Volume 133, June 2016, Pages 390–398

Amide proton signals as pH indicator for in vivo MRS and MRI of the brain—Responses to hypercapnia and hypothermia

  • Biomedizinische NMR Forschungs GmbH am Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany


First in vivo evidence of pH-dependent concurrent changes of amide proton signals.

During hypercapnia, amide proton signals increase by at least 50% at 37 °C and 22 °C.

Amide proton signal increases are due to reduced proton exchange with water.

Z-spectra indicate dipolar coupling of aliphatic/water protons as dominant factor.

Saturation of aliphatic protons identifies tissue compounds associated with myelin.


Using proton MRS and MRI of mouse brain at 9.4 T, this work provides the first in vivo evidence of pH-dependent concurrent changes of three amide signals and related metabolic responses to hypercapnia and hypothermia. During hypercapnia, amide proton MRS signals of glutamine at 6.8–6.9 ppm and 7.6 ppm as well as of unspecific compounds at 8.1–8.3 ppm increase by at least 50% both at 37 °C and 22 °C. These changes reflect a reduced proton exchange with water. They are strongly correlated with intracellular pH which ranges from 6.75 ± 0.10 to 7.13 ± 0.06 as determined from a shift in creatine phosphokinase equilibrium. In MRI, saturation transfer from aliphatic as well as aromatic and/or amide protons alters slightly during hypercapnia and significantly during hypothermia. The asymmetry in magnetization transfer ratios decreased slightly during hypercapnia and hypothermia. Regardless of pH or temperature, saturation transfer from aliphatic protons between − 2 and − 4 ppm frequency offset to water protons is significantly greater than that from aromatic/amide protons at corresponding offsets between + 2 and + 4 ppm. Irradiation of aliphatic compounds at − 3.5 ppm frequency offset from water predominantly saturates lipids and water associated with myelin. Taken together, the results indicate that, for the B1 power used in this study, dipolar coupling between aliphatic and water protons rather than proton exchange is the dominant factor in Z-spectra and magnetization transfer ratio asymmetry of the brain in vivo.


  • ADP, adenosine diphosphate;
  • Ala, alanine;
  • ATP, adenosine triphosphate;
  • CEST, chemical exchange saturation transfer;
  • CHESS, chemical-shift selective;
  • Cr, creatine;
  • FLASH, fast low-angle shot;
  • Glc, glucose;
  • Gln, glutamine;
  • Glu, glutamate;
  • HC, homocarnosine;
  • His, histidine;
  • HE and HZ, amide protons of glutamine;
  • Lac, lactate;
  • MR, magnetic resonance;
  • MRI, magnetic resonance imaging;
  • MRS, magnetic resonance spectroscopy;
  • MTR, magnetization transfer ratio;
  • MTRasym, magnetization transfer ratio asymmetry;
  • NAA, N-acetylaspartate;
  • ‐NHX, unspecific amide protons;
  • NOE, nuclear Overhauser enhancement;
  • Phe, phenylalanine;
  • PCr, phosphocreatine;
  • R1, longitudinal relaxation rate;
  • RF, radiofrequency;
  • STEAM, stimulated-echo acquisition mode;
  • Tau, taurine;
  • Trypt, tryptophan


  • Creatine phosphokinase;
  • Glutamine;
  • Hypercapnia;
  • Hypothermia;
  • Proton exchange;
  • Saturation transfer


Magnetic resonance (MR) spectroscopy (MRS) and imaging (MRI) play important roles in translational biomedical research and in particular allow for a biochemical phenotyping of the brain. Complementing the neurochemical profiling by in vivo MRS, there have been manifold investigations of pH dependencies, mainly with the use of 31P MRS. Exploiting the higher sensitivity of 1H MRS at 4.7 T, amide signals in brain spectra were found to change with pH, because they exchange protons with water in base-catalyzed reactions ( Kanamori and Ross, 1997; Mori et al., 1998 ;  Zhou and van Zijl, 2006). While two amide resonances of glutamine (Gln) at 6.8–7.6 ppm were studied by nuclear Overhauser enhancement (NOE) through 15N excitation after administration of exogenous 15NH4+ (Kanamori and Ross, 1997), amide signals of proteins and peptides around 8.3 ppm were identified by MRS sequences with minimized saturation transfer from water ( Mori et al., 1998 ;  Zhou and van Zijl, 2006).These base-catalyzed exchange reactions are also responsible for the contrast described in chemical exchange saturation transfer (CEST) MRI (Zhou and van Zijl, 2006).

Here, we report saturation transfer MRS and MRI studies of proton exchange in mouse brain in vivo at 9.4 T, which detect all aforementioned amide protons without the use of 15NH4+ or the need for special pulse sequences. At a high field of 9.4 T, the application of a common MRS technique with effective water pre-saturation yields a significant increase of amide signals, i.e., reduced amide–water proton exchange, in response to hypercapnia and hypothermia.

Materials and methods

Animals and anesthesia

A total of 11 female C57BL/6N mice (4–5 months, 23–29 g) were studied in accordance with German animal protection laws after approval by the responsible governmental authority. As shown in Fig. 1 ;  Fig. 5 mice underwent MRS of the forebrain and acquisition of the Z-spectra before and during hypercapnia at 37 °C (protocol 1-1) followed by MRS of the forebrain and of the striatum before, during, and after hypercapnia at 22 °C one day later (protocols 1–2). Another 6 mice underwent MRS of the striatum, T1, T2, and/or Z-spectra measurements of the striatum at 37 °C (n = 6), 32 °C (n = 4), 27 °C (n = 5), and/or 22 °C (n = 5) under 1.5 or 0.5% isoflurane anesthesia (protocol 2). After induction of anesthesia with 5% isoflurane, animals were intubated with a purpose-built polyethylene endotracheal tube (0.58 mm inner diameter, 0.96 mm outer diameter) and artificially ventilated using an animal respirator (TSE, Bad Homberg, Germany) with a respiratory rate of 25 breaths per minute and an estimated tidal volume of 0.35 ml as previously described ( Schulz et al., 2002; Watanabe et al., 2004 ;  Boretius et al., 2013). The animals were then placed in a prone position on a purpose-built palate holder equipped with an adjustable nose cone. The Göttingen animal bed (Tammer et al., 2007) secured a reproducible and reliable fixation of the mouse head and receiver coil in the magnet isocenter. Respiratory movement of the abdomen as well as rectal temperature were monitored by a unit supplied by the manufacturer (Bruker Biospin MRI GmbH, Ettlingen, Germany). Thirty minutes after the rectal temperature reached 37 ± 1 °C, 32 ± 1 °C, 27 ± 1 °C, or 22 ± 1 °C with the use of a heating system (i.e., a water blanket and animal bed), the respective chemical shifts of the N-acetylaspartate (NAA) amide signal at 7.83–7.84, 7.87–7.88, 7.91–7.92, and 7.95–7.96 ppm confirmed the brain temperature to be within the target range (Arús et al., 1985). MRS and MRI data were acquired only after the equilibrium was established.