1. Leakage and water exchange characterization of gadofosveset in the myocardium
Octavia Bane a,b,d
, Daniel C. Lee c
, Brandon C. Benefield c,1
, Kathleen R. Harris c,1
, Neil R. Chatterjee b,c,d
,
James C. Carr c,d
, Timothy J. Carroll b,d,
⁎
a
Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA
b
Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA
c
Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
d
Department of Radiology, Northwestern University, Chicago, IL, USA
a b s t r a c ta r t i c l e i n f o
Article history:
Received 14 February 2013
Revised 2 August 2013
Accepted 22 October 2013
Keywords:
Gadofosveset
Ferumoxytol
Myocardial vascular fraction
Water exchange
Extracellular contrast agent
Blood pool contrast agent
Purpose: To determine the compartmentalization of the blood pool agent gadofosveset and the effect of its
transient binding to albumin on the quantification of steady-state fractional myocardial blood volume (fMBV).
Methods: Myocardial vascular fraction measurements were simulated assuming the limiting cases (slow or
fast) of two-compartment water exchange for different contrast agent injection concentrations, binding
fractions, bound and free relaxivities, and true cardiac vascular fractions.
fMBV was measured in five healthy volunteers (4 males, 1 female, average age 33) at 1.5 T after
administration of five injections of gadofosveset. The measurements in the volunteers were retrospectively
compared to measurements of fMBV after three serial injections of the ultra-small, paramagnetic iron oxide
(USPIO) blood pool agent ferumoxytol in an experimental animal. The true fMBV and exchange rate of water
protons in both human and animal data sets was determined by chi square minimization.
Results: Simulations showed an error in the measurement of fMBV due to partial binding of gadofosveset of
less than 30%. Measured fMBV values over-estimate simulation predictions, and approach cardiac
extracellular volume (22%), which suggests that the intravascular assumption may not be appropriate for
the myocardium, although it may apply to more distal perfusion beds. In comparison, fMBV measured with
ferumoxytol (5%, with slow water proton exchange across vascular wall) agree with published values of
myocardial vascular fraction. Further comparison between myocardium relaxation rates induced by
gadofosveset and by other extracellular and intravascular contrast agents showed that gadofosveset behaves
like an extracellular contrast agent.
Conclusions: The distribution of the volunteer data indicates that a three-compartment model, with slow water
exchange of gadofosveset and water protons between the vascular and interstitial compartments, and fast
water exchange between the interstitium and the myocytes, is appropriate. The ferumoxytol measurements
indicate thatthis USPIO isanintravascular contrastagent that can be usedtoquantify myocardial blood volume,
with the appropriate correction for water exchange using a two-compartment water exchange model.
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
Quantification of absolute myocardial blood volume (MBV, ml/
100 g tissue) and perfusion (ml/min/100 g tissue) has the potential
to improve the diagnosis and management of cardiac diseases. In
patients with coronary artery disease (CAD), absolute quantification
of myocardial perfusion during pharmacologic vasodilatation can
detect and characterize coronary stenoses with greater accuracy
than the qualitative or semi-quantitative techniques commonly
employed to evaluate perfusion images in clinical practice [1–3].
Quantification of MBV can potentially characterize compensatory
dilation of microvessels that occurs distal to a coronary stenosis at
rest, raising the possibility of CAD detection without the need of a
provocative stressor [4]. Another disease for which MBV can improve
diagnosis and management is cardiac allograft vasculopathy (CAV).
CAV is the greatest risk factor for mortality in the first five years after
heart transplant, accounting, along with late graft failure, for 30% of
the deaths in the first year [5]. It is characterized by progressive,
concentric intimal thickening of all myocardial vasculature, starting
with the small vessels. This leads to stenosis and occlusion of small
vessels, which leads to small stellate infarcts. Once CAV is detected,
treatment with short-term augmentation of immunosuppressive
therapy has demonstrated some efficacy in reversing CAV progres-
sion [6–8]. Notably, the success rate of this treatment is improved
when CAV is detected within the first year after transplant [6].
Magnetic Resonance Imaging 32 (2014) 224–235
⁎ Corresponding author at: Biomedical Engineering and Radiology, 737 N Michigan
Ave, Suite 1600, Chicago, IL 60611, USA. Tel.: +1 312 926 1733; fax: +1 312 926 5991.
E-mail address: t-carroll@northwestern.edu (T.J. Carroll).
1
These authors contributed equally to this work.
0730-725X/$ – see front matter © 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.mri.2013.10.014
Contents lists available at ScienceDirect
Magnetic Resonance Imaging
journal homepage: www.mrijournal.com

2. However, despite the efficacy of immunosuppressive augmentation,
the risks from such a regimen (infection and neoplasm) preclude its
routine use in transplant patients without CAV diagnosis.
CAV is currently detected by invasive coronary angiography or
intravascular ultrasound (IVUS) of the coronary arteries [9]. While
angiography and IVUS are sensitive for detecting CAV once it has
progressed to the coronary arteries, they do not directly assess the
microvasculature where CAV originates. A diagnostic test capable of
detecting CAV in the myocardial microvasculature could potentially
serve as an early marker of CAV, leading to targeted changes in
clinical management and ultimately improved survival. However,
such a test has so far remained elusive.
Previous SPECT studies of the myocardium have shown that
perfusion reserve decreases in CAV, but prognostic results have been
variable, with one recent (2012) study reporting a sensitivity of only
13% [10]. Less well understood is the effect of CAV on local
intravascular volume (MBV). In theory, MBV should decrease in
areas of the myocardium affected by CAV, since diffuse stenosis and
small stellate infarcts would lead to capillary loss, and previous work
has shown that MBV decreases are associated with capillary closure
on histological section [11–13]. Decreases in intravascular volume
should therefore result from capillary loss in CAV. However, this has
not been extensively studied and remains an open question. Should
MBV decrease as a result of CAV, it may serve as a novel marker of
CAV progression.
Thus, noninvasive assessment of MBV has tremendous potential
to improve our understanding of the pathophysiologic mechanisms
governing coronary microcirculatory regulation in health and
disease [11–14]. It is well known that for an intravascular
gadolinium-based T1 shortening contrast agent, the parenchymal
T1 change reflects tissue blood volume. However, to accurately
quantify MBV, we must test whether available contrast agents are
truly intravascular, and characterize the intra-to-extra-vascular
water proton exchange in the myocardium.
2. Theory
2.1. Intravascular Contrast Agent
In quantifying cerebral blood flow and volume, the most widely
used MRI contrast agent, Gd-DTPA (gadopentetate dimeglumine,
Magnevist, Bayer Health Care, Wayne, NJ) behaves like an intravas-
cular contrast agent because it does not cross the blood–brain
barrier, although the effect of water compartmentalization must be
accounted for, as well. However, in the myocardium, Gd-DTPA
exhibits tissue wash-in and wash-out with a transfer constant in
the heart of K1 of 0.45 +/− 0.19 ml/min/g tissue [15]. In fact, the
distribution of Gd-DTPA into the extracellular space (i.e. the
extracellular volume, or ECV) is known to be a biomarker of a
variety of cardiac diseases [16]. In this paper, we develop a model to
quantify myocardial blood volume, the steady-state distribution
volume of an intravascular contrast agent.
MS-325 (gadofosveset trisodium; Ablavar, Lantheus Medical
Imaging; AngioMark, Vasovist; EPIX Pharmaceuticals, Schering AG) is
a small molecule, T1-shortening contrast agent with a long intravas-
cular half-life. Unlike other gadolinium (Gd) chelates, it binds
reversibly to serum albumin, in a proportion of 80–90% for humans
[17]. It has recently obtained FDA approval for MR angiography in the
United States and has been used for over a decade in Europe as an
intravascular agent for angiographic applications [17–19]. Albumin
binding slows down the leakage of gadofosveset into extra-vascular
space, and increases its half-life, so that the T1 shortening effect is
observed up to four hours post-injection, and interpretable steady-
state images can be obtained up to an hour post injection [17]. The
albumin bond also slows down the rotation rate of the complex, which
enhances its relaxivity (T1-shortening effect, in mmol/L/sec) 6–10
times compared to other non-binding Gd-chelates [17], and results in a
4–10fold lower relaxivity of unbound gadofosveset, dependingon field
strength [18]. The reversible binding allows excretion through the
kidneys, or uptake by hepatocytes [18].
The pharmacokinetics of gadofosveset has been well character-
ized in distal vascular beds such as the peripheral vasculature. Given
the high proportion of gadofosveset bound to human serum albumin
(HSA), its long half life in the blood pool (48 minutes), and increased
T1 relaxivity of the bound fraction, we hypothesized that gadofo-
sevset is a suitable intravascular agent for quantification of fractional
myocardial blood volume (fMBV).
We compare our results to similar measurements made using
the ultra-small paramagnetic iron oxide (USPIO) ferumoxytol
(Feraheme, AMAG Pharmaceuticals, Lexington, MA). Ferumoxytol
consists of carbohydrate-coated elemental iron and has been
approved by the FDA as an intravenously administered iron
supplement for patients with severe anemia. Its large size
(30 nm measured by laser light scattering [7]) and molecular
weight (731 kDa [20], compared to 68 kDa for albumin-bound
gadofosveset and 500–700 Da for Gd-DTPA and free gadofosveset,
[21]) ensure a long intravascular half-life (10–14 hours) [22].
Ferumoxytol clears the blood vessels by macrophage uptake,
observable by MRI of vessel walls at 1–5 days after injection [23].
Published work shows its off-label use as a blood pool agent for MR
angiography (MRA) [22,24], in ten-fold diluted doses (3 mg/ml Fe
from label single dose of 30 mg/ml Fe). The T1 relaxivity at 1.5 T in
vitro, in human blood at 39.50
C has been previously measured as
12 mM−1
sec−1
, about half the relaxivity of the HSA-bound
fraction of gadofosveset [25].
2.2. Compartmentalization
Quantification of tissue blood volume requires a careful treat-
ment of contrast agent compartmentalization and water exchange.
Even in the case of intravascular contrast agents, the T1-shortening
effect on protons that move freely between the vascular and extra-
vascular compartment is not uniform. Fractional myocardial blood
volume (fMBV), the fraction of a pixel in a myocardium image that
represents blood, depends not only on signal intensity, but also on
the T1 and exchange rate of water protons between the vascular and
the extra-vascular spaces.
The two compartment water exchange model (Fig. 1a) devel-
oped by Hazlewood [26] and applied by Donahue [27] to an IR
signal is a simplified representation of the tissue voxel as a two
compartment space. The intravascular compartment includes the
plasma and red blood cells, while the extravascular compartment
is comprised of the interstitium and the cells. While blood pool
agents are restricted to the intravascular compartment, water can
diffuse freely between the two compartments and contributes to
the signal [27]. As described by Hazlewood [26], residence times τ
of the protons in each of the two compartments links the true and
apparent vascular fractions and T1 relaxation times. The equations
describing the effect of compartmentalization on signal are given
in Appendix I.
In the "slow exchange" limit (exchange rate 1/τ goes to zero),
protons do not cross the compartment barrier, so that the T1
shortening effect of the contrast agent is confined to the vessel. In the
slow exchange limit, the apparent and true volume fractions and T1's
are equal, and the vascular fraction or fMBV equals the distribution
volume of an intravascular MRI contrast agent, which is measured by
signal change in tissue with respect to signal change in blood
(Eq. (1))[26–28]. In the "fast water exchange" limit (the exchange
rate 1/τ goes to infinity, or residence time τ in each compartment
goes to zero), protons can be assumed to be moving very fast
225O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

3. between the compartments, so that they experience the same T1
shortening with the passage of contrast agent (Eq. (2)) [27,29,30].
fMBVslow exchange ¼
Spost contrast
myocardium
−Spre contrast
myocardium
Spost contrast
blood
−Spre contrast
blood
ð1Þ
fMBVfast exchange ¼
1
T post contrast
1 myocardium
− 1
T pre contrast
1 myocardium
1
T post contrast
1 blood
− 1
T pre contrast
1 blood
ð2Þ
The limiting values of the exchange rate are not realistic for in-
vivo measurements, which is why choosing one of the limiting cases
would under or over-estimate fMBV. To obtain the best quantitative
MBV for calibration, the water exchange rate must be estimated from
measurements and simulations [29].
2.3. Effect of contrast agent extravasation on measured distribution
volume (fMBV)
If we assume the reported steady-state (more than two minutes
after injection [31,18]), the unbound fraction of gadofosveset,
although small (10–15%), can distribute into the ECV. This makes
determination of the effects of unbound gadofosveset on fractional
myocardial blood volume (fMBV) measurements important for its
use in quantitative MBV. Separately from water exchange, we sought
to isolate and quantify the effect that the extravasation of the
unbound fraction of gadofosveset has on signal and on T1 values in
the myocardium measured at steady-state. To quantify the effect of
extravasation, we model the myocardium as a three compartment
environment, in which the unbound fraction of gadofosveset has
extravasated to the interstitial space at equilibrium (Fig. 1b). Our
model includes the effect of variable vascular volume, variable
binding, variable relaxivities of bound and unbound fractions for the
steady state. We did not model the approach to steady-state (time
after injection less than 1 minute). We use this model to predict T1
values in the myocardium and the error in fMBV in the slow and fast
exchange limits induced by extravasation of contrast agent.
We initially varied the binding fraction (bf) in the published
range of binding fractions of gadofosveset to HSA (80%–90%, and
100% for the ideal non-leakage case). We also studied the
dependence of bf on the concentration of injected gadofosveset,
the concentration of HSA, and the association constant between
albumin and gadofosveset. For this set of simulations, we used
previously published findings [18,31,32], which identified a main
binding site of gadofosveset to albumin [18], and measured the
association constant of gadofosveset to albumin in vitro [18]. Binding
fraction was calculated as a function of HSA association constant
(Ka) [18], vascular albumin concentration ([HSA]) values from
literature [33, 18], and gadofosveset concentration in the vessel after
each injection. This model for gadofosveset binding to albumin was
developed for the in vitro setting, but we extended it to in vivo by
assuming that the unbound fraction of gadofosveset extravasates
entirely after each injection. The error in fMBV was calculated with
respect to the ideal non-leakage case. Both iterations of our model
are described in Appendix II.
Fig. 1. a) Two-compartment model of an intravascular contrast agent. While the contrast agent (CA) is non-diffusible, water molecules can move freely through the vessel wall.
The parameters associated with MRI signal in each compartment are described in Appendix I. b) Model of extravasation of gadofosveset. The Gd complex bound to human serum
albumin (HSA) in proportion (binding fraction) bf stays in the blood vessel, whereas the free complex extravasates. At steady-state, we assumed the free gadofosveset to have
extravasated into the interstitium. Gadofosveset does not enter the cellular space. The parameters that described MRI signal in each compartment are described in Appendix II.
226 O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

4. 3. Methods
We simulated expected results using our theoretical model based on published characteristics of the widely used blood pool contrast agent
gadofosveset. Simulated results were compared to in vivo measurements made in healthy volunteers. As an ancillary study, we compared our
expectations to retrospective animal data acquired with a USPIO contrast agent. Table 11 summarizes the models presented in each study, in
simulations or data analysis.
3.1. Transient Binding Simulations
We performed simulations in MATLAB according to our model of the myocardium, which assumes that the unbound fraction of gadofosveset is
uniformly distributed in the interstitial space at steady-state. The r1 relaxivity of the gadofosveset fraction bound to albumin (r1bd) was assumed to
be in the range of 20 mM-1
sec-1
–30 mM sec-1
, while the relaxivity of free gadofosveset (r1f) was assumed to vary from 4.5–8 mM-1
sec-1
, as
measured in vitro by other groups [18,34].
The empirical simulations were performed in MATLAB and calculated fMBVslow exchange (Eq. (1)) and fMBVfast exchange (Eq.(2)) from
inversion recovery signals simulated for literature values of r1bd, r1f, bf, injected contrast agent concentration and true vascular fraction (fv) in
tissue.
3.2. In-vivo Study of Gadofosveset Compartmentalization
To validate our results, we characterized contrast agent leakage and water proton exchange in the myocardium in a pilot study of 5 healthy
volunteers (4 males, 1 female, ages 21–48, average age 33). Prior to the imaging procedures all subjects gave written informed consent to the
study protocol, which was approved by the institutional review board (IRB).
3.2.1. T1 measurement with Modified Look Locker Inversion Recovery (MOLLI)
Our current T1 measurement method uses the previously reported Modified Look-Locker Inversion Recovery (MOLLI) pulse sequence
[35,36]. MOLLI is based on the classical Look-Locker experiment, in which the magnetization re-growth after an inversion pulse is interrupted
and sampled by several small angle RF pulses. The T1 can be calculated from a six to ten point Look-Locker re-growth curve by a three-
parameter non-linear fit [37]. The conventional IR-LL sequence was not suitable for mapping T1 values in the myocardium because the
myocardium T1 of 1000 ms at 1.5 T exceeds the average duration of the cardiac cycle (600–700 ms), and the typical patient breath hold
interval of 20 seconds allows acquisition of only four to five IR-LL data points [36].
Interleaved acquisition, as well as the merging of data from several LL experiments with different inversion times into a single slice allows
the mapping of myocardial T1 in a single-breath hold with MOLLI. MOLLI allows T1 measurement with errors comparable to those seen in brain
studies (5–10%), and with a larger image matrix in a shorter acquisition time, compared to cardiac T1 mapping by arterial spin labeling (ASL)
[36]. However, heart-rate dependence observed in phantom-ECG simulator studies is a limitation of the sequence. Although a subsequent
study addressed heart rate dependence and validated the sequence in healthy volunteers and patients, arrhythmia in subjects remains a
limiting factor [35]. Given our model of compartmentalization, in which fMBVslow exchange is calculated by ratios, uniform 5–10% perturbations
introduced by error in the T1 measurements will not affect the fMBV measurements.
Steady-state T1 values in myocardium and blood pool (left ventricle) were measured using the MOLLI pulse sequence (slice thickness
8 mm, FOV 300 x 400 mm2
, matrix 256x172, TE 1.14 ms, TR 2.25, 11 acquisitions, minimum effective TI 100 ms, TI incremented by 80 ms for
the first three acquisitions, and by the RR interval for the subsequent acquisitions). The five healthy volunteers underwent imaging with MOLLI
on a 1.5 T Espree scanner (Siemens Medical Systems, Erlangen, Germany), during a short breath-hold, before and 2 minutes after
administration [38] of five injections of 0.006 mmol/kg (a fifth of a single dose) gadofosveset. The repeated injection protocol has previously
been used by other workers for assessment of contrast agent distribution volume and compartmentalization [27,28,39,40]. Injections were
Table 1
Summary of models used in simulations and data analysis.
Study Effect of water exchange Effect of partial binding of gadofosveset
Transient Binding
Simulations
Hazlewood–Donahue two-compartment
model: calculation of fMBV according to
the slow and fast water exchange limit
(Eqs. (1), (2))
Simulations of fMBV and error in fMBV introduced by leakage with:
1) independently modified binding fraction (Appendix II, Fig. 3)
2) binding fraction dependency on albumin concentration
(Appendix II, Figs. 4,5)
In-vivo Study of
Gadofosveset
Compartmentalization
Hazlewood–Donahue two-compartment
model: calculation of fMBV according to
the slow and fast water exchange limit
(Eqs. (1–3))
Comparison of data with Hazlewood–Donahue
simulations (Appendix I, Fig. 6)
Comparison of fMBV measurements with simulations
Comparison with an
ultra-small paramagnetic
iron oxide (USPIO)
Hazlewood–Donahue two-compartment
model: calculation of fMBV according to
the slow and fast water exchange limit
(Eqs. (1–3), Fig. 2)
Comparison of data with Hazlewood–Donahue
simulations (Appendix I, Fig. 7)
N/A
Comparison with other
contrast agents
Comparison of gadofosveset data with Judd
three-compartment water exchange model
and of USPIO data with the two-compartment
water exchange limits
N/A
227O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

5. administered via power injector as 4–6 ml boluses, at rate of 4 ml/sec, followed by 8–10 ml saline flush. We chose this high injection rate
because it is typical for dynamic contrast enhanced cardiac perfusion protocols, and our aim is to quantify MBV at steady state in the context of
a perfusion protocol. The contrast agent was diluted in saline to obtain uniform dose/weight and injection volume for all subjects.
3.2.2. Two-Compartment Water Exchange Analysis
The T1 changes were estimated from the signal map by fitting the MOLLI signal to the Look-Locker re-growth curve (Signal(t) = M0(1-
invF*(exp(-t/T1)) in MATLABR2009 (The Mathworks, Natick, MA) (5). T1 was also quantified from T1 maps generated by inline post-
processing (Siemens).
fMBV in the slow exchange limit was calculated pixel-by-pixel from signal maps calculated from the magnetization (M0), inversion factor
(invF) and Siemens T1 maps at 260 ms (the point of maximum image contrast), according to Eq. (3).
fMBVslow ¼
bM0
post
à 1−invFpost
à exp − 260
Tpost
1
−M0
pre
à 1−invFpre
à exp − 260
Tpre
1
NROI myo
bM0
post
à 1−invFpost
à exp − 260
Tpost
1
−M0
pre
à 1−invFpre
à exp − 260
Tpre
1
NROI blood
ð3Þ
Regions of interest (ROIs) were selected manually off the fMBV maps, covering the entire myocardium. For these regions of interest, we
plotted the measured (ΔR1, fMBV) data pairs against simulated curves of vascular fraction vs ΔR1 of blood for different exchange rates 1/τ. ΔR1
blood was calculated from T1 values measured in the left ventricular blood pool. Simulations for the myocardium were conducted in MATLAB,
according to the two-compartment water exchange model presented by Hazlewood and Donahue [26,27].
3.3. Comparison with an ultra-small paramagnetic iron oxide (USPIO)
A 16 kg instrumented dog was imaged using the USPIO contrast agent ferumoxytol, with the purpose of observing the evolution of
myocardial and blood T1 up to 12 minutes post injection. The ferumoxytol was diluted from label single dose of 30 mg elemental iron (Fe)/ml
solution to 5.3 mg Fe/ml. Studies were conducted using procedures and protocols in accord with the position of the American Heart
Association on Research Animal Use. One injection bolus of 6 ml (2 mg/kg), followed 13 minutes later by two 3 ml injections (1 mg/kg)
4 minutes apart, was administered via power injector at the rate of 4 ml/sec. MOLLI images in the short-axis view (slice thickness 8 mm, FOV
340 x 143 mm2
, matrix 256x108, TE 1.14, TR 2.25, 11 acquisitions, minimum effective TI 100 ms, TI increment 80 ms for the first three
acquisitions, and RR interval increment for subsequent acquisitions) were acquired every minute up to 13 minutes after the first injection,
3 minutes after the second, and 3 and 8 minutes after the third. T1 was measured off T1 maps, generated in MATLAB by a three-variable fit
(Fig. 2a). fMBV slow exchange (Fig. 2b) was mapped retrospectively from T1, M0 and invF by the same procedure as used in the human
volunteers, outlined in Eq. (3). fMBV fast exchange was calculated from T1 values, according to Eq. (2).
3.4. Comparison with other Contrast Agents
The (ΔR1 blood, ΔR1 myocardium) values 3 minutes following each injection of ferumoxytol were compared to the (ΔR1 blood, ΔR1
myocardium) pairs for gadofosveset at 1.5 T, as well as to published ΔR1 values for the extracellular contrast agent gadoteridol (ProHance,
Bracco Diagnostics, Princeton, NJ; Bristol-Myers-Squibb, Princeton, NJ) [41] and the intravascular contrast agent feruglose (NC100150,
Clariscan, Nycomed Amersham Imaging, Oslo, Norway) [42].
4. Results
4.1. Simulations
From simulations of the independent effects of bf, r1bd, r1f and
serial injections, we found that the error introduced by partial
binding of gadofosveset to human serum albumin (HSA) in the
measurement of fMBV slow exchange is less than 20% (Fig. 3a). Thus,
for a true vascular volume of 10%, one would measure a vascular
volume of 8%–12% from MRI signal change pre and post contrast
(Eq. (1)). The error in fMBV fast exchange is more pronounced, at
almost 50% for a binding fraction of 80%, r1bd = 20 mM-1
sec-1
and
r1f = 4.5 mM-1
sec-1
(Fig. 3b). In the fast exchange limit, the
measured vascular fraction would be 5%–15% for a true vascular
fraction of 10%.
By accounting for the dependence of bf on the concentrations of
albumin and gadofosveset present in blood, we found that bf
decreases with each additional injection of gadofosveset (from 74%
bound after first injection to 69% bound after fifth injection, for
[HSA] = 0.3 mM, Ka = 11 mM-1
, see Fig. 4). Binding fractions
increase, as expected, for all injections in the series when there is
an increased concentration of albumin in the blood (Fig. 4). The error
introduced by leakage in fMBV slow and fast exchange decreases
with increasing r1bd, and increases with increasing r1f (Fig. 5a and b),
as expected. The error in fMBV slow exchange introduced by leakage
in the case of binding fraction dependence on concentration is under
30%. The errors in fMBV fast exchange due to leakage are more
considerable, between 50% and 150%.
4.2. Volunteer Results
Fig. 6 shows water exchange in the myocardium to exhibit a
characteristic shape that we would expect if slow exchange were
present. By performing Chi Square minimizations between the data
and the two compartment water exchange simulations for different
ranges of residence time τ, we obtained true fMBV = 22% and true
τ = 1 msec, which is inconsistent with our predictions based on the
transient binding simulations.
4.3. Comparison with USPIO
To verify our models, we compared to retrospective measure-
ments of fMBV with a truly intravascular contrast agent. Measure-
ments of T1 in the blood pool agreed with literature values for the
cumulative dose of 4 mg Fe/kg, and r1 relaxivity measured in the left
ventricle blood pool after the first injection was 12.14 mM-1
sec-1
, in
agreement with the literature. For the relaxivity calculation, the
injection volume was assumed to be mixed with the total blood
volume, assuming blood is 7% of the canine's body weight [25].
Measurements of fMBV in the slow and fast exchange limits for the
228 O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

6. canine subject, after serial injections of ferumoxytol show that slow
exchange is the appropriate compartmentalization model for the
USPIO (Fig. 7). After Chi Square minimization, the fMBV data
calculated according to the slow exchange model (Eq. (1)) yielded
a true vascular fraction of 5% and true τ = 70 msec. For the fMBV
fast exchange measurements, the slow exchange model with a true
vascular fraction 5% and τ = 602 msec minimized chi square.
4.4. Comparison with Three Compartment Model
Inspection of Fig. 8 shows that the gadofosveset ΔR1 data,
averaged across all 5 volunteers at 1.5 T, follows the same linear
distribution as published for the extracellular agent gadoteridol [41].
Comparison of gadofosveset data to the linear distribution of the
gadoteridol data of Judd et al. yields a sum of squared errors of 0.05,
for 4 degrees of freedom. The ferumoxytol data aligns within the
bounds for (ΔR1 blood, ΔR1 myocardium) distributions predicted by
the fast and slow exchange limits of the Hazlewood–Donahue model
for an intravascular contrast agent, assuming a true vascular fraction
of 10% in the myocardium. ΔR1 myocardium after administration of
ferumoxytol injections stays flat (ΔR1 myocardium = 0.08 *ΔR1
blood, R2
= 0.84) similar to previously published ΔR1 myocardium
measurements after administration of another USPIO, feruglose.
5. Discussion
Although other groups have previously measured the plasma
distribution volume of the compound HSA-(Gd-DTPA)30 in animals
using MRI [39], our study is the first attempt to use gadofosveset for
the quantification of steady-state blood volume in the human
myocardium. We have found that gadofosveset, when not bound
to albumin before injection, has similar pharmacokinetic properties
to extracellular contrast agents. Since gadofosveset behaves like an
extracellular contrast agent in the myocardium, a three-compart-
ment model, with slow exchange between the intravascular space
and the interstitium, and fast exchange between the interstitium and
the intracellular space, as proposed by Judd et al. [41], is more
appropriate to describe its compartmentalization than a two-
compartment model.
Previous studies of albumin-bound, gadolinium-based contrast
agents showed that the prototype compound HSA-(Gd-DTPA)30 [43]
has the properties of an intravascular contrast agent. In animal
studies, HSA-(Gd-DTPA)30 displayed high signal enhancement, had a
distribution volume in the healthy myocardium corresponding to
the intravascular volume [39], and negligible microvascular perme-
ability [44]. However, this compound is irreversibly bound to
albumin, so it cannot be excreted through the kidneys, which
makes it unsuitable for human use.
Studies of first-pass cardiac perfusion with gadofosveset in
animals were qualitative [45] or semi-quantitative [46], and do not
discuss the extravasation of gadofosveset [45]. However, a recent
study [47] comparing absolute myocardial perfusion measurements
with gadofosveset and the protein-binding agent gadobenate
dimeglumine (Gd-BOPTA) found that gadofosveset did not display
the expected first-pass profile for an intravascular contrast agent
[47]. Published in vitro work has also shown that gadofosveset
violates the assumption of complete binding in some animal
models [32], due to its variable and moderate binding affinity to
albumin and plasma proteins across species, which results in
variable T1 relaxation rates [18]. While the model of one-to-one
association of gadofosvet with albumin is appropriate at steady-
state, it may break down when the concentration of gadofosveset
far exceeds that of albumin, such as during and right after bolus
first pass. Port et al. have traced the pharmacokinetic profile of
gadofosveset in rabbits (Ka = 11 mM-1
, equal to that measured in
vitro for HSA), and found that as little as 30% of plasma
gadofosveset was bound to albumin at 5 seconds after injection.
In rabbits, the binding fraction stabilizes at about 75%, 120 seconds
after injection [31]. Since the bound fraction is low in the first
30 seconds after injection (even when using a slow injection rate of
0.5 ml/sec), gadofosveset can extravasate like a conventional
extracellular agent in this time frame.
Our partial binding simulations at steady state in a repeated
injection protocol predict smaller binding fractions (and therefore
more extravasation) for incrementally increasing gadofosveset dose,
Fig. 2. a) T1 (ms) and b) fMBV slow exchange maps, before and 3 minutes after repeated injections of ferumoxytol in a canine subject.
229O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

7. and uniform increase of binding fractions with higher blood albumin
concentrations. This is in accordance with the one-to-one association
of gadofosveset to albumin presented by previous work in vitro [18].
However, the errors in fMBV slow exchange predicted by our
simulations (under 30%) far under-estimate the difference between
our data and established values of vascular fraction in the
myocardium (100%). Although more considerable, the errors in
fMBV fast exchange still under-estimate the discrepancy between
the data (measured fMBV fast exchange values between 30%–50%, or
300%–500%) and expected fMBV (8–12%).
Measured fMBV values in the volunteers, even after correction for
partial binding of contrast agent to albumin, over-estimate published
values for myocardial blood volume in animal and human subjects
[2,3,42,48]. The true vascular fraction obtained after accounting for
leakage and two-compartment water exchange approaches the
value of ECV in healthy volunteers (23–25%) [49], which suggests
that the intravascular assumption may not be appropriate for
gadofosveset. Also, the intravascular-to-extravascular exchange
rate of water protons, determined by chi-square minimization (1/
τ = 1/1 ms = 1000 sec-1
) over-estimates the published value in
human myocardium (1/τ = 0.48 sec-1
) for the feruglose study of
Wacker, Bauer et al. [42]. Since the 1/τ we observe is 2000 times
higher than previously measured, what we observe is not slow intra-
to-extravascular exchange of the water protons, but fast water
Fig. 3. Simulations of apparent vascular fraction (fMBV) for five step increases in concentration (five injections), assuming true vascular fraction is 10%. (a) In the slow exchange
limit (fMBV slow exchange = ΔS myocardium/ΔS blood), the size of the leakage effect is under 20%. (b) In the fast exchange limit (fMBV fast exchange = ΔR1 myocardium/ΔR1 blood),
the size of the leakage effect increases up to 40%.
Fig. 4. Variation of bf values with total concentration of Gd present in the blood after each injection, for different physiologic concentrations of HSA.
230 O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

8. proton exchange between the extracellular and intracellular spaces,
as observed by Judd et al. for an extracellular agent [41]. Thus, the
discrepancy in exchange rate can be construed as further evidence
that gadofosveset extravasates more than expected from literature
and from our simulations. The T1 shortening behavior of gadofosve-
set in the myocardium (Fig. 6) further confirms its behavior as an
extracellular agent.
Our results contradict the initial studies of gadofosveset in
humans, which focused on dynamic and steady-state peripheral
angiography [31]. Gadofosveset demonstrated a five-fold increase
in signal-to-noise ratio in the peripheral arteries, compared to the
leg muscle up to an hour post injection [31], which suggests that
gadofosveset is largely retained into the vasculature because of
the strong albumin binding. Stronger albumin binding of
gadofosveset in angiography applications is achieved due to a
slow injection rate (0.6 ml/sec) and longer transit time of the
contrast bolus between the injection site and the peripheral
vascular bed of interest (~30 seconds). Thus, in angiography
protocols, the contrast agent has more time to bind to albumin. In
our study, we used an injection rate of 4 ml/sec, appropriate for
Fig. 5. Simulations of apparent vascular fraction (fMBV), for stepped injections of C = 0.05 mM, assuming true vascular fraction is 10%, and accounting for dependence of binding
fraction on the ratio of serum albumin to gadofosveset concentrations. The association constant is Ka = 11 mM-1
. (a) In the slow exchange limit (fMBV slow exchange = ΔS myocardium/
ΔS blood), the size of the leakage effect is under 30%, for [HSA] = 0.3 mM, r1bd = 30 mM-1
sec-1
and r1f = 8 mM-1
sec-1
. (b) In the fast exchange limit (fMBV fast exchange = ΔR1
myocardium/ΔR1 blood), the size of the leakage effect increases up to 150%, for lowest [HSA] = 0.3 mM, r1bd = 30 mM-1
sec-1
and r1f = 8 mM-1
sec-1
.
Fig. 6. Volunteer myocardial fMBV measurements, corrected for leakage with the assumption of 80% binding to HSA, bound fraction relaxivity of 20 mM-1
sec-1
, and free fraction
relaxivity of 4.5 mM-1
sec-1
at 1.5 T. The true fMBV and water proton residence Tau, obtained by chi square minimization between the data and Hazlewood model simulations of
water exchange indicate that the distribution volume of gadofosveset is the extracellular volume.
231O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

9. first-pass cardiac perfusion applications, since our initial goal was
to use steady state measurements of myocardial blood volume to
calibrate first-pass perfusion measurements, in a quantitative
perfusion measurement technique similar to the one validated by
our research group in the brain [29,50,51]. The myocardial
vascular bed is also closer to the injection site, which makes for
a shorter transit time of the contrast bolus (3–4 sec) during
which gadofosveset can bind to albumin. Thus, the over-
estimation of fMBV and water exchange rate constitutes evidence
that most of gadofosveset extravasates during first-pass of the
injection bolus through the heart chambers and the pulmonary
circulation, and that a smaller fraction of injected dose than
expected is bound to albumin at steady-state.
5.1. Comparison with USPIO
The results in the animal subject point to ferumoxytol as a truly
intravascular alternative to gadofosveset for the quantification of
vascular volume fraction in the myocardium. The true vascular
fractions obtained from the slow and fast exchange limit data agree
with published measurements of MBV in canines equivalent to a
myocardial vascular fraction of 8.7 ± 1.8% [3].
5.2. Limitations
Our study was not without limitations. In developing our leakage
model, we used relaxivity and binding fraction values measured in
vitro by other investigators. The r1bd, r1f, and bf in vivo for
gadofosveset are not known. We also used several simplifying
assumptions in our leakage model. We neglected extraction of the
unbound fraction of gadofosveset by the kidneys. However, since
imaging is performed 2 minutes after injection, and the timing
between the first injection and the last steady-state T1 measurement
is no more than 20 minutes, we expect clearance of the agent by the
kidneys to be minimal in that time interval. The mean plasma half-
life of gadofosveset, for humans with normal glomerular filtration
Fig. 7. True vascular fractions with ferumoxytol, obtained from chi square minimization between water exchange simulations and fMBV calculated in the a) the slow exchange
limit and b) fast exchange limit.
Fig. 8. Comparison of gadofosveset with other contrast agents. Gadofosveset aligns with the model developed by Judd et al. (1999) for an extracellular agent undergoing limited
contrast agent and water exchange between the intravascular and interstitial space, and fast interstitial–intracellular water exchange.
232 O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

10. rate (GFR), is 48 ± 11 minutes, and interpretable steady-state
angiograms have been obtained up to an hour after injection [52].
We also assumed that the injected bolus (5 ml) is fully mixed with
the blood volume (5 L) at steady state, so that the concentration that
reaches the tissue of interest is injected concentration downscaled
by 1000 (e.g. for a full dose of 0.25 mol/L gadofosveset, 0.25 mmol/L
reach the tissue of interest).
6. Conclusions
Our observations show that, although the extravasating fraction
of gadofosveset is thought to be small at steady-state (5–20%), the
effect of first-pass extravasation of the contrast agent on quantitative
blood volume measurements at steady-state in the myocardium is
significant. Also, the relaxivity of the unbound fraction of gadofos-
veset in the interstitium might be higher than previously thought
due to possible binding to proteins in the interstitium. Thus,
gadofosveset is less suitable for quantification of myocardial blood
volume than the USPIO intravascular contrast agent ferumoxytol.
Acknowledgments
The authors would like to thank Marie Wasielewski, MRI
technologist, and the cardiovascular imaging staff of the Center for
Translational Imaging at Northwestern University, for technical
training and clinical assistance with recruiting and imaging the
volunteers. The volunteer study was supported by the US National
Institute of Healthy grants T32 EB005170, R01 NS0493395, and R01
HL088437. The animal data presented here are part of a study
supported by an award from the American Heart Association and the
Northwestern Memorial Foundation.
Appendix I
As shown by Donahue et al, the signal behavior in time from an
inversion-recovery sequence in a voxel containing tissue and blood
depends on the apparent intra-vascular and extra-vascular fractions
(fiv
’
and fev
’
), and the apparent T1's in the intra and extra-vascular
compartments (Eq. (4), Fig. 1). Apparent T1's are dependent on true
T1's and on proton residence times in each compartment (Eqs. (5),(6),
(9) and (10)). The apparent intra-vascular and extra-vascular fractions
can be expressed in terms of true fractions, longitudinal relaxation
times and residence times (Eq. (7)), and sum up to one (Eq. (8)).
S tð Þ ¼ f
0
iv M0 1−2 e
− t
T
0
1iv
þ f
0
ev M0 1−2 e
− t
T
0
1ev
ð4Þ
1
T
0
1iv
¼ C1 þ C2 ð5Þ
1
T
0
1ev
¼ C1−C2 ð6Þ
f
0
iv ¼
1
2
−
1
4
fev−fivð Þ 1
T1 iv
− 1
T1 ev
þ 1
τiv
þ 1
τev
h i
C2
ð7Þ
f
0
ev ¼ 1−f
0
iv ð8Þ
C1 ¼
1
2
1
T1 iv
þ
1
T1 ev
þ
1
τiv
þ
1
τev
ð9Þ
C2 ¼
1
2
1
T1 iv
þ
1
T1 ev
þ
1
τiv
þ
1
τev
2
þ
4
τivτev
1
2
ð10Þ
Appendix II
In order to estimate the size of the effect of partial binding of
gadofosveset with albumin on fMBV calculated in the slow and fast
exchange limits (Eqs. (1) and (2)), we performed mathematical
simulations based on our model of partial binding (Fig. 1, bottom). T1
shortening in the blood will depend on whether the contrast agent is
fully bound (no leakage, Eq. (11)) or partially bound (Eq. (12)) to
human serum albumin (HSA). T1 shortening will thus depend on the
binding fraction bf and on the bound r1 relaxivity, r1bd. At steady state,
we assume that all unbound contrast is uniformly distributed in the
interstitium, where it causes signal enhancement with relaxivity r1
free (r1f), Eq. (13).
1
T1vnl
¼
1
T1v0
þ r1bd à C ð11Þ
1
T1vl
¼
1
T1v0
þ bf à r1bd à C ð12Þ
1
T1ev post
¼
1
T1ev0
þ 1−bfð Þ Ã r1f à C ð13Þ
where T1v0 =1500 ms (T1 of blood) and T1ev0 = 1000 ms (T1 of
myocardium) at 1.5 T.
Assuming an inversion recovery preparation for the pulse sequence
used to measure T1, the apparent signal of a myocardium voxel (Fig. 1
b) will have three components: intravascular, interstitial and intracel-
lular. In the case of leakage, the intravascular and interstitial
components of signal will be affected by the T1-shortening properties
of gadofosveset (Eq. (14)), while the intracellular compartment
maintains the native T1 of the myocardium, since gadofosveset does
not enter the cells. With no leakage, only the T1 in the vessels will be
shortened by the contrast agent (Eq. (15)). Left ventricle blood pool
signal enhancement will also be different, depending on whether the
contrast agent is fully bound to HSA or not (Eqs. (16) and (17)).
fMBVslow exchange values were calculated (Eqs. (18) and (19)) from the
simulated signals, for both the leakage and no leakage scenario, at
the time point tmax of maximum signal difference (contrast) between
the blood pool and the myocardium. fMBVfast exchange was calculated
from the apparent T1's of the blood pool and myocardium, obtained by
three variable fitting of the simulated signals, in the leakage and no
leakage scenario (Eqs. (20) and (21)). The error introduced by leakage
in the measurement of fMBV was calculated for both the two-
compartment slow and fast exchange limits (Eqs. (22) and (23)).
S
post leakage
myo tð Þ ¼ fv 1−2e
− t
T1vl
þ ECV−fvð Þ 1−2e
− t
T1evpost
þ 1−ECVð Þ 1−2e
− t
T1ev0
ð14Þ
S
post no leakage
myo tð Þ ¼ fv 1−2 e
− t
T1vnl
þ 1−fvð Þ 1−2e
− t
T1ev0
ð15Þ
S
post leakage
blood tð Þ ¼ 1−2e
− t
T1vl
ð16Þ
S
post no leakage
blood tð Þ ¼ 1−2e
− t
T1vnl
ð17Þ
where fv is the true vascular fraction in the tissue, ECV is
extracellular volume
fMBV
leakage
slow exchange ¼
Spost leakage
myo t maxð Þ−Spre
myo t maxð Þ
Spost leakage
blood
t maxð Þ−Spre
blood
t maxð Þ
ð18Þ
233O. Bane et al. / Magnetic Resonance Imaging 32 (2014) 224–235

11. fMBV
no leakage
slow exchange ¼
Spost no leakage
myo t maxð Þ−Spre
myo t maxð Þ
Spost leakage
blood
t maxð Þ−Spre
blood
t maxð Þ
ð19Þ
fMBV
leakage
fast exchange ¼
1
T1post;leakage
apparent;myocardium
− 1
T1pre
apparent;myocardium
1
T1post;leakage
apparent;blood
− 1
T1pre
apparent;blood
ð20Þ
fMBV
no leakage
fast exchange ¼
1
T1post;no leakage
apparent;myocardium
− 1
T1pre
apparent;myocardium
1
T1post;no leakage
apparent;blood
− 1
T1pre
apparent;blood
ð21Þ
fMBVslow exchange ¼
fMBV leakage
slow exchange
−fMBVno leakage
slow exchange
fMBVno leakage
slow exchange
ð22Þ
fMBVfast exchange ¼
fMBV leakage
fast exchange
−fMBVno leakage
fast exchange
fMBVno leakage
fast exchange
ð23Þ
A second iteration of our model studied the dependency of
binding fraction on gadofosveset concentration in the vessel. Other
investigators have shown that the degree of binding varies with
concentration of gadofosveset and of albumin in the compartment
to be studied (Eqs. (24–27)).
C ¼ Cbd þ Cf ¼ bf à C þ 1−bfð Þ Ã C ð24Þ
HSA½ Š ¼ HSA½ Šbf þ HSA½ Šf ð25Þ
HSA½ Šbd ¼ bf à C ð26Þ
Ka ¼
Cbd
Cf à HSA½ Šf
ð27Þ
where [HSA], [HSA]bd, [HSA]f are total, bound and free albumin
concentrations, respectively, and Ka is the association constant of
albumin and gadofosveset.
Substituting Cbd and Cf from Eq. (24), expressing [HSA]f as a
function of total and bound albumin concentration, and dividing
both numerator and denominator by total gadofosevset concentra-
tion, we obtain:
Ka ¼
bf
C Ã 1−bfð Þ HSA½ Š
C −bf
ð28Þ
The second order equation can be solved for bf, for each post-
injection concentration of gadofosveset (Eq. (29)).
bf ið Þ ¼
ið ÞKa C ið Þ þ Ka C ið Þ þ 1ð Þ
−
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
α ið ÞKaC ið Þ þ KaC ið Þ þ 1ð Þ2
−4α ið ÞK a
2
C ið Þ2
q
2Ka C ið Þ
ð29Þ
where i is the injection concentration, and α(i) is the ratio of total
albumin concentration to gadofosveset concentration C(i) post each
injection.
Assuming extravasation of the free fraction of the agent after each
injection, then concentration C(i) after each injection (starting with
the second injection) that determines bf (i) is iteratively calculated
by Eq. (30). Injections were assumed to be of equal volume (5 ml)
and concentration (0.05 M), which, when mixed in 5 L of blood,
reach the tissue of interest as 0.05 mM.
C ið Þ ¼ bf i−1ð ÞC i−1ð Þ þ C 1ð Þ; i≥2 ð30Þ
where C(1) = 0.05 mM represents the first injection.
With each binding fraction dependent on the ratio of albumin
and gadofosveset concentrations in the vessel, the effect on T1
relaxation in the vascular and extracellular compartments, account-
ing for extravasation after each injection is described by Eqs. (31)
and (32), with r1bd and r1f varied independently of concentration and
binding fraction. The ideal non-leakage case relaxation is described
by Eq. (11), and Eqs. ((14)–(23)) describe the effect on signal and
measured fMBV.
1
T1vl
ið Þ ¼
1
T1v0
þ bf ið Þ Ã r1bd à C ið Þ ð31Þ
1
T1ev post
ið Þ ¼
1
T1ev0
þ 1−bf ið Þð Þ Ã r1f à C ið Þ ð32Þ
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