Am J Physiol Heart Circ Physiol 297: H1010H1019, 2009.

First published June 26, 2009; doi:10.1152/ajpheart.01195.2008.

Novel strategy for measuring creatine kinase reaction rate in the in vivo heart
Qiang Xiong,1 Qinglu Li,1 Abdul Mansoor,1 Mohammad Nurulqadr Jameel,1 Fei Du,2 Wei Chen,2
and Jianyi Zhang1,2
1

Cardiovascular Division, Department of Medicine, and 2Center for Magnetic Resonance Research, Department of Radiology,
University of Minnesota Medical School, Minneapolis, Minnesota
Submitted 14 November 2008; accepted in final form 24 June 2009

Xiong Q, Li Q, Mansoor A, Jameel MN, Du F, Chen W, Zhang

J. Novel strategy for measuring creatine kinase reaction rate in the in
vivo heart. Am J Physiol Heart Circ Physiol 297: H1010 H1019, 2009.
First published June 26, 2009; doi:10.1152/ajpheart.01195.2008.In the
heart, the creatine kinase (CK) system plays an important role in the
cascade of ATP production, transportation, and utilization. The forward pseudo-first-order rate constant for the CK reaction can be
measured noninvasively by the 31P-magnetic resonance (MR) spectroscopy magnetization saturation transfer (MST) techniques. However, the measurement of MST in the in vivo heart is limited by the
lengthy data acquisition time, especially for studies requiring spatial
localization. This technical report presents a new method for measuring ATP production rate via CK that can reduce the MST data
acquisition time by 82%. This method is validated using an in vivo pig
model to evaluate the forward pseudo-first-order rate constant of
myocardial CK reaction noninvasively.
magnetic resonance spectroscopy; metabolism; high-energy phosphates; adenosine 5-triphosphate

as a compensatory response to
various stressors, including pressure overload, volume overload, and myocardial infarction. After a prolonged period of
compensatory hypertrophy with restored ventricular wall
stress, severe ventricular dysfunction often occurs with signs of
congestive heart failure (CHF). The exact mechanisms underlying this transition from compensated ventricular hypertrophy
to CHF are likely multifactorial, but alterations in myocardial
bioenergetics have been considered to be important factors in
this pathway (14, 15). The creatine kinase (CK) enzyme
catalyzes the reaction that converts adenosine diphosphate
(ADP) and phosphocreatine (PCr) to adenosine triphosphate
(ATP) and creatine (Cr) (13). This maintains high ADP levels
in the mitochondria, where ATP is generated, and low ADP
levels at the contractile apparatus, where ATP is utilized, thus
facilitating the production and utilization of ATP (26, 28). Our
laboratory has previously reported that the myocardial CK forward flux rate is significantly reduced in hypertrophied hearts (20,
29 31). The severity of this reduction is linearly related to the
severity of the hypertrophy and left ventricular (LV) dysfunction
(29). Recently, using a low-field 1.5-T magnet, Weiss and associates examined heart failure patients and found that the CK flux
was reduced in patients with LV hypertrophy and CHF (23, 27).
Importantly, the studies demonstrate that the severity of the
reduction in CK flux rate is related to the severity of LV contractile dysfunction (23) and suggest that the CK flux rate may be a
more sensitive myocardial energetic indicator for the severity of
LV dysfunction compared with the PCr-to-ATP ratio in heart

CARDIAC HYPERTROPHY OCCURS

Address for reprint requests and other correspondence: J. Zhang, Univ. of

Minnesota, 268 Variety Club Research Center, 401 East River Rd., Minneapolis, MN 55455 (e-mail: zhang047@umn.edu).
H1010

failure patients (27). However, the quantitative assessment of CK

forward flux rate at low field magnet may be limited by the low
signal-to-noise ratio (SNR).
Transmurally differentiated measurements of ATP production rate
via CK require longer data acquisition time for spatial localization. In
a dog model, the CK flux rate was reported to be heterogeneous
across the LV wall of normal heart, with the lowest rate in inner
layers of the LV wall (21). These CK flux measurements with
transmural differentiation took 6 h to complete the data acquisition
for one experimental condition, suggesting unlikely to perform experimental interventions, using a conventional approach.
Here, we demonstrate a novel strategy for magnetization
saturation transfer (MST) study of ATP production rate via CK
forward reaction. By employing numerically optimized presaturation delays, this strategy results in a 82% and a 56%
reduction in total data acquisition time and saturation pulse
duration, respectively. The novel strategy was validated using
an in vivo porcine heart model, which generated identical
forward pseudo-first-order rate constant (kf) values compared
with that measured with the conventional approach from the
same heart. The significant reductions in acquisition time, as
well as saturation pulse duration provided by this novel approach can potentially facilitate the future high field cardiac
CK MST studies with spatial localization on patients with heart
failure in high field magnet.
METHODS

All experiments were performed in accordance with the animal use

guidelines of the University of Minnesota, and the experimental protocol
was approved by the University of Minnesota Research Animal Resources Committee. The investigation conformed to the Guide for the
Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).
Open-chest surgery preparation for 31P-magnetic resonance spectroscopy. Details of the open-chest surgery preparation for 31Pmagnetic resonance spectroscopy (MRS) have been described previously (12, 32). Briefly, young Yorkshire swine (30 kg) were
anesthetized with pentobarbital sodium (initial dose 30 mg/kg iv,
maintenance dose 4 mg kg1 h1 iv), intubated, and ventilated with
supplemental oxygen on a respirator. Polyvinyl chloride catheters
(3-mm outer diameter) were inserted into the ascending aorta, superior
vena cava, and LV for hemodynamic monitoring. The ascending aorta
catheter was placed via the left internal carotid artery, and the two
intravenous catheters were placed through the left external jugular vein.
The heart was exposed via a sternotomy and suspended in a pericardial
cradle. The LV catheter was introduced through the apical dimple. Aortic
and LV pressures were measured by pressure transducers positioned at
midchest level and recorded on an eight-channel recorder. Ventilation
rate, volume, and inspired oxygen content were adjusted to maintain
physiological values for arterial PO2, PCO2, and pH. Aortic and LV
pressures were continuously monitored throughout the study.
31
P-MRS. Measurements were performed in a 40-cm bore 4.7-T
magnet interfaced with a SISCO (Spectroscopy Imaging Systems, Fre-

0363-6135/09 $8.00 Copyright 2009 the American Physiological Society

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mont, CA) console. 31P- and 1H-NMR frequencies were 81 and 200
MHz, respectively. Radio-frequency transmission and signal detection
were performed with a 28-mm-diameter double-tuned surface coil sutured directly to the epicardium of anterior LV wall. The coil was
cemented to a sheet of silicone rubber 0.7 mm in thickness and 20%
larger in diameter than the coil itself. A capillary containing 15 l of 3 M
phosphonoacetic acid was placed at the coil center to serve as a reference.
The proton signal from water detected with the surface coil was used to
homogenize the magnetic field and to adjust the position of the animal in
the magnet so that the coil was at or near the magnetic isocenter. This was
accomplished using a spin-echo experiment and a readout gradient. The
information gathered in this step was also utilized to determine the spatial
coordinates for spectroscopic localization (10, 17). Chemical shifts were
measured relative to PCr, which was assigned a chemical shift of 2.55 ppm
relative to 85% phosphoric acid at 0 ppm. Baseline myocardial high-energy
phosphate data acquisition was achieved with a 90 adiabatic half-passage
pulse, and selective saturation on ATP- was achieved with B1-insensitive
train to obliterate signal (BISTRO), as previously reported (16).
kf calculation of conventional MST methods. Table 1 summarizes
all of the abbreviations and symbols.
A conventional MST method consists of two experiments, namely,
progressive and steady-state saturations. The CK enzyme catalyzes
the reaction PCr ADP H k^f Cr ATP. The progressive
saturation transfer experiment employs multiple data acquisitions with
progressively prolonged saturation on ATP- to retrieve the apparent
relaxation time constant of PCr by fitting its signal intensity to the
following equation (2):

app
M PCrt M0,PCr T 1,PCr
kf exp

app
T 1,PCr

app
T 1,PCr
int
T1,PCr

(1)

where M0,PCr is the thermal equilibrium signal intensity of PCr detected

int
is the intrinsic spin-lattice relaxation time of PCr; and
by MRS; T1,PCr
app
T1,PCr
is the apparent relaxation time of PCr when ATP- is continuously saturated and defined according to the following equation (2):

Table 1. Abbreviations and symbols

Abbreviation

MST
MRS
CK
d1
d2
FID
kf
M0
Mshort
Mss
MTR,PCr

nt
T app
1
T mix
1

TR

Actual Physical Meaning

Magnetization saturation transfer

Magnetic resonance spectroscopy
Creatine kinase
Presaturation delay employed in the MST experiment
Duration of saturation pulse in the MST experiment
Free induction decay
Pseudo-first-order rate constant to characterize the ATP
production rate via CK
Magnetization measured by 31P-magnetic resonance
spectroscopy under fully relaxed condition
Magnetization measured with very short repetition time.
This measurement, in combination with M0, can yield an
approximate value of Tint
1 , according to Eq. 14
Steady-state magnetization of a spin (e.g., PCr) when the
counterpart spin (e.g., ATP-) is continuously saturated,
as dictated by Eq. 1
Magnetization measured with ATP- continuously saturated.
The TR can be arbitrary. This, in combination with
app
Mss,PCr, can yield accurate value of T1,PCr
, according to
Eq. 10
Number of averaged FIDs for one spectrum
Apparent longitudinal relaxation time constant of a spin
(e.g., PCr) when the counterpart spin (e.g., ATP-) is
continuously saturated, as dictated by Eq. 1
Apparent longitudinal relaxation time constant when
chemical exchange is involved. Tmix
is an approximation,
1
because the actual recovery of spin magnetization under
chemical exchange is not an exact exponential curve
Repetition time of pulse sequence in MST experiment
AJP-Heart Circ Physiol VOL

p31

Gx
Gy
Gz
d2

d1

Fig. 1. A general pulse sequence design for magnetization saturation transfer

(MST) study. d1 is the duration of presaturation delay, d2 is the duration of
saturation process, and pwat is the duration of readout pulse plus acquisition
time. Total repetition time TR d1 d2. FID, free induction decay; G,
gradient (Gx, X-direction gradient strength).
app1
int1
T 1,PCr
T 1,PCr
kf

(2)

The steady-state saturation transfer experiment employs infinitively

app
) to find the
long saturation on ATP- (usually 35 times of T 1,PCr
steady-state signal intensity of PCr (Mss,PCr), and then the kf can be
calculated according to the following equation (2):
kf

MPCr
M0,PCr

app
T1,PCr

(3)

where MPCr M0,PCr Mss,PCr.

A steady-state saturation transfer experiment alone can determine
the kf, according to the following equation (22):
kf

MPCr
Mss,PCr

int
T1,PCr

(4)

The exact value of kf requires knowledge of intrinsic spin-lattice

relaxation time of PCr. In the presence of chemical exchange, the
progressive saturation experiment is the gold standard for measuring
int
T1,PCr
. However, since it has been suggested by numerous studies that
int
is constant among subjects with different physiological and
the T1,PCr
pathological conditions (1, 2, 20, 30), and even among different
species (8), valid comparisons of CK kinetics between subjects can
still be achieved based on the MPCr-to-Mss,PCr ratio. Moreover, a
recent method developed by Spencer and colleagues (11, 24) allows
fast measurement of intrinsic spin-lattice relaxation times in the
presence of chemical exchange, without using multipoint progressive
saturation experiment and curve fitting.
Numerical analysis of MST governing equations and novel MST
strategy. A standard pulse sequence for CK MST experiment is
composed of three segments (Fig. 1): a presaturation delay (d1),
selective saturation on ATP- (d2), and 90 readout pulse followed
by free induction decay (FID) acquisition (pwat). The duration of
the readout pulse plus acquisition is usually 100 ms and neglectable; thus total repetition time (TR) is approximated by d1 d2.
The change of PCr and ATP- magnetizations along the z direction
[M(t)] during this saturation pulse sequence could be described by
the modified Bloch-McConeell equations (3, 18) shown below:

dMPCrt M0,PCr MPCrt

(5)
kfMPCrt k rMATPt

int
dt
T1,PCr
t 0, d1
dMATPt M0,ATP MATPt
kfMPCrt krMATPt

int
dt
T1,ATP
(6)

dMPCrt M0,PCr MPCrt

kfMPCrt

int
t d1, d1d2
dt
T1,PCr
MATPt 0

(7)
(8)

A numerical analysis of the above equations is shown in Fig. 2, where

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SATURATION TRANSFER EXPERIMENTS USING MR SPECTROSCOPY

Fig. 2. The numerical analysis of governing

Eqs. 5 8. Time dependence of PCr and
ATP- magnetizations in z direction during
MST pulse sequence (see in Fig. 1) is plotted
with different presaturation delays: 13.5 s
(A), 2 s (B), 1.4 s (C), and 0 s (D). Dotted
lines indicate 5% deviations from theoretical Mss,PCr value as given by Eq. 9, from
which the minimal saturation time d2,min is
defined, such that MPCr at the end of saturation process just lies between those lines.
Simulation parameters are as follows: kf
int
int
0.44 s1, T1,PCr
4.5 s, T1,ATP
1.5 s, and
M0,PCr/M0,ATP 2. The simulation demonapp
strated that the values of Mss,PCr and T1,PCr
are
independent of d1. See text for definition of
terms.

the time dependence of PCr and ATP- magnetizations is plotted

against different presaturation delays (d1). This figure suggests two
important points: 1) the MPCr relaxation is a single exponential
app
as long as
curve (decay or recovery) characterized only by T1,PCr
ATP- is fully saturated [i.e., MATP(t) 0]; and 2) the same
steady-state value of PCr magnetization (Mss,PCr) is achieved regardless of d1. In fact, the explicit expression of Mss,PCr can be obtained by
solving Eq. 7:
M ss,PCr M0,PCr

Tapp
1,PCr
int
T 1,PCr

M0,PCr
int
1T 1,PCr
kf

(9)

Based on the numerical analysis of Eqs. 5 8, which are the governing

equations for CK MST experiments, we have derived the following
equations (Eqs. 10 13) and proposed a novel strategy for MST
experiment that can be applied to any solid organ. This novel approach has the following significant benefits.
1) For progressive saturation experiment, short d1 (d1 0) can be
app
as measured by convenemployed (Fig. 2D) to yield the same T1,PCr

deviation

deviation

deviation

deviation

tional method, where long d1 would be employed (Fig. 2A). The short
d1 strategy can offer great reduction of TR. In case of a very limited
time window for MST experiment, only one spectrum with ATP-
saturated is needed in addition to Mss,PCr measurement to unambiguapp
, according to the following equation:
ously solve the T1,PCr
app
TR/ln1 MTR,PCr /Mss,PCr
T 1,PCr

where MTR,PCr is the PCr signal intensity acquired with a TR and

continuous saturation on ATP-.
2) An optimal d1 (d1,opt) can be employed (Fig. 2B) to yield the
same Mss,PCr as measured by conventional method, where long d1
followed by long saturation time (d2) would be employed. The d1,opt
strategy not only reduces the TR but also shortens the duration of
saturation pulse required for Mss,PCr quantification (Fig. 3). This
reduction of the saturation pulse duration results in a minimal specific
absorption rate, which is often an Food and Drug Administration
concern in clinical studies.

Fig. 3. d1,opt seeking by numerical solving

of Eqs. 5 8. Parameters for numerical simulation are as follows: kf (0.2, 0.8) s1,
int
int
int
4.5 s, T1,PCr
/T1,ATP
3, and M0,PCr/
T1,PCr
M0,ATP 2. A: three-dimensional plot
showing the dependence of minimal saturation time (d2,min) on kf and d1. B: a twodimensional view of A. The solid line (d1
2 s) represents d1,opt condition because the
maximum value of d2,min (defined as d2,opt)
throughout the preset kf range is the smallest.
These simulations demonstrated that the duration of saturation time (d2) would be minimal when d1,opt is applied compared with all
other cases. See text for definition of terms.

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int
Fig. 4. Spectra obtained for T1,PCr
calculation.
int
T1,PCr
was estimated from Spencers method
(11) using two spectra: Mshort,PCr [TR 0.3 s,
number of averaged FIDs for one spectrum
(nt) 480] (A) and M0,PCr (TR 12 s, nt
12) (B). The vertical scale of A is 15 times that
of B, and a larger data acquisition number was
employed in A for better illustration of the
spectra. Both spectra were obtained with a total
acquisition time of 2.4 min. The spectra
int
yielded a T1,PCr
value of 4 s, according to Eq.
14. 2,3-DPG, 2,3-diphosphoglycerate. See text
for definition of terms.

mix
int
lnT1,PCr
kf1 1
d 1,ideal T 1,PCr

RESULTS

d1,opt Calculation. As shown in Fig. 2, in principle, for each

CK system with known parameters, there exists an ideal
presaturation delay (d1,ideal, shown in Fig. 2C) such that the
PCr signal intensity after presaturation delay just equals the
steady-state value after a long saturation on ATP-, i.e.
M PCrd1,ideal Mss,PCr

(11)

When a d1,ideal is applied, an accurate Mss,PCr can be measured

without any saturation process; thus the total TR will be the
shortest. The relaxation process of PCr signal intensity during
presaturation delay can be approximately described by the
following equation (16):
mix
M PCrt M0,PCr 1expt/T 1,PCr

(12)

mix
1,PCr

where T
is the approximate apparent relaxation time of PCr
undergoing chemical exchanges with ATP- and depends on
int
int
T1,PCr
, T1,ATP
, M0,PCr/M0,ATP, and kf. By combining Eqs. 10
12, the ideal presaturation delay can be calculated as:

(13)

Equation 13 is not helpful for practical use, since d1,ideal

calculation requires a kf value, which is the purpose of the
experiment itself. However, based on literature and/or prior
experiences, a confident estimate of kf range for a specific
study can be made, then an optimized presaturation delay
(d1,opt) can be derived (in contrast to d1,ideal, which requires the
exact value of kf), such that, when d1,opt is applied, the required
saturation time for accurate quantification of Mss,PCr is minimal
for all possible kf values within the estimated range.
Based on previous CK reaction rate studies, kf ranges from
0.2 to 0.8 s1 under physiological conditions (1, 5, 20, 22, 23,
int
25, 29, 30), and T1,PCr
ranges from 2 to 6 s (1, 5, 20, 29, 30).
Other parameters that are required for calculation of d1,opt
int
include T1,ATP
and M0,PCr-to-M0,ATP ratio. Under physiological conditions, M0,PCr/M0,ATP in the heart is 2 (25, 27, 33),
int
int
while the ratio of T1,PCr
to T1,ATP
is 3 (2, 5, 6, 9). An example
int
of d1,opt calculation for T1,PCr
of 4.5 s is illustrated in Fig. 3. The
dependence of minimal saturation time (d2,min, defined as the

Fig. 5. Representative steady-state saturation

transfer spectra for d1,opt strategy validation.
Thick arrows indicate the frequency of saturation pulse. Spectrum A1 is the M0,PCr measurement (TR 12 s and no saturation);
spectrum A2 is the control spectrum with
saturation pulse set downfield from PCr symmetrically opposite from ATP- (d1 12 s,
d2 8 s). Spectrum A3 A1 A2. Spectra
B2 and B1 are Mss,PCr measurements using
d1,opt (d1 2 s and d2 3.5 s) and conventional (d1 12 s and d2 8 s) approaches,
respectively. Spectrum B3 B1 B2. All
spectra were acquired with 12 averaged FIDs,
and the total acquisition times for spectra A1,
A2, B1, and B2 were 2.4, 4, 4, and 1.1 min,
respectively. The spectra demonstrated an
identical Mss,PCr measurement obtained by
d1,opt and conventional approaches and no
spillover effect on PCr signal provided by
B1-insensitive train to obliterate signal
(BISTRO) saturation pulse.
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Fig. 6. Typical progressive saturation spectra

from same heart with different presaturation
delays of 7 s (A), 2 s (B), and 0 s (C). Spectra
17 (bottom to top) represent prolonged saturation on ATP- of 0.5, 1, 1.5, 2.5, 4, 6, and
8 s, respectively. Total data acquisition times
for A, B, and C are 14.5, 7.5, and 5.6 min,
respectively. These spectra confirmed the simulation results as shown in Fig. 2.

minimal saturation time giving rise to a PCr magnetization that

is within 5% deviation from theoretical Mss,PCr value, as
given by Eq. 9) upon d1 and kf can be obtained by numerically
solving Eqs. 5 8 (Fig. 3A). Among the d2,min vs. kf curves
obtained from different d1 values (Fig. 3B), there will be one
that has the smallest maximum value throughout the physiological kf range. This curve represents the optimal value of
presaturation delay (d1,opt) and the corresponding optimal duration of saturation pulse (d2,opt) required for the physiological
kf range.
Validation of the novel MST strategy in in vivo heart. We
firstly validated the d1,opt strategy. To calculate the d1,opt and
d2,opt for MST experiment, we have to estimate the intrinsic
int
spin-lattice relaxation time of PCr (T1,PCr
). Using the method
proposed by Horska et al. (11), a short-TR spectrum (see in

Fig. 4A, TR 0.3 s) and M0,PCr (Fig. 4B, TR 12 s) were

int
used to calculate the T1,PCr
, according to Eq. 14:

int
TRshort ln 1
T 1,PCr

Mshort,PCr
M0,PCr

(14)

where TRshort and Mshort,PCr stand for the TR and PCr signal
intensity of spectrum 4A, respectively. The calculation resulted
int
int
in an estimated T1,PCr
of 4 s; therefore, a T1,PCr
value of 4.5 s
(10% overestimation based on the mathematical simulation to
int
compensate the possible errors in T1,PCr
measurement) was
employed to calculate the d1,opt and d2,opt. Other parameters
used in calculation are as follows: kf (0.2, 0.8) s1, M0,PCr/
int
int
M0,ATP 2, and T1,PCr
/T1,ATP
3. The calculation yielded a
d1,opt value of 2 s and a d2,opt of 3.3 s. To compensate possible
int
int
deviations in M0,PCr/M0,ATP and T1,PCr
/T1,ATP
estimation, a
saturation time of 3.5 s was applied based on the mathematical
simulation. The spectra obtained with d1,opt and conventional
strategies are shown in Fig. 5. There was no detectable difference in Mss,PCr measurement between the d1,opt and the conventional strategies (Fig. 5, B1B3). However, substantial
reductions in saturation time (56%) and total TR (73%) were
granted by using the d1,opt strategy. A control spectrum (Fig. 5,
A2) with saturation pulse set downfield to PCr symmetrically
opposite from ATP- was also acquired. By comparing it with
Table 2. Comparison of measurements between novel and
conventional MST strategies from the same hearts of
four pigs

Fig. 7. Quantification of progressive saturation experiments (typical set of

spectra illustrated in Fig. 6). PCr peak integrals obtained from each progressive
saturation experiment (d1 of 0, 2, and 7 s) were normalized by the M0,PCr value
of each heart and plotted against the saturation pulse duration. Data shown
were from four independent pig hearts, with error bars representing the
standard deviation. Also shown in the figure were simulation results (lines)
int
using the kf and T1,PCr
values from Table 2. This figure provided a quantitative
app
.
validation of our novel MST strategy. See text for definition of T1,PCr
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Strategy

M0,PCr
Mss,PCr/M0,PCr

app
T1,PCr
,s

app
T1,PCr
,s

kf, s

Conventional
Novel

0.650.01
0.650.01

1.530.05
1.560.08

4.30.2
4.40.2

0.420.01
0.420.03

In the conventional approach, progressive saturation transfer experiment

app
with long d1 was employed to obtain T1,PCr
, and long d1 plus long d2 was
app
employed for Mss,PCr measurement. kf was calculated using Eq. 3, and T1,PCr
was calculated using Eq. 2. The novel approach used d1,opt plus d2,opt to obtain
app
the Mss,PCr measurement. T1,PCr
was calculated from two spectra (B2 in Fig. 5
app
and C3 in Fig. 6), according to Eq. 10: T1,PCr
TR/ln(1 MTR,PCr/Mss,PCr).
Statistical analysis showed no significant difference in any measurement
obtained by novel and conventional strategies.

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SATURATION TRANSFER EXPERIMENTS USING MR SPECTROSCOPY

Table 3. Comparison of total data acquisition time required

for MST measurements between novel and
conventional strategies
Strategy

M0 and Mss,
TR nt, s

app
T1,PCr
, TR
nt np, s

kf, min

Conventional
Novel

(12 20) 12
(12 5.5) 12

14.6 12 6
1.5 30 1

23.9
4.3

app
T1,PCr
was calculated from curve fitting of 6 time points (np 6). In the
conventional approach, 14.6 is the average TR for 6 points. All points were
acquired with 12 averaged FIDs (nt 12). In the novel approach, only TR
app
1.5 s spectrum (nt 30, Fig. 6C3) was used to calculate T1,PCr
, together with
Mss measurement (nt 12, Fig. 5B2), according to Eq. 10.

2A, A1

3A, A

M0,PCr measurement (Fig. 5, A1), there was no detectable

spillover effect on PCr peak with a saturation time of 8 s.
A further validation of our novel MST strategy was performed using progressive saturation experiments with long,
short, and optimal presaturation delays (Fig. 6). As saturation
times prolonged, PCr peak intensities from all three different
presaturation delays approached toward the same Mss,PCr value.
However, in contrast to the long or short presaturation delays
(Fig. 6, A and C), an initial value of MPCr very close to Mss,PCr
was granted when d1,opt was applied (Fig. 6, B). This further
demonstrated the underlying mechanism of saturation time
reduction of our d1,opt strategy. A quantification of these
progressive saturation experiments was performed and shown
in Fig. 7. The PCr peak areas obtained in progressive saturation
experiments from four independent pig hearts were integrated,
normalized to M0,PCr of each heart, and then fitted into the
analytic solution of Eq. 7 as follow:
M PCrt

M0,PCr

MPCr0 Mss,PCr exp

M0,PCr

app
1,PCr

Mss,PCr
(15)
M0,PCr

where MPCr(0) is the initial value of MPCr(t) at the start of

app
saturation. Least squares fitting yielded T1,PCr
values of 1.53
0.05 s from d1 7 s curve and 1.54 0.05 s from d1 0 s
app
curve, respectively. The almost identical T1,PCr
values retrieved
Table 4. CK kinetic measurements reported from
previous studies
Ref. No.

Species and Condition

int
T1,PCr
,s

int
T1,ATP
,s

kf, s1

5
2
9
6
20
29
30
19
27

Isolated rat heart

Isolated rat heart
Isolated rat heart
Isolated rat heart
Open-chest pig heart
Open-chest pig heart
Open-chest dog heart
Closed-chest human heart
Closed-chest human heart

2.5
2.06
2.7
2.78
2.2
2.02
3.75
5.3
NA

0.8
0.75
0.9
0.99
NA
NA
NA
2.7
NA

0.67
0.271.3
0.84
NA
0.20.41
0.310.49
0.57
NA
0.210.32

Study based on porcine left ventricular remodeling model secondary to

myocardial infarction. A significant decrease in kf value was observed in
congestive heart failure group (0.21 0.03 s1) compared with normal group
(0.41 0.03 s1). Study based on porcine heart failure model secondary to
aortic banding. A significant decrease in kf value was observed in congestive
heart failure group (0.31 0.03 s1) compared with normal group (0.49
0.07 s1). Study based on human heart with normal, stressed, and failing
conditions. The kf value of failing human hearts (0.21 0.07 s1) was
significant smaller compared with normal subjects (0.32 0.07 s1). NA, not
applicable.
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A3, B

Fig. 8. Flow chart showing each data acquisition step in implementing the
novel MST strategy. Bold boxes indicate the actual measurements from
spectra. MST protocol: the implement of novel MST strategy for noninvasive creatine kinase (CK) flux rate measurement of any solid organs. All
spectra are acquired with 90 flip angle. (1) Take scout images (Fig. A1, left)
to guild the spatial localization of the spectroscopy. Choose the region of
interest, and take a scout spectrum (Fig. A1A). Number of averaged FIDs for
each spectrum is determined here according to signal-to-noise ratio (SNR). The
scout spectrum also provides the frequency offset information for the saturation pulse. The TR for scout spectra can be varied according to best SNR per
unit acquisition time. (2) Take M0,PCr measurement (Fig. A2A1) with the same
spatial localization parameters as used in scout spectrum. The TR for M0,PCr
measurement is usually 918 s, depending on the allowed data acquisition
window. (3) Take Mshort,PCr measurement with very short repetition time
int
using
(TR 0.3 s). (4) Combine step 2 and 3 measurements to calculate T1,PCr
Eq. 14. Note that we only need to make this measurement for an initial few
int
subjects, because the T1,PCr is considered as a constant and is independent of
physiological conditions. (5) Estimate a viable kf range, then combine it with
int
value to calculate d1,opt and d2,opt via numerical simulation of Eqs.
the T1,PCr
5 8. TR d1,opt d2,opt. (6) Take Mss,PCr measurement (Fig. A3A) using TR
from step 5. (7) Take MTR,PCr measurement (Fig. A3B) with ATP- continuously saturated. The TR here can be arbitrary according to Eq. 10. The SNR
per unit acquisition time can be maximized if the TR is chosen to be around
app
T1,PCr
. (8) Calculate M/M0 from step 2 and 6 measurements. (9) Calculate
app
from steps 6 and 7 measurements using Eq. 10. (10) Combine results
T1,PCr
from steps 8 and 9 to calculate kf using Eq. 3.

from both curves demonstrate that the short-d1 progressive

app
saturation can be employed to measure the T1,PCr
instead of the
conventional long-d1 counterpart, as stated earlier. The compiled data from four pigs comparing the conventional and
novel approaches from the same heart are summarized in
Table 2. Here a TR 1.5 s spectrum instead of the whole d1 0
app
curve was used to calculate the T1,PCr
, according to Eq. 10, so
that the maximum reduction in TR could be achieved. The
detailed reductions in data acquisition time using the novel
strategy are summarized in Table 3.
DISCUSSION

This technical report demonstrates a novel approach in MST

experiments that results in a significant reduction in total data
Table A1. Parameters used for d1,opt and d2,opt calculation
Parameters

int
T1,ATP
,s

int
T1,ATP
,s

kf,CK, s1

Values

4.0

1.33

0.15 0.6

See Ref. 7.

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Fig. A1. Demonstration of spatial localization

from 3D-ISIS pulse sequence on rat brain. Localized spectra (TR 9 s, nt 128) from brain
(A) and skeletal muscle (B) were obtained with
corresponding regions of interest shown on top
of the axial image of rat brain (turbo FLASH
sequence). Each spectrum was acquired with
19.2 min. The smaller SNR of muscle spectrum
is due to farther distance from the surface coil
that was positioned next to the brain region.

acquisition time, as well as the duration of saturation pulse.

Although the concept has been validated only on myocardial
CK reaction in the heart, this approach can be applied to other
enzyme reactions (such as ATPase) in any solid organs.
Superior performance compared with conventional MST
strategy. The most important advantage of the novel strategy is
the reduction of TR: the d1,opt approach in steady-state saturation transfer experiment would reduce TR by 73% (5.5 s in
d1,opt approach vs. 20 s in conventional approach), and the total
acquisition time for a complete kf measurement could be
reduced by 82% (4.3 min in novel approach vs. 23.9 min in
conventional approach, Table 3).
This reduction in TR is significant and will have several
future applications. First, it will allow us to determine myocardial CK forward flux rate with transmural differentiation to
separate inner layers vs. outer layers in open-chest preparation.
In an early study using a model of normal dog heart, the CK
flux measurements with spatial localization required 6 h of
data acquisition time for one experimental condition (21). Such
a long data acquisition time excludes the CK kinetic measure-

ment of a diseased heart, or CK flux rate changes in response

to experimental interventions. Second, the novel strategy will
facilitate the high field human MST studies using an external
coil, in which case similar spatial localization is required. The
data acquisition time would be prohibitively long, should a
conventional approach be applied. The strategy presented in
this work is compatible with various spatial localization techniques (such as 3D-ISIS and 3D-CSI) and thus would greatly
facilitate applications, such as CK forward flux rate mapping.
We have currently applied this strategy to investigate the ATP
production rate via CK forward reaction of rat brain in combination with 3D-ISIS spatial localization techniques. A complete kf measurement using this novel strategy required only 43
min compared with 3 h in the conventional approach (7) (see
APPENDIX).
Another advantage brought by this novel strategy is the
significant reduction in the duration of saturation pulse train
required for Mss,PCr measurement (56% reduction, 3.5 s in
d1,opt approach vs. 8 s in conventional approach). The first
beneficial effect from this reduction is the minimal spillover

Fig. A2. Experimental validation of d1,opt strategy on rat brain. All spectra were acquired with 3D-ISIS pulse sequence and 128 averaged FIDs. A1 is M0,PCr
measurement (TR 9 s); A2 is the control spectrum with saturation pulse set downfield from PCr symmetrically opposite from ATP-; spectrum A3 A1
A2, from which we can see there is no spillover effect of the BISTRO saturation pulse. Spectra B1 and B3 are Mss,PCr measurements using conventional (d1
9 s, d2 8 s) and novel (d1 d1,opt 2.1 s, d2 d2,opt 3.4 s) strategies, respectively. Spectrum B2 was acquired with TR d1,opt 2.1 s and saturation
pulse off. Spectrum B4 B3 B1. The bold arrows indicate the frequency of saturation pulses. Total acquisition times for spectra A1, A2, B1, B2, and B3 were
19.2, 36.3, 36.3, 4.5, and 11.7 min, respectively.
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app
Fig. A3. T1,PCr
measurement from two spectra. Spectrum A is the Mss,PCr measurement
(same as spectrum B3 in Fig. A2). Spectrum
B was acquired with ATP- continuously
saturated and relatively shorter TR (TR
1.4 s, nt 512). Total acquisition times for
spectra A and B were 11.7 and 11.9 min,
app
respectively. T1,PCr
is calculated using Eq.
app
10: T1,PCr
TR/ln(1 MTR,PCr/Mss,PCr).

effect on PCr peak of the saturation pulse train and thus better
SNR for quantification. In the present study, the novel strategy,
together with BISTRO saturation pulse train, yielded no detectable spillover effect on PCr peak (Fig. 5A). Consequently,
the control spectrum usually employed in other MST studies
(25, 29) can be eliminated. A second benefit from the reduced
saturation time is related to the specific absorption rate issue
when human subjects are involved. The minimal saturation
time provided by d1,opt strategy can, to a large extent, relieve
the Food and Drug Administration concerns on heat generation
by long duration of saturation pulse train employed in conventional MST strategy.
Other merits of the novel strategy. Recently, an important
four-angle saturation transfer method has been developed and
utilized for measuring CK reaction rates in humans (4, 23, 27).
app
This method employs a short TR (TR T1,PCr
) and different
app
flip angles to calculate the M0,PCr, Mss,PCr, and T1,PCr
. This
method is very time efficient, and better SNR within a given
data acquisition time can be achieved by flip angle optimizaapp
tion. However, the M0,PCr, Mss,PCr, and T1,PCr
calculations of the
four-angle saturation transfer method heavily rely on the accuracy of designed flip angles. This could potentially be
difficult at a higher field because of the characteristics of B1
penetration in high field magnet. A homogeneous flip angle
may not be achieved at high field, even though adiabatic pulses
such as BIR4 or BIRP are employed. The novel strategy
demonstrated in the present study, as an alternative method,
can provide the following advantages, in addition to reductions in TR and saturation time. Both M0,PCr and Mss,PCr are
directly measured, which enables the simple calculation of
app
kf and insensitivity to flip angle variation. For T1,PCr
calculation, the novel strategy requires only one ATP--saturated
spectrum (Fig. 6, C3) in addition to Mss,PCr measurement,
according to Eq. 10. The TR for ATP--saturated spectrum can
be arbitrary, and maximum SNR per unit time can be achieved
app
by a simple 90 readout pulse, if TR is chosen to be around T1,PCr
.
(When ATP- is saturated, PCr magnetization relaxes accordapp
app
ing to T1,PCr
, and thus Ernst angle will be 90 if TR T1,PCr
).
Validity of the methodology. The validity of the new methodology was tested in a pig heart model. The results from the
novel strategy were the same as for the conventional MST
app
method with long presaturation delay (Table 2). The T1,PCr
values retrieved from d1 of 0- and 7-s curves were identical
AJP-Heart Circ Physiol VOL

(Fig. 7). This is consistent with our numerical analysis. The kf

value calculated by using our new strategy yielded a value of
0.42 0.03 s1 that is similar to our laboratorys previous
report of 0.41 0.03 s1 (20).
Although we have provided two equations (Eqs. 3 and 4) for
conventional MST methods, all of the kf calculations described
in this work utilized Eq. 3. A number of previous studies
int
indicated that the T1,PCr
is independent of physiological condiint
tions (1, 2, 20, 30); however, variations of T1,PCr
values are
observed in the literature (Table 4). This is likely due to
different animal models and different experimental preparations. Therefore, we would recommend direct determination of
app
T1,PCr
value and use of Eq. 3 for kf calculation instead of Eq. 4,
int
together with T1,PCr
obtained from the literature. The major
reason that Eq. 4 was preferred in some studies is because it
eliminates the lengthy acquisition time used for progressive
app
saturation experiment to retrieve T1,PCr
. However, by using the
app
novel strategy, T1,PCr
can be calculated from one extra ATP-saturated spectrum with arbitrary TR. The advantage of this kf
calculation strategy is that it requires no approximation or
assumption.
For the purpose of this novel strategy being readily used by
other investigators, and especially those doing patient studies,
we summarized a flow chart in Fig. 8 to clearly specify each
steps of a magnetic resonance protocol that one could follow in
practice to obtain the noninvasive kf measurements.
Conclusion. Here, we have demonstrated a novel strategy for
MST experiments that provides 82% reduction in total data
Table A2. Rat brain CK MST measured by in vivo
31
P-magnetic resonance spectroscopy
app
T1,PCr
,s

Source

This work (n 6)
Literature (7)

int
T1,PCr
,s

1.90 0.09 3.30.3

NA
3.680.50

kf,CK, s1

Acquisition
Time, min

0.220.02
0.240.02

43*
180

*Acquisition time is based on TR 9 s for M0,PCr measurement, TR 5.5

app
for Mss,PCr measurement and TR 1.4 s for T1,PCr
measurement. Both M0,PCr
and Mss,PCr measurements were acquired with 128 averaged FIDs, while
short-TR measurement was acquired with 512 averaged FIDs for comparable
signal-to-noise ratio. Acquisition time is based on mean TR of 12 s, 5-point
app
progressive saturation transfer experiment for T1,PCr
measurement plus TR
9 s for M0,PCr and TR 15 s for Mss,PCr measurement. All measurements were
obtained from 128 averaged FIDs.

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SATURATION TRANSFER EXPERIMENTS USING MR SPECTROSCOPY

acquisition time compared with the conventional MST method.

The new strategy is based on the numerical analysis of the
governing equations of magnetization relaxation and was validated using an in vivo swine model. This MST strategy can be
readily applied to study of other enzyme kinetics (such as
ATPase) and in other important organs.
APPENDIX

Objective
This appendix serves to demonstrate that the novel MST strategy is
compatible with spatial localization of 3D-ISIS pulse sequence for
noninvasive measurement of ATP production rate via CK. The total
data acquisition time was decreased by 76% compared with conventional MST methods.

Methods
Animal preparation. The experiments were performed using a rat
brain model, and details of animal preparation for 31P-MRS have been
described previously (7). Briefly, rats were anaesthetized via inhalation of 2% isofluorane, intubated, and ventilated by a mechanical
respirator. The femoral artery and vein were catheterized for blood
sampling and monitoring of physiological parameters. The rectal
temperatures were maintained at 37 1C throughout the experiment
with an external heating patch.
In vivo 31P-MRS experiments. All in vivo 31P-MRS experiments
were conducted on a 9.4-T horizontal animal magnet (Magnex Scientific) interfaced with a Varian INOVA console. A multinuclear
radio-frequency surface-coil probe, consisting of a butterfly-shaped
1
H surface coil and an elliptical-shaped 31P surface coil with axes of
12 mm and 10 mm, was used. The 1H surface coil was used to acquire
anatomical images of the brain to guide choosing the regions of
interest and for shimming magnetic field homogeneity. For 31P spectroscopy, a 3D-ISIS pulse sequence was used for spatial localization.
BISTRO pulse train was used to achieve frequency selective saturation, and an adiabatic half-passage pulse was used for readout. To help
visualize whether the spatial localization was adequate, two capillaries
filled with 10% phosphoric acid were inserted into the skeletal muscle
surrounding the skull.
The optimized MST strategy. The d1,opt strategy was employed
for Mss,PCr measurement. The parameters used for calculating d1,opt
and d2,opt were from literature (7) and are summarized in Table A1.
Those parameters gave rise to a d1,opt value of 2.1 s and d2,opt of 3.4 s.
app
measurement, a short-TR (TR 1.4 s) spectrum with
For T1,PCr
continuous saturation of ATP- was performed and used in combiapp
nation with Mss,PCr measurement to calculate the T1,PCr
, according to
Eq. 10.
Results
Spatial localization of 3D-ISIS sequence. A demonstration of
spatial localization of 3D-ISIS pulse sequence is shown in Fig. A1.
The spatial localization from 3D-ISIS pulse sequence is demonstrated
by the following observations: 1) no detectable phantom signal in
brain region (spectrum A); 2) the obvious low and high PCr-to-ATP
ratios (PCr/ATP) in spectra A and B that are characteristic of brain
and skeletal muscle, respectively.
Validation of novel MST strategy in rat brain model. First,
the d1,opt strategy was validated. The spectra with d1,opt and conventional strategies for M0,PCr and Mss,PCr measurements are shown in
Fig. A2. There was no detectable difference in Mss,PCr measurement
between the d1,opt and the conventional strategies (Fig. A2, B). An
extra spectrum (Fig. A2, B2) with TR d1,opt and saturation pulse off
was also acquired to further confirm the d1,opt strategy. Spectrum B2
generated a PCr signal very close to Mss,PCr measured in B1 and B3,
AJP-Heart Circ Physiol VOL

which is consistent with our d1,opt hypothesis that MPCr(d1,opt)

Mss,PCr. For M0,PCr measurement (Fig. A2, A), the spillover effect of
BISTRO saturation pulse train on PCr peak was again negligible.
app
The T1,PCr
measurement using the novel strategy is shown in
Fig. A3. Here, a two-spectrum method using Eq. 10 was employed for
app
T1,PCr
calculation. All of the measurements obtained from the novel
strategy are summarized in Table A2, along with data obtained from
a previous study (7) for comparison. The novel strategy generated a
mean kf value of 0.22 0.02 s1 from six rats, which is similar to the
reported value of 0.24 0.02 s1 under the same condition. Moreover, a 76% reduction in total acquisition time was achieved [43 min
for novel strategy vs. 180 min for conventional strategy (7)].

Conclusion
We have successfully demonstrated the compatibility of the novel
MST strategy with the 3D-ISIS spatial localization and an external
coil for completely noninvasive MST measurement. The strategy was
validated using a rat brain model, which generates a kf measurement
similar to the reported value with only 24% of total data acquisition
time compared with the conventional approach.
GRANTS
This work was supported by US Public Health Service Grants. M. N.
Jameel was supported by American Heart Association Greater Midwest Predoctoral Award no. 0810015Z.
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