiology

Transcription

1 Metabolites in Proton Spectroscopy of the Brain: Neurochemistry and Physiology Josef Pfeuffer Max-Planck Institute for Biological Cybernetics, Department of Neurophysiology, Tübingen, Germany Aims chemical composition of the brain and biochemistry in vitro cell 1H MRS assays what can we measure in vivo? in humans? to identify metabolite resonances in 1H spectra of the brain neurochemistry of major metabolic paths

2 Chemical composition of the human brain whole brain (%) gray matter (%) white matter (%) Water Inorganic salts Soluble organic subst Carbohydrate Protein Lipids: simple fats cholesterol phosphatides cerebrosides Entry of substances from bloodstream to brain Glucose Amino acids Monocarboxylic acids (lactate, pyruvate, ketone) Anions (thiocyanate, chloride) Cholesterol / Uncharged nonmetabolized comp. / Large molecules (e.g. proteins, inulin) entry promoted by carrier-mediated transport carrier-mediated active transport carrier-mediated transport concentration gradient concentration gradient intracellular ligands cerebral concentration limited by capacity of influx mechanism; utilization capacity of influx mechanism; active extrusion; utilization capacity of influx and efflux mechanism; utilization electrochemical gradient; membrane permeability membrane permeability; availability of ligand sites

3 Free amino acids and related compounds of the brain Content (µmol/g) H NMR detectable Compounds [brain/plasma ratio] Glutamate, N-Acetylaspartate, Phospho-/ Creatine Choline-containing compounds, myo-inositol, Lactate, Taurine, Glucose, Glutamine, Gluthathione, GABA, Phosphoethanolamine, N-Acetylaspartylglutamate, ( Urea, Glycine, Serine ) Alanine, Aspartate, scyllo-inositol, ( Ammonia, Arginine, Asparagine ) ( Arginine, Citrulline, Cystathionine, Cystine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Ornithine, Proline, Phenylalanine, Threonine, Tryptophan, Tyrosine, Valine ) 1 H NMR on extracts and in-vitro cell assays Preparation of cell cultures: glial, neuronal, glioma... - body fluids (plasma, serum, urine, CSF etc.) - extracts ( tissue, biopsates, cells) Incubation & treatment: e.g. osmotic stress, anoxia, ischemia, labeled substrates (glucose, acetate), pharmaceuticals perchloric acid extracts: hydrophilic lipophilic phase high-resolution NMR ( 13 C, 1 H, 31 P) in solution - lipid extraction - protein extraction

4 DOH Cerebrospinal Fluid (CSF) Lac β Glc Leibfritz et al Glc α Glc Lac Cr/Cn Pyruvate Acetate Cit Gln Ala Lys Val, Leu, Ile (ppm) 1 High resolution 1 H NMR spectra of PCA extracts of rat brain de Graaf Cr/PCr NAA 300 MHz Cr/PCr Cho Lac

5 Brand 1 H spectra from cell extracts 360 MHz Glutamate ( Glu ) and Glutamine ( Gln ) Glutamate Glutamine C2 C3 C4

6 13 C spectra from cell extracts incubated with [2-13 C] acetate Brand 360 MHz Bachelard 13 C spectra of tissue extracts 3h depolarization 4h control

7 1H NMR on extracts and in-vitro cell assays Preparation of cell cultures: glial, neuronal, glioma... - body fluids (plasma, serum, urine, CSF etc.) - extracts ( tissue, biopsates,, cells) Incubation & treatment: e.g. osmotic stress, anoxia, ischemia, labeled substrates (glucose, acetate), pharmaceuticals perchloric acid extracts: hydrophilic lipophilic phase high-resolution NMR ( 13 C, 1 H, 31 P) in solution in-vitro perfused cells / tissue: gel threads, beads fully metabolically intact system over hours / days Perfusion system Flögel, Leibfritz et al

8 Pfeuffer/ Leibfritz cell colume change hypotonic time / h Osmotic Stress temporal changes in cellular water content (diffusion-weighted 1 H NMR of water in perfused cell threads) changes in metabolite content 2 hours incubated under anisosmotic conditions (PCA extracts) Treatment of primary astrocytes with immunosuppressiva Cyclosporine A rel. volume / % time / h

9 Apoptosis ( KB cells in-vitro) HePC ( Miltefosine ) rel. volume / % time / h Spectroscopic Considerations Spectral localization: outer-volume contamination, esp. lipids Resonance assignment: by chemical shift compare with other methodologies, e.g. 1H-13C, human-animal by spectral editing metabolic perturbation, e.g. glucose infusion Resonance Quantification from peak integration to multi-parameter/peak fitting

10 'From in-vitro to in-vivo MRS' 'from high to low resolution' or dominating water signal magnetic field and spectral resolution echo time dependence: T 2 decay and J-coupling metabolite concentration in different tissues / different locations in the brain RF coils: sensitivity, voxel volume, and signal-to-noise The dominating water signal water: 55 M metabolites 1-10 mm Roell

11 Magnetic field and spectral resolution: line width, spectral dispersion Gruetter glutamate glutamine echo time dependence: T2 decay and J-coupling degraaf lactate in D 2 O rat brain anti-phase n*72ms, n=1,3,5

12 Different metabolite concentrations: in tissues (GM, WM, CSF) - spatial & dynamic changes Ross RF coil sensitivity, voxel volume, and signal-to-noise Bluml TE 30 ms STEAM (1.5 T) standard GE head coil (birdcage) quadrature half-volume coil >3-fold increase in SNR

13 N-Acetyl Aspartate ( NAA ) CH 3 CH CH 2 N-Acetyl Aspartate ( NAA ) 2.02 ppm methyl resonance biggest peak in brain present in neurons: specific marker for neuronal damage glial cells mostly little or no NAA concentration higher in GM than in WM: higher neuronal density measure for axon density in WM: independent of demyelination

14 Creatine and Phosphocreatine ( Cr, PCr ) CH 3 : 3.03 ppm CH 2 : 3.91/3.93 ppm (Cr, PCr) marker for brain cell density in glial and neuronal cells Creatine kinase activity 31P MRS studies, [Cr+PCr] Cr-biosynthesis pathway [PCr], [Pi], [Lac] Brain energetics Choline containing compounds ( Cho ) (CH 3 ) 3 : 3.21 ppm marker for membrane damage phospholipid bound choline groups membrane synthesis and degradation indistinguishable trimethylamine resonance: betaine, choline (Cho), phosphorylcholine (PC) glycerophosphorylcholine (GPC) 31 P and 1 H MRS from same volume helpful!!! significant overlap at 3.2 ppm with resonances of taurine, glucose, phosphoethanolamine

15 myo-inositol ( Ins ) 4,6-CH 2-CH 1,3-CH 5-CH scyllo-inositol: 3.35 ppm (singulet, 6H) myo-inositol ( Ins ) 1-CH,3-CH: 3.54 ppm most pronounced peak simple sugar-alcohole: dominant in infant brain 3 times decreased in adults recognized as osmolyte believed to be marker for astrocytes decreased in pathologies: hepatic encephalopathy, ornithine transcarbomylase deficiency in the center of a complex metabolic pathway (intracell. signalling) inositol-polyphosphate messengers, inositol-1-phosphate, glucose-6-phosphate, glucuronic acid

16 Lactate ( Lac ) brain lactate level: sensitive indicator of impaired oxygen supply elevated lacate in tumor, stroke,... brain energy Taurine ( Tau ) strong variation in different brain regions and species neonatal brain ~6 mm with brain maturation ~2 mm functions: osmoregulator, anti-oxidant, calcium regulator, biological sink for sulfur

17 γ-aminobutyrate ( GABA ) 4-CH 2 2-CH 2 3-CH 2 inhibitory neurotransmitter - decarboxylation of Glu - synthesis partly from Gln - 1H MRS editing - direct 15N MRS Glutamate ( Glu ) and Glutamine ( Gln ) Glutamate Glutamine 2-CH 4-CH 2 3-CH 2

18 Glutamate: higher order spin system separation of glutamate and glutamine

19 Neurotransmitter one of the major excitatory neurotransmitter: glutamate in all brain cell types, higher in neurons TCA cycle - neurotransmitter rate ammonia detoxification liver function, liver cycle glycine CH ppm overlap with inositol, editing at long TE GABA - derived from decarboxylation of Glu - synthesis partly from Gln aspartate structure identical to NAA (except N-acetyl group) glutamine synthetase rate Glucose ( Glc ) 1 central role in energy metabolism, glycolysis direct detection complicated 1-CH(α) 1-CH(β) 2,3,4,5,6-CH(α,β) anomeric forms α,β 2:3 in brain 1-2 mm in blood plasma 5-10 mm important for 13 C-isotope labeling and products

20 1 H MRS in rat brain (400 MHz) automated frequency-domain fitting full pattern of 18 metabolite model spectra Cr+PCr NAA Cr PCr Gln Glu Ins Tau Cho Gln Glu Macromolecule background Lac ppm Toward an in vivo neurochemical profile: quantification of 18 metabolites in short-echo-time 1 H NMR spectra of rat brain (Pfeuffer, J Magn Reson 1999) myo-ins mm (2%) Glu mm (2%) scyllo-ins Gln mm (4%) NAAG Asp ppm

21 phosphoethanolamine mm (6%) GABA mm (12%) GSH mm (9%) ppm Cho Ins PE PCr Cr Glc Ins Ins Tau InsTauPE PCr Cr ppm

22 1 H MRS - quantification of glucose PCr Cr highly resolved spectral features full pattern used for quantification H1 α-glc water suppression ppm Determination of Metabolic Fluxes: Normal Metabolism and Physiology Glucose transport TCA cyclic rate Glycolysis Glucose metabolic rate NMR studies of metabolic changes upon visual stimulation changes of V TCA and CMR Glc

23 NMR studies of visual stimulation increase of lactate changes of glucose Frahm V TCA CMR Glc Glc Lac

24 neuron glia cells / astrocytes 24

25 Cytological relationship of astrocytes with neurons and capillaries Glutamate-induced glycolysis in astrocytes during physiological activation Magistretti 13 C detection of label incorporation in human brain Gruetter 4 Tesla 3 x 3 x 5 cm

26 13 C detection of label incorporation in rat brain Glc C1β Glc C1α Gln C2 Glu C2 Gln C4 Glu C4 Glu C3 Gln C3 Glycogen C1 Asp C3 Lac C3 Ala C ppm 0 Gruetter: non-localized 13C MRS 1 H detection of 13 C label: Adiabatic Carbon Editing and Decoupling (ACED) [1-13 C] glucose 70% enriched H-1 α 13 C 12 C 13 C ACED inversion signal of protons bound to 13 C J CH fi ppm 5.02 S off S on S off S on 13 C Decoupling

27 ACED subspectrum - S off ( x 1 ) Cr+PCr NAA TE = 7.9 ms nt = 1280 PCr Cr Glx Tau Gln NAA Asp Glu Glx Lac Ala ppm ACED S off S on ( x 20 ) Glu C4 TE = 7.9 ms summed over 3h Cr tot + Asp Glx C2 Cr tot + GABA Gln C4 Asp C3 Glu C3+ Gln C3 GABA Lac C3 Ala ppm

28 ACED detection of 13 C label in glutamate and glutamine C3 Glu C4 Glu C3 Gln C3 NAA Glu C2+ Gln C2 Gln C ppm ppm Glx C2 Glu C4 Gln C4 Glx C3 Lac min C turnover during infusion of [1-13 C] glucose TE = 7.9 ms 15 min averaged 192 scans ppm

29 Temporal evolution of 13 C enrichment 13 C (µmol/g) glutamate C glutamine C4 5 min averaged min 13 C labeling and fractional enrichment 13 C (µmol/g) FE of glucose 0.50 Lac C3 FE(Glc) FE(Glc) Lac C3 Asp C3 GABA C min averaged

30 Selected Literature Bachelard H, Badar-Goffer R (1993) J Neurochem 61(2) Brand A, Leibfritz D (1997) Cell Mol Biol 43(5), de Graaf (1998) in vivo NMR spectroscopy, Wiley Gruetter R (2002) Neurochem Int 41(2-3), Magistretti PF, Pellerin L (1999) Phil Trans R Soc Lond B 354, McIlwain H, Bachelard HS (1985) Biochemistry and the Central Nervous System Pfeuffer J et al (1999) J Magn Reson 141, ; Magn Reson Med 41, Ross B, Michaelis T (1994) Magn Reson Q 10(4), Ross B, Bluml S (1996) NMR Biomed 9, ; (2001) Anat Rec 265, Rothman DL (1994), Ch 21, NMR in Physiology and Biomedicine Siegel GJ et al (1999) Basic Neurochemistry, Lippincott-Raven Ugurbil K et al (2000) Annu Rev Biomed Eng 2,