³¹P Spectral Peaks

What peaks are seen in the ³¹P spectrum and what do they mean?
The ³¹P NMR spectrum appears quite different from that of ​¹H, as seen in the normal brain below.
Normal brain 31P spectrum
³¹P spectrum from normal brain
          Abbreviations: 
Phosphomonoesters (PMEs), Phosphorylethanolamine (PE), Phosphorylcholine (PC), Inorganic Phosphate (Pi), Phosphodiesters (PDEs), Glycerylphosphorylethanol-amine (GPE), Glycerophospho-choline (GPC), Phospho-creatine (PCr), Adenosine Triphosphate (ATP), Nicotine adenine dinucleotide (NAD), Uridine diphosphate glucose (UDPG)
The central (and usually) tallest peak is from the energy metabolite phosphocreatine (PCr). Because of its prominence, biological importance, and relative stability, PCr is assigned a chemical shift (δ) = 0 for in vivo studies. Metabolites to the right of PCr have negative chemical shifts; those to the left are positive. ³¹P metabolites are widely separated, spanning a range of about 30 ppm. By comparison, the ¹H peaks used in clinical MRS are clustered in the narrow interval between 0 and 5 ppm.
PCr and metabolites to its right are all critical for cellular oxidative metabolism and can be considered "energy peaks". Besides PCr, distinct resonances for the three phosphate groups of ATP (adenosine triphosphate) are reliably visible, denoted α, β, and γ. Small notches on the right shoulder of the α-ATP peak correspond to the energy-related diphosphonates (NAD and UDPG).
PCr reversibly donates its high energy phosphate group to adenosine diphosphate (ADP) creating ATP and Creatine (Cr).  This reaction is catalyzed by the enzyme creatine kinase and can be written: 
PCr + ADP  ↔  ATP + Cr
When ATP is catabolized for energy, it is reconverted to ADP with release of inorganic phosphate (Pi) according to the following reaction: 
ATP →  ADP + Pi + Energy
A small Pi peak can usually be seen near δ = +5 ppm. (Its exact position depends on tissue pH, and this pH can even be calculated by measuring the chemical shift difference between PCr and Pi.)  ADP stores are too low to generate a detectable spectral peak, but the concentration of ADP can be estimated by calculating the ratio [Pi]/[PCr]. 
With the exception of Pi, all the peaks to the left of PCr derive from metabolites related to membrane phospholipids. These "membrane peaks" can be divided into two major groups: Phosphomonoesters (PMEs) and Phosphodiesters (PDEs). Although not completely correct, PMEs are often thought of as primarily reflecting membrane synthesis, with PDEs reflecting membrane breakdown. The PME/PDE ratio is therefore sometimes used as an index of net phospholipid metabolism.
Adenosine Triphosphate (ATP)
  • Principal energy storage and transport chemical
  • Resonances from 3 phosphate groups (α, β, γ) at δ ≈ −7.5, −16.0, and −2.5 ppm
  • P-P coupling constant (J) is ~16 Hz
  • α and γ are doublets, each split by the ³¹P at β 
  • β is a 1:2:1 triplet, split by α and γ
  • In brain, up to 25% of ATP signal comes from other nucleotide triphophosphates (NTPs), including guanosine, uridine, and cytidine triphosphates
  • ADP and AMP resonances too small to detect
Phosphocreatine (PCr)
  • Called the "ATP replenisher", PCr stores high energy phosphate for conversion of ADP to ATP
  • Assigned chemical shift δ = 0.0 ppm
  • Tallest peak in most spectra, especially skeletal muscle
  • Not present in liver or kidney
Inorganic Phosphate (Pi)
Picture

HPO4−2
Picture

H2PO4−
  • Ionic reservoir for ³¹P in biologic systems
  • ​H2PO4:HPO4 ratio is pH dependent; due to fast exchange only single average value observed 
  • Sub-peaks at δ ≈ 4.8 and 5.2 may be seen, representing extracellular and intracellular Pi
  • Δδ between Pi and PCr can estimate tissue pH
Phosphodiesters (PDE)
Picture
Glycerylphosphorylcholine (GPC)
Picture
Glycerylphosphorylethanolamine (GPE)
  • A group of resonances in the δ = 2.5−3.5 range, including a broad peak for cross-linked membrane phospholipids plus discrete peaks for mobile forms:
  • Glycerophosphocholine (GPC) at δ = 3.0 is a major source of choline storage in the cytoplasm and an important renal osmolyte   
  • Glyceryl-phosphorylethanoamine (GPE), a smaller PDE peak just to the left of GPC at δ = 3.5, is a membrane breakdown product​
Phosphomonoesters (PME)
Picture
Phosphorylcholine (PC)
Picture
Phosphorylethanolamine (PE)
  • Group of resonances in the the δ = 6−7  range reflecting membrane synthesis, especially prominent in brain and liver spectra. At least two discrete peaks can be regularly identified
  • Phosphorylethanolamine (PE) at δ = 6.8 ppm, an intracellular compound frequently the tallest peak in the immature brain
  • Phosphorylcholine (PC) at δ = 6.2 ppm, the primary precursor and storage form for choline
Diphosphates (NAD, UDPG)
NAD
Nicotinamide adenine dinucleotide (NAD)
UDPG
Uridine diphosphoglucose (UDPG)
  • Nicotine adenine dinucleotide (NAD) is a coenzyme for cellular respiration, serving as an electron (hydrogen) carrier by being alternately oxidized (NAD+) or reduced (NADH)
  • In good quality decoupled spectra can be seen as a small notch on the right shoulder of the α-ATP peak at δ = − 8.2 ppm
  • Uridine diphosphate glucose (UDPG) is a key intermediate in carbohydrate metabolism, a precursor of glycogen, polysaccharides, and glycosphingolipids.
  • Small peak seen especially in liver and brain just to right of NAD at at δ = −​ 9.7 ppm

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References  
     Daly PF, Lyon RC, Faustino PJ, Cohen JS. Phospholipid metabolism in cancer cells monitored by ³¹P spectroscopy. J Biol Chem 1987; 262:14875-14878. (early paper characterizing in detail the chemical nature of PME and PDE resonances)
     Ha D-H, Choi S, Oh JY, et al. Application of ³¹P MR spectroscopy to the brain tumors. Korean J Radiol 2013; 14:477-486.
     Jung W-I, Staubert A, Widmaier S, et al. Phosphorus J-coupling constants of ATP in human brain. Magn Reson Med 1997; 37:802-4.
     Lanza IR, Bhagra S, Nair KS, Port JD. Measurement of human skeletal muscle oxidative capacity by ³¹P-MRS: a cross-validation with in vitro measurments. J Magn Reson Imaging 2011; 34:1143-1150.
     Murphy-Boesch J, Stoyanova R, Srinivasan R, et al. Proton-decoupled ³¹P chemical shift imaging of the human brain in normal volunteers. NMR Biomed 1993; 6:173-180.    
     ten Hove M, Neubauer S. Evaluating metabolic changes in heart disease by magnetic resonance spectroscopy. Heart Metab 2006;32:18-21.  
     Thomas G. 31P Spectroscopy intracellular pH calculator. Philips NetForum Community, 2013. Download calculator here as Excel file. Download instructions here.
Related Questions
     What is meant by a chemical shift?  
     What must you do differently to perform ³¹P spectroscopy in lieu of ¹H spectroscopy? 
     How do ³¹P spectra differ among the various organs?​