NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning 333
Proton NMR in Biological Objects Subjected to Magic
Angle Spinning
R A Wind and J Z Hu, Pacific Northwest National
procedure is time consuming, introduces spectral
Laboratory, Richland, WA, USA
artifacts, and cannot be applied to study intact tis-
sues and organs. Another method of improving the
& 2005, Elsevier Ltd. All Rights Reserved.
spectral resolution is to increase the external magne-
tic field B0, as the separation between the lines in an
NMR spectrum increases linearly proportional with
Introduction
B0. However, the susceptibility-induced broadening
Magnetic resonance imaging (MRI) of the spatial is linearly proportional to B0 as well, and in many
distribution of water in biological objects has cases zero or only marginal improvements in the
developed as one of the major tools to diagnose le- spectral resolution have been reported when higher
sions and diseases and follow the therapy response in field strengths were employed.
animals and patients in a minimally invasive way. In In principle, the susceptibility broadening can be
addition, in vitro and in vivo localized magnetic reso- averaged to zero by the technique of magic angle
nance spectroscopy (MRS) and spectroscopic ima- spinning (MAS), where the sample is rotated about
ging or chemical shift imaging (CSI) are increasingly an axis making an angle of 541440 relative to the
used in biochemical and biomedical studies in cells, external magnetic field. In a standard MAS experi-
tissues, animals, and humans. With these techniques ment, where the NMR signal is observed after a
the resonance lines of several relatively small molec- single 901 rado frequency (RF) pulse (to be called
ular weight chemical compounds such as amino ac- single-pulse MAS or SP-MAS hereafter), the spinning
ids, lipids, and other key mobile metabolites are frequency must be larger than the broadening in ord-
measured, and their presence and intensities have er to avoid the occurrence of spectral spinning side-
been linked to tumor phenotype, tumor formation, bands (SSBs) surrounding the various resonance
tumor size, increased cell proliferation, and cell death peaks, which can overlap with other resonance lines,
pathways. However, a major problem associated rendering the interpretation of the spectra difficult
with nuclear magnetic resonance (NMR) spectro- again. In fact, in practice often the spinning fre-
scopy in intact biological tissues is that relatively quency is chosen larger than the spectral width, i.e.,
large resonance line widths are observed, often one a kilohertz or more, in order to avoid SSBs arising
to two orders of magnitude larger than the widths from the water signal, which occurs in biological
1
measured in liquids using established NMR tech- objects in a concentration of B30 mol l , and
1
niques. This is especially a problem for H NMR, which is often much stronger than the metabolite
which is the most widely used nucleus because of its signals, arising from compounds with two to three
relatively large NMR sensitivity, but has a relatively orders of magnitude smaller concentrations, even
small chemical shift range of B10 parts per million when water suppression is applied. Figure 1 shows
1
(ppm). As usually many metabolites contribute to the H water-suppressed NMR metabolite spectra of ex-
NMR signal, the result is a spectrum with severely cised rat liver, obtained in a 7 T external field (i.e.,
overlapping spectral lines, which seriously hampers a 300 MHz proton frequency) on a stationary sample
quantitative analysis of the spectra, and sometimes and with SP-MAS at different spinning speeds, illu-
even makes it impossible to assign the spectral lines strating the appearance of SSBs at low spinning
unambiguously. speeds. In fact, at 1 Hz spinning the side bands are so
In biological samples, the main mechanisms for dense that the spectrum is virtually the same as the
this broadening are the local magnetic field gradients static spectrum. In these experiments, water suppres-
arising from variations in the isotropic bulk magnetic sion was achieved by preceding the 901 pulse by
susceptibility near boundaries of intra- and extracel- a DANTE (delays alternating with nutations for
lular structures, such as the various intracellular tailored excitation) water suppression sequence, con-
compartments, air tissue interfaces near the lungs sisting of a train of equally spaced small-tip-angle
and sinuses, and bone tissue interfaces. Then, chemi- hard pulses.
cally equivalent nuclei experience different local A serious problem associated with fast SP-MAS is
magnetic fields, depending on their spatial locali- the large centrifugal force, Fc, induced in the sample
zation, giving rise to line broadening. Using cell by the spinning, which destroys tissue structures and
extracts can eliminate this broadening, but this even individual cells at high spinning rates. Fc is
334 NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning
(B)
(A)
9
6
8
7
1
3
4
5
4 3 2 1 0 4 3 2 1 0
(C) ppm
ppm (D)
1
Figure 1 300 MHz H NMR spectra of freshly excised rat liver samples obtained with different methods: (A) static sample; (B) 1 Hz
SP-MAS; (C) 40 Hz SP-MAS; (D) 4 kHz SP-MAS. The external field was 7 T. Line assignments: 1 (B0.88 ppm), triglycerides CH3
terminal, or neutral amino acid methyl, valine, leucine, isoleucine methyl; 2 (B1.28 ppm), triglycerides (CH2)n, lactate methyl,
threonine methyl; 3 (B2.04 ppm), triglycerides CHQCH2CH22CH2; 4 (B2.24 ppm), triglycerides CH2 CH2 CO; 5 (B2.8 ppm),
triglycerides CHQCH2CH22CHQCH; 6 (B3.2 ppm), choline methyl, phosphocholine methyl, b-glucose, trimethylamine-N-oxide
methyl; 7 (3.4 ppm) 8 (3.6 ppm), glucose, glycogen; and 9 (3.8 4.0 ppm), glucose, glycogen, amino acids. (Reproduced with per-
mission from Wind RA and Hu JZ (2003) Magnetic susceptibility effects in nuclear magnetic resonance spectroscopy of biological
objects. In: Recent Research Developments in Magnetism and Magnetic Materials, vol. 1, pp. 147 169.)
given by Fc ź mo2r, where m is the mass, o ź 2pF, F additional magnetic field strength induced by M is
being the spinning frequency, and r the distance from much less than H0 so that wEM/H0. With this defi-
the rotation axis to the point of interest. For exam- nition, in the material the net magnetic field induc-
ple, when F ź 2 kHz and r ź 1 cm, Fc ź 1.6 105 tion B, which is the fundamental magnetic field
times the gravitational force Fg. Therefore, the responsible for NMR, is given by B ź m0H0 þ m0ME
SP-MAS method is not viable for MRS or CSI in (1 þ w)B0 ź mrB0, where mr is the relative magnetic
large intact biological samples or in vivo studies, and permeability. M can arise from several sources: (1)
methods are needed that yield high-resolution, SSB- The nuclear magnetization. Although this magnet-
free spectra at reduced MAS frequencies. In the re- ization is responsible for the MR signal, its contri-
mainder of this article, two such methods and some bution to M can usually be neglected. (2) The
applications will be discussed, following short back- magnetization associated with slight changes in the
ground introductions about magnetic susceptibility angular velocities of paired electrons in their orbitals,
and MAS. induced by B0. According to Lenz s law this will
cause a magnetic field at the center of the orbital that
opposes B0, resulting in the well-known chemical
Magnetic Susceptibility
shift. Outside the orbital at a distance large com-
The magnetic susceptibility factor w arises from the pared with the dimensions of the orbital, the effect of
magnetization M induced in a material exposed to B0 on an electron orbital can be approximated by a
local magnetic field arising from a magnetic dipole
an external magnetic field H0, corresponding to a
magnetic induction B0 ź m0H0, in the absence of the associated with a ring current in the orbital. This
7 1
material, where m0 ź 4p 10 (T m A ) is the results in a negative value of w, the diamagnetism. (3)
magnetic permeability in vacuum. When the magneti- Paramagnetic susceptibility arises in materials con-
taining unpaired electrons, resulting in a positive
zation is oriented in the same direction as B0, w is a
scalar and the susceptibility is called isotropic. w is value of w. (4) Ferromagnetism occurs in materials
called the volumetric susceptibility and is a unitless possessing permanent magnetic moments aligned in
quantity defined as w ź M/H, where M is the magne- Weiss domains.
tic dipole moment per unit volume and H is the total Most biological objects are diamagnetic. Water
magnetic field strength. w is equal to 0 in free space and organic compounds possess susceptibility fac-
6
and is practically 0 for air. In biological systems, the tors w of the order of 4p 10 . In liquids, this
2
NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning 335
susceptibility gives rise to an induced additional where g is the angle between R and the direction of
homogeneous magnetic field in the sample, resulting the magnetic moment, defined as the z direction. It
in a shift of the resonance lines rather than a line follows that the dipolar field component along the
broadening. However, in biological samples, which z-axis, Bz, is given by
contain many intra- and intercellular structures, the
m0m
various compounds are distributed heterogeneously, Bz ź Brz þ Bgz ź ð3 cos2 g 1Þ ½1Š
4pR3
and often close to a boundary of a material with a
different susceptibility factor. Then, the susceptibility
Figure 2B shows the case that the sample is rotated
differences between the various compartments in-
with an angular frequency or about an axis making
duce magnetic field gradients in the sample. As a re-
an angle b with the external field. Then, the angle g
sult the resonance shifts become space dependent,
and the azimuth angle f become time dependent, and
resulting in a line broadening. Often, line widths of
cos g(t) is given by
the order of 0.5 ppm are observed, at least one order
of magnitude larger than their intrinsic line widths. cos gðtÞ Åº cos a cos b þ sin a sin b cos½fðtÞŠ
These values are in accordance with estimations
ź cos a cos b þ sin a sin b cosðort þ f0Þ ½2Š
based on the susceptibility differences one can en-
counter at interfaces in biological objects.
a is the angle between the rotation axis and R.
Equation [1] becomes
m0m
Magic Angle Spinning
Bz ź fð3 cos2 a 1Þð3 cos2 b 1Þ
8pR3
In order to illustrate the effect of MAS we consider
þ 3 sin 2a sin 2b cos ðort þ f0Þ
the magnetic moment m arising from a sphere of a
þ 3 sin2 a sin2 b cos½2ðort þ f0ÞŠg ½3Š
stationary diamagnetic material (see Figure 2A). At a
distance R equal to or larger than the radius a, the
Hence, Bz contains a static term and two time-
magnetic field generated by this moment has two
dependent terms. Then, in a standard SP-MAS ex-
components Br and Bg, oriented in the direction and
periment after Fourier transformation the NMR
perpendicular to R, respectively. These components
spectrum arising from the interactions between the
are given by
nuclear spins and these local fields consists of a
m0 m cos g m0 m sin g center-band line, located at the frequency determined
Br ź and Bg ź
2p R3 4p R3 by the static term, and SSBs, located at frequency
B0 B0
Brz
(t)
Br
m
R
m
R
(t)
Z B
B
z
(A)
(B)
Figure 2 Magnetic dipole field outside a sphere of a diamagnetic material with dipolar moment m induced by the external field B0:
(A) static sample; (B) rotating sample. (Reproduced with permission from Wind RA and Hu JZ (2003) Magnetic susceptibility effects in
nuclear magnetic resonance spectroscopy of biological objects. In: Recent Research Developments in Magnetism and Magnetic
Materials, vol. 1, pp. 147 169.)
336 NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning
distances 7nor, n ź 1, 2, 3,y, from the center-band in biological materials the (dia)magnetic susceptibi-
line and with amplitudes depending on the static line lity is the main source of the line broadening
width and or. Hence, by choosing the angle observed in stationary samples.
pffiffiffi
b ź cos 1 3 ź 54:741, the effect of the static term
becomes zero, irrespective of the angle a, and for
Slow-MAS Techniques
spinning speeds much larger than the spectral line
widths the SSBs have very small amplitudes and can
In solid-state NMR, several methods have been
often be neglected. As a result, the susceptibility shift
developed where slow MAS is combined with spe-
in a homogeneous liquid and the susceptibility line
cial RF pulse sequences to suppress the spinning
broadening in heterogeneous samples are eliminated.
sidebands or to separate them from the isotropic
Moreover, MAS eliminates other line broadenings as
spectrum so that a sideband-free high-resolution
well, arising from other interactions with a similar
isotropic spectrum is obtained. Examples of such
angular dependence as eqn [1]. Table 1 summa-
methodologies are total suppression of spinning side-
rizes the various interactions that can occur in a
bands (TOSS), phase-adjusted spinning sidebands
material and the effectiveness of MAS in averaging
(PASS), and phase-corrected magic angle turning
out these interactions. As already mentioned above,
(PHORMAT). While TOSS cannot be used at low
spinning speeds because of serious spectral distor-
tions, it was found that PASS and PHORMAT can be
Table 1 The impact of magic angle spinning on the various spin
modified successfully for studies of biological sam-
interactions playing a role in NMR
ples at low speeds, an order of magnitude or more
a
Interaction Impact of MAS
lower than the speeds typically used in solid-state
NMR experiments. The basic RF pulse sequences
Indirect spin spin or J-coupling N
Static spin spin dipolar coupling Y
used in SP-MAS, PASS, and PHORMAT are shown
Isotropic chemical shift N
in Figure 3. In the following paragraphs PASS and
Static anisotropic chemical shift Y
PHORMAT will be described briefly (SP-MAS is
First-order quadrupolar coupling Y
self-explanatory).
Second-order quadrupolar coupling P
Isotropic susceptibility Y
PASS
Anisotropic susceptibility P
Spin lattice relaxation time T1b N
PASS is a one-rotor-period (Tr), constant evolution
Intrinsic spin spin relaxation time T2b N
time 2D experiment, during which five p pulses are
a
Y: MAS averages the interaction to zero, N: MAS does not affect
applied, with time intervals tm1 tm6 (Figure 3B). In
the interaction, P: MAS partially averages the interaction.
b PASS, the center-band spectrum and the SSB spectra
If determined by interactions rendered time-dependent by
are separated by order. This is achieved by acquiring
molecular motions.
acq
(A)
acq
tm1 tm2 tm3 tm4 tm5 tm6
Tr
0
(B)
r1 p1 r2 p2 r3 p3 r4
t1/3 t1/3 t1/3 t2
L L L
Åš1 Åš2
Åš3
0 2Tr /3 Tr
Tr /3
(C)
Figure 3 Three RF pulse sequences used in combination with MAS. The 901 pulses are black while the 1801 pulses are gray: (A) SP-
MAS; (B) PASS; (C) the prototype PHORMAT. The various timing parameters are explained in the text. (Reprinted with permission
from Hu JZ and Wind RA (2002) The evaluation of different MAS techniques at low spinning rates in aqueous samples and in the
presence of magnetic susceptibility gradients. Journal of Magnetic Resonance 159: 92 100; & Elsevier)
NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning 337
the signal after a series of PASS experiments with magnetic susceptibility fields, the sum of the preces-
different values of the time intervals tm1 tm6. Each sion angles F1, F2, and F3 averages to the isotropic
combination of time intervals has been chosen in values of the interactions, i.e., F1 þ F2 þ F3 ź
such a way that the contribution of the signal in the oisot1. With a proper phase cycling of the projection
observed free induction decay (FID) in the acquisi- pulses, p1, p2, p3 and the receiver, the FID can be
tion dimension (t2), arising from the center band and expressed as
SSBs, is proportional to a phase factor given by
FIDðt2; t1Þ Åºexpð ioisot1ÞFIDðt2Þ½4Š
exp( ikY), where k denotes the sideband order and
y is a variable called pitch . Then after 2D Fourier
After Fourier transformation as a function of t2 and
transform with respect to t2 and y a series of spectra
then as a function of t1 a pure absorption-mode 2D
is obtained that separates the contributions for each
spectrum is obtained. Figure 4C shows an example of
k value, i.e., it separates the center-band and side-
1
water-suppressed H PHORMAT metabolite spectra
band spectra. In practice, it suffices to use n discrete
of excised rat liver tissue, obtained at a MAS spin-
values of y, varying from 0 and 2p in steps of 2p/n,
ning frequency of 1 Hz and in a 7 T field. Figure 4C
where n denotes the total number of center-band and
displays the 2D plot together with the projections
sideband spectra that have to be resolved. Figure 4A
1
along the isotropic F1 (t1) and anisotropic F2 (t2) di-
shows the stacked H PASS water-suppressed spectra
mensions. By making slices parallel to the F2 axis, the
obtained on excised rat liver in a 7 T field using a
anisotropic line shapes of each isotropic peak can be
spinning speed of 40 Hz. Water suppression was ac-
determined separately, nine of which are plotted in
hieved by preceding the PASS sequence by a DANTE
Figure 4D. In this way the susceptibility gradients
sequence. In this PASS experiment, 16 different com-
surrounding individual metabolites can be deter-
binations of delay times tm1 tm6 are used, which
mined, which could be of diagnostic value. The spec-
makes it possible to separate the center-band and 15
tral line widths in the isotropic projection are
sideband spectra without spectral aliasing. Figure 4B
comparable to, albeit slightly larger than, the widths
shows the center-band spectrum separately. It follows
obtained with 4 kHz SP-MAS (Figure 1D) and 40 Hz
that the spectral resolution is at least the same as that
PASS (Figure 4B). As will be discussed in the next
obtained in the standard fast SP-MAS experiment
section, this slight increase (a few hertz) is attributed
shown in Figure 1D, and similar results have been
to the diffusion of the metabolites in the susceptibi-
obtained in other excised tissues and organs.
lity gradients.
PHORMAT
Limitations of SP-MAS, PASS,
PHORMAT is a regular 2D experiment, with a
and PHORMAT
variable evolution time and a (fixed) acquisition time
(Figure 3C). The PHORMAT methodology is based The question arises to what values the MAS fre-
on the so-called magic angle hopping (MAH) experi- quency can be reduced with the various MAS meth-
ment, where the sample is hopped over angles of odologies, as this will determine the size and type of
1201 about an axis at the magic angle. In PHOR- biological object that can be studied with these tech-
MAT, the sample is spun slowly and continuously niques. The answer depends on the MAS methodo-
instead of hopped. The prototype PHORMAT se- logy used and the NMR properties of the magnetic
quence is shown in Figure 3C. The parameter Tr de- nuclei under investigation. Moreover, the NMR sen-
notes the rotation period of the sample, t1 is the sitivity per unit measuring time and the total
variable evolution time, and t2 is the acquisition measuring time itself are different for the various
1
time. The 901 pulses labeled r1, r2, r3, and r4 are techniques. Figure 5 shows 300 MHz H SP-MAS,
synchronized to 1/3 of the rotor period, and rotate PASS center-band, and PHORMAT isotropic spectra
the magnetization into the transverse plane. Then of a mixture of water and spherical glass beads with
during the evolution periods, t1/3, the magnetization diameters of B230 mm as a function of the spinning
precesses through angles F1, F2, andF3, respectively. frequency F. The susceptibility difference between the
The 901 pulses labeled p1, p2, and p3 are the storage beads and water, broadened the static water line to
pulses, which project a component of the precessing 3.7 kHz (12.5 ppm), at least a factor 20 larger than
magnetization after the corresponding t1/3 period to the line widths observed in biological systems. For
the z-axis, where it remains during the storage pe- SP-MAS it follows that F has to be at least of the
riods labeled L. A FID is acquired following the last order of the static line width in order to reduce the
901 pulse (r4). For local fields arising from second- SSBs. In contrast, it follows from Figure 5 that PASS
rank interactions, such as interactions with the produces a nearly sideband-free isotropic chemical
338 NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning
k = +4
k = 0
4 3 2 1 0
k = -4
k = -8
43210
(A)
ppm (B)
1
2
0 3
4
1
5
6
2
7
3
8
9
4
4 3 2 1 0
4 3 2 1 0
F2 (ppm)
(C) F2 (ppm) (D)
1
Figure 4 Top: The water suppressed H 2D-PASS spectra of freshly excised rat liver acquired at a spinning rate of 40 Hz. The
external field was 7 T. (A) The stacked 2D plot. The parameter k denotes the kth sideband; k ź 0 corresponds to the center band; (B)
1
The center-band spectrum. Bottom: The water suppressed H PHORMAT spectra of a freshly excised rat liver sample, acquired at a
sample spinning rate of 1 Hz. The external field was 7 T; (C) The contour plot of the 2D PHORMAT spectrum along with its anisotropic
(F2) and isotropic (F1) projections. The F1 projection was obtained by summing only the data inside the dotted box; (D) The stacked plot
of the anisotropic line shapes corresponding to nine isotropic peaks, which were obtained by taking a slice parallel to the F2 axis at the
center of each isotropic peak (F1). ((A) and (B) are reproduced with permission from Wind RA and Hu JZ (2003) Magnetic susceptibility
effects in nuclear magnetic resonance spectroscopy of biological objects. In: Recent Research Developments in Magnetism and
Magnetic Materials, vol. 1, pp. 147 169. (C) and (D) are reproduced with permission from Hu JZ, Rommereim DN, and Wind RA
1
(2002) High-resolution H NMR spectroscopy in rat liver using magic angle turning at a 1 Hz spinning rate. Magnetic Resonance in
Medicine 47: 829 836.)
shift spectrum at a spinning frequency as low PASS data, whereas at this spinning rate the spectrum
as 50 Hz. (The peaks marked by the symbol in contained B90 visible sidebands, requiring at least
Figure 5 are aliased sidebands, arising because only 90 increments.) Moreover, the isotropic line width
32 evolution increments were used to acquire the observed at 50 Hz is similar to that measured at
1
2
3
4
1
F
(ppm)
5
6
7
8
9
ppm
NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning 339
SP-MAS PASS
PHORMAT
1 kHz
ND
5 4
250 Hz
ND
50 Hz
" "
1 Hz
ND
20 10 -10 20 10 0 -10 20 10 -10
0 0
ppm ppm ppm
1
Figure 5 H MAS spectra obtained at different spinning rates by SP-MAS, PASS, and PHORMAT on a mixture of H2O and spherical
glass beads with diameters of 230720 mm. The PASS spectra are the center-band spectra, the PHORMAT spectra are the isotropic
projections. (Reprinted with permission from Hu JZ and Wind RA (2002) The evaluation of different MAS techniques at low spinning
rates in aqueous samples and in the presence of magnetic susceptibility gradients. Journal of Magnetic Resonance 159: 92 100;
& Elsevier.)
higher spinning frequencies. However, at this speed metabolite T2 values are of the order of 30 ms, with
the signal is seriously attenuated. This is caused by PASS spinning frequencies of B40 Hz or larger
the decay of the magnetization during the rotor pe- should be used.
riod Tr prior to the acquisition (cf. Figure 3B). The T2 attenuation is avoided in a PHORMAT
During this period the magnetization dephases in experiment. Here, the magnetization is stored paral-
the transverse plane, and is partially refocused by the lel to the main field direction with a maximum
1801 pulses. Hence, the time constant governing the duration of 2/3 times the rotor period, which means
decay is approximately given by the intrinsic spin that the spinning frequency has to be large compared
1
spin relaxation time T2, which means that the mini- with the spin lattice relaxation rate (T1) of the
1
mum spinning frequency should be larger than spins rather than (T2) in order to avoid signal
1
(T2) , i.e., the isotropic line width, in order to attenuation. Hence, for the water/bead mixture,
avoid serious signal losses. This has as a consequence where the water T1 is several seconds, the spinning
that for biological samples, where the minimum speed can be made as low as 1 Hz without causing
340 NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning
serious signal attenuation, and the same is true for chronic centrifuging experiments, and dB/dt is
1 1
biological samples, where the metabolite T1 values B10 Ts , well below the threshold of B90 Ts
are at least an order of magnitude larger than the for which nerve stimulations have been reported. In
T2 values. However, it follows from Figure 5 that fact, 15 mice were spun in a 2 T field at frequencies
at ultralow spinning speeds the isotropic line up to 8 Hz and for durations up to 60 min without
width increases. This is due to the diffusion of the causing any apparent short-term or long-term health
water molecules in the susceptibility gradients. This effects. Hence, it is possible to use ultraslow PHOR-
diffusion-induced broadening, which cannot be elimi- MAT for in vivo studies on live animals. Figure 6
nated with MAS techniques, is proportional to shows the first result of such an experiment, obtained
pffiffiffiffiffiffiffiffiffi
ffi
G D=F, where G is the susceptibility gradient, D on the middle section of the body of an (anesthetized)
is the diffusion coefficient, and F is the MAS fre- female BALBc mouse between the dotted lines shown
quency. It follows that this broadening is much less in Figure 6A and the arrows shown in Figure 6B.
1
in biological samples, where both G and D are at Figures 6C and 6D shows the H 85 MHz water-
least an order of magnitude less than in the water/ suppressed metabolite spectra obtained in a station-
bead mixture. Comparing the SP-MAS, PASS, and ary mouse and a mouse subjected to 1.5 Hz MAS,
PHORMAT experiments of the excised rat liver respectively. Even in this relatively low field a signi-
shown above, it was estimated that the diffusion- ficant increase in spectral resolution is obtained.
induced line broadening in a 1 Hz PHORMAT ex-
periment is B2 Hz at 7 T, considerably less than the
Future Perspectives
intrinsic line width.
Finally, it is worth noting that compared to PASS, It can be concluded that it is possible to significantly
PHORMAT has a considerably lower NMR sen- increase the resolution in the proton NMR metabo-
sitivity and often requires a longer measuring time. lite spectra in intact biological samples by PASS and
The sensitivity loss is mainly due to an intrinsic loss PHORMAT. It was found that for small samples,
of a factor of 4 resulting from the use of two storage with sizes of a few millimeter or less, where spinning
pulses p1 and p2 in the PHORMAT sequence, cf. speeds of 40 Hz or more can be tolerated, PASS is the
Figure 3C (the storage pulse p3 is omitted in a regular
method of choice because of its superior sensitivity
PHORMAT experiment). Also, PHORMAT often and short measuring time. For larger biological sam-
requires a relatively large number of evolution steps, ples, including animals, ultraslow-MAS PHORMAT,
resulting in a long measuring time, an hour or more. allowing spinning speeds as low as 1 Hz, has to be
Although the performance of PHORMAT can be used. In a 7 T field with PASS the spectral line widths
improved, e.g., by applying multiple-echo acquisi- are reduced by an order of magnitude or more to
tion, PASS should be considered as the method of values determined by the intrinsic T2, originating
choice if the sample can tolerate spinning speeds of from the various spin spin dipolar interactions, ren-
tens of hertz. PHORMAT should be reserved for re- dered time-dependent by the molecular motions.
search in larger biological samples, including in vivo
With 1 Hz PHORMAT slightly larger isotropic line
applications. widths are observed, resulting from the molecular
diffusion in the susceptibility gradients. Increasing
the spinning speed, if allowed, will reduce this con-
In Vivo PHORMAT
tribution. In external fields larger than 7 T the line
Two effects associated with spinning a live animal width reductions are expected to become even larger,
can cause harm: the centrifugal forces induced in the as the residual isotropic line widths are essentially
animal, and the effects induced by the external field-independent, whereas the susceptibility broad-
magnetic field, rendered partially time-dependent ening increases linearly proportional to the field.
from the rotating animal s point of view when spin- Hence, with PASS or PHORMAT the full benefits of
ning inside the magnet. This time-dependent field high-field NMR are obtained.
dB/dt can cause nerve and cardiac stimulations in a It is worth noting that the slow-MAS methodology
similar way as pulsed field gradients. However, both is a new area of research, and that several further
effects can be small in a PHORMAT experiment. For improvements are necessary to make PASS and
instance, when a mouse is placed in a cylinder with a PHORMAT more viable methods for biochemical
1 cm radius, and rotated around the magic angle in a and biomedical research. Particularly, specific com-
2T field and with a spinning speed of 1 Hz, the maxi- binations of RF and pulsed-field-gradient sequences
mum centrifugal force at the periphery of the animal need to be developed so that PASS or PHORMAT
is 0.04 times the gravitational force Fg, about two spectra can be obtained of a select volume in the
orders of magnitude below the forces applied in sample or the animal rather than the whole sample or
NMR SPECTROSCOPY APPLICATIONS / Proton NMR in Biological Objects Subjected to Magic Angle Spinning 341
the main part of the body. If these implementations
are successful, it can be expected that PASS and
PHORMAT will significantly increase the utility of
proton metabolite NMR spectroscopy for biochem-
ical and biomedical studies in cells, tissues, organs,
and animals.
Finally, the question arises whether the slow-MAS
approach will ultimately reach the hospital to
investigate patients. Obviously in this case PHOR-
MAT is the only candidate. Although spinning a pati-
ent at a frequency of (e.g., 1 Hz) may cause no phys-
ical harm, it is likely that it will induce unacceptable
(A)
stress in many patients. An alternative approach
might be to rotate the external magnetic field instead
of the patient, either mechanically or electronically,
or by rotating both the magnetic field and the patient
in opposite directions. In this way the spinning
speed of the patient can be reduced. The future will
tell whether this approach will become a viable
option.
See also: Nuclear Magnetic Resonance Spectroscopy:
Overview; Principles. Nuclear Magnetic Resonance Spec-
troscopy Applications: Pharmaceutical. Nuclear Magne-
tic Resonance Spectroscopy Techniques: Solid-State.
(B)
Further Reading
Andrew ER (1996) Magic angle spinning. In: Grant DM
and Haris RK (eds.) Encyclopedia of Magnetic Reso-
nance, pp. 2891 2901. New York: Wiley.
Antzukin ON (1999) Sideband manipulation in magic-
angle-spinning nuclear magnetic resonance. Progress in
NMR Spectroscopy 35: 203 266.
(C)
Boxerman JL, Weisskopf RM, and Rosen BR (2000) Sus-
ceptibility effects in whole body experiments. In: Young
IR (ed.) Biomedical Magnetic Resonance Imaging and
× 5
Spectroscopy, pp. 654 661. New York: Wiley.
Callaghan PT (1993) Principles of Nuclear Magnetic Reso-
nance Microscopy. Oxford: Clarendon Press.
4.0 3.0 2.0 1.0 0 Gadian DG (2000) Animal methods in MRS. In: Young IR
ppm
(ed.) Biomedical Magnetic Resonance Imaging and Spec-
(D)
troscopy, pp. 898 904. New York: Wiley.
Figure 6 (A, B) The mouse-MAS NMR probe: (A) an anest-
Hu JZ, Wang W, and Pugmire RJ (1996) Magic angle
hetized female BALBc mouse placed in a mouse-shaped cavity in
turning and hopping. In: Grant DM and Haris RK (eds.)
one-half of the rotor; (B) top part of the probe with the mouse and
Encyclopedia of Magnetic Resonance, pp. 2914 2921.
the mold mounted in place. The area between the arrows is the
New York: Wiley.
NMR-sensitive area of the NMR coil; (C) The anisotropic (F2)
projection of the 2D PHORMAT spectrum, obtained on the part of Mukherji SK (ed.) (1998) Clinical Applications of MR
the mouse body between the dotted lines shown in (A); (D) The Spectroscopy. New York: Wiley.
isotropic (F1) projection of the 2D PHORMAT spectrum, obtained
Slichter CP (1990) Principles of Magnetic Resonance,
on the same part of the mouse body. ((A) and (B) are reproduced
pp. 392 406. Berlin: Springer.
with permission from Wind RA, Hu JZ, and Rommereim DN
Smith ICP and Bezabeh T (2000) Tissue NMR ex vivo. In:
1
(2003) High resolution H NMR spectroscopy in a live mouse
Young IR (ed.) Biomedical Magnetic Resonance Imaging
subjected to 1.5 Hz magic angle spinning. Magnetic Resonance
and Spectroscopy, pp. 891 897. New York: Wiley.
in Medicine 50: 1113 1119. (C) and (D) are reproduced with
VanderHart DL (1996) Magnetic susceptibility & high
permission from Wind RA and Hu JZ (2003) Magnetic suscepti-
resolution NMR of liquids & solids. In: Grant DM and
bility effects in nuclear magnetic resonance spectroscopy of
Haris RK (eds.) Encyclopedia of Magnetic Resonance,
biological objects. In: Recent Research Developments in Magnet-
ism and Magnetic Materials vol. 1, pp. 147 169.) pp. 2938 2946. New York: Wiley.
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