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DOI: 10.1161/CIRCRESAHA.120.317970 1
EIIect oI Do[orXEicin on Myocardial BicarEonate 3rodXction IroP 3yrXYate
DeKydrogenase in :oPen witK Breast Cancer
-ae Mo Park1,2,3, Galen D. Reed1,4, -eff Liticker1
, :illiam C. Putnam5, Alvin Chandra6,7, .atarina <aros8,
Aneela Afzal1,6, -ames MacNamara6
, -affar Raza5, Ronald G. Hall5, -eannie Baxter1, .elley Derner1,
Salvador Pena1, Raja Reddy .allem5, Indhu Subramaniyan5, Vindhya Edpuganti5, Crystal E. Harrison1,
Alagar Muthukumar10, Cheryl Lewis7,10, Sangeetha Reddy7,9, Nisha Unni7,9, Dawn .lemow7,9, Samira
Syed7,9, Hsiao Li7,9, Suzanne Cole7,9, Thomas Froehlich7,9, Colby Ayers6, -ames de Lemos6, Craig R.
Malloy1,3,6,7,11, Barbara Haley7,9, Vlad G. =aha1,6,9
1
Advanced Imaging Research Center 3Radiology 6Cardiology, Department of Internal Medicine
8
Internal Medicine 9Hematology and Oncology, Department of Internal Medicine 10Pathology,
University of Texas Southwestern Medical Center 2 Electrical and Computer Engineering, University of
Texas at Dallas 4General Electric Healthcare 5Pharmacy Practice, -erry H. Hodge School of Pharmacy,
Texas Tech University, Dallas Campus, Dallas, Texas 7 Harold C. Simmons Comprehensive Cancer
Center, Dallas, Texas, and 11 Veterans Affairs North Texas Healthcare System, Dallas, Texas.
Running title: Myocardial Pyruvate Metabolism in Cancer Patients
SXEMect TerPs:
Biomarkers
Clinical Studies
Magnetic Resonance Imaging (MRI)
Metabolism
Myocardial Biology
Address corresSondence to:
Dr. Vlad G. =aha
5323 Harry Hines Blvd.
Cardiology Division
Department of Internal Medicine
University of Texas Southwestern Medical Center
Dallas, T; 75390
vlad.zaha#utsouthwestern.edu
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DOI: 10.1161/CIRCRESAHA.120.317970 2
Keywords:
Doxorubicin, cardiotoxicity, dynamic nuclear polarization, pyruvate dehydrogenase, bicarbonate,
magnetic resonance spectroscopy diagnosis, metabolism, mitochondria, biomarker.
Clinical Trial Registration: NCT03685175
DATA AND MATERIALS AVAILABILITY
Datasets are available from the corresponding author on reasonable request, reviewed by the Institutional
Review Board and the Data Safety Monitoring Committee of the Harold C. Simmons Comprehensive
Cancer Center, University of Texas Southwestern Medical Center.
More than 5% of women treated for breast cancer with anthracyclines develop cardiotoxicity1
. The
mechanism of injury is not fully understood, but mitochondrial damage may play a causal role. Early
detection of myocardial damage due to anthracyclines is an important but unrealized objective.
Hyperpolarized (HP) 13C MR spectroscopy detects fluxes through reactions essential for normal energy
metabolism including pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH)2. This study tested
the hypothesis that exams of cardiac metabolism using HP [1-13C1]pyruvate are feasible before and after
conventional neoadjuvant doxorubicin chemotherapy in women with breast cancer, and that production of
HP [13C]bicarbonate or HP [1-13C1]lactate may be sensitive to chemotherapy.
The study (clinicaltrials.gov/ct2/show/NCT03685175) was approved by the relevant institutional
committees (STU 072016-058), under an Investigational New Drug approval (133229). All patients
provided written informed consent, were > 18 years old, without diabetes, with biopsy-proven breast cancer
requiring neoadjuvant doxorubicin (cumulative 240mg/m2
). Patients with metastatic lesions, significant
kidney, liver, cardiovascular or pulmonary disease and MR safety restrictions were excluded. Participants
continued their ordinary nutrition, activity and medications and received standard care for breast cancer
including screening with echocardiography.
Experimental procedures were similar to recently published exams in human subjects 3-5. Following
an overnight fast, participants arrived for study at 9:00 AM, and thirty minutes prior to the exam ingested
48 grams of glucose. Sterile [1-13C1]pyruvate was prepared in a laminar flow hood by a licensed pharmacist.
Polarization and quality assurance testing were performed using a SPINlabTM (GE Healthcare). Prior to
injection, the solution passed through a 0.22μm filter. MR studies were performed on a wide-bore 3T
clinical scanner (GE Discovery 750w). The positioning of the 13C coils, a transmit Helmholtz and 8-channel
receive array, was determined by participant comfort and body habitus with one or both coils anterior to
the heart (Figure). 13C data were acquired from a 10-cm long-axis slice ECG-triggered in mid diastole. The
excitation RF pulse was 10° every 2.8-3.8 seconds for 80 timepoints, total 3.7-5 minutes. 13C data were
reconstructed and analyzed using MATLAB. Data from each coil were weighted according to the distance
from the center of the LV cavity based on fiducials in a 1
H image acquired with the body coil. HP 13C
spectra were averaged over 90 seconds from injection for peak quantification and normalized to the total
13C signal (TC). Second study was completed 11 ± 0.5 days after doxorubicin. Data analysis was completed
in a blinded fashion, using objective measurement criteria. Statistical significance was evaluated using a
paired t-test (D= 0.05, two-tailed analysis). The sample size was 9 except echocardiography (n = 8) due to
missing post-doxorubicin test from Patient #4. Data are presented as mean ± standard error.
Ten patients were enrolled. One patient discontinued because of technical issues related to
polarization. Of the nine participants (age 47 ± 5 years, 3 Black, 6 non-Hispanic white) who completed
exams before and after therapy, none developed congestive heart failure. After therapy, patients had small
but significant changes in hemoglobin, high-sensitivity troponin (hs-cTnT) and peak left ventricular global
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DOI: 10.1161/CIRCRESAHA.120.317970 3
longitudinal strain (LVGLS) (Figure). All participants tolerated the HP exam well, without any adverse
effects. Baseline 13C NMR spectra were similar for all participants. The fraction of [13C]bicarbonate, [1-
13C1]lactate, and [1-13C1]alanine, relative to TC was 0.036 ± 0.005, 0.448 ± 0.023, and 0.045 ± 0.004,
respectively. After 4 cycles of doxorubicin and cyclophosphamide, there was a small decrease in HP
[
13C]bicarbonate relative to TC (bicarbonate/TC = 0.032 ± 0.005, p = 0.037) but no significant change in
HP [1-13C1]lactate (lactate/TC = 0.44 ± 0.04, p = 0.9) or [1-13C1]alanine (alanine/TC = 0.045 ± 0.006, p =
0.9). To examine the reproducibility of HP data acquisition during the same session, five patients before
chemotherapy and two patients after chemotherapy had two injections of HP [1-13C1]pyruvate separated by
30 min. Total signal at the first and second exam in a single session for [13C]bicarbonate/TC (0.037 ± 0.007
0.038 ± 0.007), [1-13C1]lactate/TC (0.061 ± 0.005 vs. 0.062 ± 0.006), and [1-13C1]alanine/TC (0.051 ± 0.005
vs. 0.050 ± 0.005) were not different.
In conclusion, myocardial HP 13C spectra acquired from patients with breast cancer were sensitive
to cardiotoxic chemotherapy. Data were reproducible within the same visit, and serial exams are feasible
and well-tolerated by patients. Doxorubicin was associated with a decrease in HP [13C]bicarbonate/TC,
consistent with subtle mitochondrial injury. Other biomarkers such as hemoglobin, hs-cTnT and LVGLS
(Figure) also changed in association with chemotherapy. Direct comparison to [18F]fluorodeoxyglucose
imaging with positron emission tomography and image-based localization of the [13C]bicarbonate signal is
desirable. The clinical relevance of HP methods awaits further evaluation.
S28RCES 2) )8NDIN*
The National Institute of Health (S10OD018468, S10RR029119, P41EB015908, R01NS107409), the
:elch Foundation (I-2009-20190330), The Texas Institute for Brain Injury and Repair, The Cancer
Prevention and Research Institute of Texas (RP180404 and RP170003) and a donation from the Ben E.
.eith Foundation.
DISCL2S8RE
G.D.R. is an employee of GE Healthcare.
RE)ERENCES
1. Mehta LS, :atson .E, Barac A, et al. Cardiovascular Disease and Breast Cancer: :here These
Entities Intersect: A Scientific Statement From the American Heart Association. Circulation.
2018 137:e30-e66.
2. Merritt ME, Harrison C, Storey C, -effrey FM, Sherry AD and Malloy CR. Hyperpolarized 13C
allows a direct measure of flux through a single enzyme-catalyzed step by NMR. Proceedings of the
National Academy of Sciences. 2007 104:19773-19777.
3. Cunningham CH, Lau -<, Chen AP, Geraghty B-, Perks :-, Roifman I, :right GA, Connelly
.A. Hyperpolarized 13C Metabolic MRI of the Human Heart: Initial Experience. Circ Res. 2016.
4. Gallagher FA, :oitek R, McLean MA, et al. Imaging breast cancer using hyperpolarized carbon13 MRI. Proc Natl Acad Sci U S A. 2020 117:2092-2098.
5. Rider O-, Apps A, Miller -, et al. Noninvasive In Vivo Assessment of Cardiac Metabolism in the
Healthy and Diabetic Human Heart Using Hyperpolarized (13)C MRI. Circ Res. 2020 126:725-736.
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DOI: 10.1161/CIRCRESAHA.120.317970 4
)I*8RE LE*END
)igXre E[SeriPental setXS KySerSolari]ed C NMR sSectra and clinical resXlts A Positioning of
8-channel paddle radiofrequency (RF) array receive coils is shown, along with an axial 1H MRI with
position of the 8-channel paddle array coils (blue arcs), with fiducial markers indicating the approximate
location of each loop. B 13C MR spectra from each participant, acquired over approximately 90 seconds
from a bolus injection of hyperpolarized [1-13C1]pyruvate is shown. C There was a significant decrease
in [13C]bicarbonate but not [1-13C1]lactate after doxorubicin treatment, normalized to the total 13C signal
(TC). D There was no significant change in left ventricular end-diastolic volume (LVEDV) or left
ventricular ejection fraction (LVEF) measured by echocardiography, but left ventricular global longitudinal
strain (LVGLS) deteriorated after therapy. E Compared to pre-treatment baseline, plasma hemoglobin
decreased and high sensitivity troponin (hs-cTnT) was slightly above the upper limit of normal. The error
bars denote standard error of the mean for a sample size of 9 for panels C and E, and 8 for panel D.
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Noninvasive In Vivo Assessment of Cardiac
Metabolism in the Healthy and Diabetic
Human Heart Using Hyperpolarized 13C MRI
研究背景
研究結(jié)果
研究對(duì)象
全球??? 2 ?????T2DM??????????T2DM ????????????????????????
??????????????????? 2-5 ?????????????????????????????
??????
??代謝??????????????????極?] 1-13C]pyruvate ???集????<2mins?, ???????
??????????????????????代謝?????
?研???? T2DM ?????極?] 1-13C]pyruvate ?????????????代謝????????????
????代謝?????????????????????????????????????????????
???極???????????MR ?????????????1
H-MRS ?????????? 31P-MRS ???
?????? /ATP ???????
???????????MR???????
???????????ǘ31P-MRS?????
????APT???????ǘ1
H-MRS???
????????????????????
????????極?13C?????????
(??)?????????????????
??
[
13C]?????[13C]???????????
????????????PDH??????
???????????????[
13C]???
[
13C]??????????????????
?[
13C]?????[13C]??????????
??????????????????PDH
?????
13?????????12?????????????18?ǖ??代謝??
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研究結(jié)論
應(yīng)用方向
??????????????????極?] 1-13C]pyruvate ???????????極? 13C 代謝??????
????????代謝??? PDH ?????????????代謝?????研???????極? 13C 磁共振
??????????????代謝???????????????????????????
???????????????代謝????ǘ????????研?ǘ
?ǘ???????????
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Circulation Research is available at www.ahajournals.org/journal/res
Circulation Research
Circulation Research. 2020;126:725–736. DOI: 10.1161/CIRCRESAHA.119.316260 March 13, 2020 725
Correspondence to: Prof Damian Tyler, PhD, BSci, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, Oxford Centre for Clinical Magnetic
Resonance Research (OCMR), University of Oxford, Oxford OX3 9DU, United Kingdom. Email damian.tyler@dpag.ox.ac.uk
*O.J.R. and A.A. contributed equally to this article.
?S.N. and D.J.T. contributed equally to this article.
The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.119.316260.
For Sources of Funding and Disclosures, see page 735.
? 2020 The Authors. Circulation Research is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article
under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is
properly cited.
ORIGINAL RESEARCH
Noninvasive In Vivo Assessment of Cardiac
Metabolism in the Healthy and Diabetic Human
Heart Using Hyperpolarized 13C MRI
Oliver J. Rider,* Andrew Apps,* Jack J.J.J. Miller, Justin Y.C. Lau, Andrew J.M. Lewis, Mark A. Peterzan, Michael S. Dodd,
Angus Z. Lau, Claire Trumper, Ferdia A. Gallagher, James T. Grist, Kevin M. Brindle, Stefan Neubauer,?
Damian J. Tyler ?
RATIONALE: The recent development of hyperpolarized 13C magnetic resonance spectroscopy has made it possible to measure
cellular metabolism in vivo, in real time.
OBJECTIVE: By comparing participants with and without type 2 diabetes mellitus (T2DM), we report the first case-control study
to use this technique to record changes in cardiac metabolism in the healthy and diseased human heart.
METHODS AND RESULTS: Thirteen people with T2DM (glycated hemoglobin, 6.9±1.0%) and 12 age-matched healthy
controls underwent assessment of cardiac systolic and diastolic function, myocardial energetics (31P-magnetic resonance
spectroscopy), and lipid content (1H-magnetic resonance spectroscopy) in the fasted state. In a subset (5 T2DM, 5 control),
hyperpolarized [1-13C]pyruvate magnetic resonance spectra were also acquired and in 5 of these participants (3 T2DM,
2 controls), this was successfully repeated 45 minutes after a 75 g oral glucose challenge. Downstream metabolism of
[1-13C]pyruvate via PDH (pyruvate dehydrogenase, [13C]bicarbonate), lactate dehydrogenase ([1-13C]lactate), and alanine
transaminase ([1-13C]alanine) was assessed. Metabolic flux through cardiac PDH was significantly reduced in the people with
T2DM (Fasted: 0.0084±0.0067 [Control] versus 0.0016±0.0014 [T2DM], Fed: 0.0184±0.0109 versus 0.0053±0.0041;
P=0.013). In addition, a significant increase in metabolic flux through PDH was observed after the oral glucose challenge
(P<0.001). As is characteristic of diabetes mellitus, impaired myocardial energetics, myocardial lipid content, and diastolic
function were also demonstrated in the wider study cohort.
CONCLUSIONS: This work represents the first demonstration of the ability of hyperpolarized 13C magnetic resonance spectroscopy
to noninvasively assess physiological and pathological changes in cardiac metabolism in the human heart. In doing so, we
highlight the potential of the technique to detect and quantify metabolic alterations in the setting of cardiovascular disease.
VISUAL OVERVIEW: An online visual overview is available for this article.
Key Words: diabetes mellitus ? diabetic cardiomyopathy ? hyperpolarized magnetic resonance spectroscopy ? magnetic resonance imaging
? metabolism ? pyruvate dehydrogenase
In This Issue, see p 705 | Meet the First Author, see p 706
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ORIGINAL RESEARCH
Rider et al Hyperpolarized 13C MRI in the Diabetic Human Heart
726 March 13, 2020 Circulation Research. 2020;126:725–736. DOI: 10.1161/CIRCRESAHA.119.316260
T
ype 2 diabetes mellitus (T2DM), even in the
absence of coronary artery disease and hypertension, is associated with a 2 to 5-fold increased risk
of heart failure through the development of diabetic
cardiomyopathy.1 With the rapid global increase in the
prevalence of obesity, and with it T2DM, it is very likely
that there will be a similar increase in the prevalence of
diabetic cardiomyopathy. As a result, there is a pressing
need to improve our understanding of the mechanisms
by which diabetes mellitus can cause heart failure and
to develop noninvasive readouts of the mechanisms
which underpin this process.
Several mechanisms have been implicated in the pathogenesis of diabetic cardiomyopathy with changes in myocardial structure, calcium signaling, and metabolism all
described in animal models.2
As the heart requires a vast
amount of ATP to maintain contractile function, it is not surprising that there are functional consequences if metabolism is altered, and in T2DM, metabolic alteration is inherent
to the underlying disease process. Although diabetes mellitus is characterized by an apparent abundance of substrate
with increased circulating levels of both free fatty acids and
glucose, the diabetic myocardium uses almost exclusively
free fatty acids for the generation of ATP, and its metabolic
flexibility is dramatically reduced.3
This arises due to the
combination of reduced glucose uptake4
and increased
fatty acid oxidation,5
which mediates an inhibition of PDH
(pyruvate dehydrogenase) as described by the Randle
cycle,6
resulting in a reduced efficiency of ATP production.
As both systole and diastole are ATP consuming processes, this leads to a proposed mechanism whereby
reduced glucose oxidation acts, via impaired ATP production, to contribute to the development of diabetic cardiomyopathy, with PDH being the central control point. In
line with this, we have recently shown that by pharmacologically increasing PDH flux, and therefore rebalancing
glucose utilization, it is possible to reverse the diastolic
impairment observable in a rodent model of T2DM.7 This
highlights the importance of PDH in this process as a
potential therapeutic target.
Mechanistic insights into diabetic cardiomyopathy to
date have, in general, been gained either in animal models, due to the need for invasive procedures or destructive methods which are not feasible in humans, or using
Nonstandard Abbreviations and Acronyms
CMR cardiac magnetic resonance
LDH lactate dehydrogenase
MR magnetic resonance
MRS magnetic resonance spectroscopy
PCr phosphocreatine
PDH pyruvate dehydrogenase
T2DM type 2 diabetes mellitus
Novelty and Significance
What Is Known?
? The way the heart turns fuels (eg, fats, glucose) into
energy, called metabolism, is altered in many types of
heart disease.
? However, we have very limited techniques available to
allow us to measure metabolism in patients.
What New Information Does This Article
Contribute?
? This article demonstrates the first use of a new technique, called hyperpolarized 13C magnetic resonance
imaging (MRI), for measuring changes in cardiac
metabolism in healthy controls and people with diabetes mellitus.
? We show here that hyperpolarized 13C MRI can detect
increases in the metabolism of carbohydrates (eg, glucose) when people go from being fasted to fed and
also that carbohydrate metabolism is significantly
reduced in the diabetic heart.
Alterations in cardiac metabolism are a hallmark of
many cardiovascular diseases, but current imaging techniques have a limited ability to study cardiac
metabolism noninvasively. The emerging technique of
hyperpolarized 13C MRI offers >10000-fold gains in
the sensitivity of MRI for the assessment of cardiac
metabolism. This work demonstrates the first step in
the clinical translation of this exciting new technology
into cardiovascular disease characterization through
the observation of metabolic flux changes in the normal
and the diabetic human heart. By showing that metabolic flux through the key regulatory enzyme, pyruvate
dehydrogenase is increased in the transition from the
fasted to the fed state and is significantly reduced in
the diabetic heart, this work represents the first demonstration of the ability of hyperpolarized 13C MRI to
noninvasively assess physiological and pathological
changes in cardiac metabolism in the human heart. As
hyperpolarized 13C MRI allows the in vivo visualization
of cardiac metabolism, it has major advantages over
current noninvasive imaging techniques. Hyperpolarized 13C MRI scans are fast (<2 minutes), have no ionizing radiation, and, due to the ability to simultaneously
acquire standard MRI acquisitions, have the potential
to directly assess perfusion, ischemia, viability, and
altered substrate selection in one imaging session.
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ORIGINAL RESEARCH
Rider et al Hyperpolarized 13C MRI in the Diabetic Human Heart
Circulation Research. 2020;126:725–736. DOI: 10.1161/CIRCRESAHA.119.316260 March 13, 2020 727
positron emission tomography and magnetic resonance
spectroscopy (MRS). Positron emission tomography
studies have revealed reductions in glucose uptake8 and
increases in fatty acid oxidation9 while MRS studies have
shown elevated myocardial triglyceride content10 and
impaired myocardial energetics,11 confirming to a large
extent the findings in animal studies. However, gaining
a window on important changes at the level of PDH has
not been possible without invasive biopsies, making this
impractical to assess as a routine biomarker.
One potential solution to this is 13C MRS. This technique
allows a direct evaluation of the activity of PDH by measuring the conversion of [1-13C]pyruvate into [13C]bicarbonate (H13CO3
?). However, although this is scientifically
attractive, conventional 13C MRS suffers from an inherently low sensitivity and low signal-to-noise ratio, making
scan times very long, and routine acquisition unfeasible
at clinical field strengths. This low sensitivity can be overcome using the recent development of hyperpolarized
magnetic resonance (MR) technology, which can amplify
the 13C MRS signal by over 10000-fold.12 Using hyperpolarized [1-13C]pyruvate, physiological changes in PDH
flux have been demonstrated in animal models of feeding
and fasting.13–15 In addition, changes in cardiac substrate
selection in a variety of pathological situations have been
observed,16–18 particularly in diabetes mellitus.7,14,19,20
The human applications of this technique are in their
infancy, with an initial clinical demonstration in a study of
patients with prostate cancer,21 and 2 smaller feasibility studies, one in the healthy heart22 and another in the
healthy brain.23 Despite its potential, the assessment of
either physiological or pathological changes in metabolic
flux using hyperpolarized MRS have not yet been undertaken in the human heart.
As such, the primary aim of the work presented here
was to provide the first noninvasive in vivo demonstration
that physiological and pathological changes in PDH flux
can be detected in the human heart using hyperpolarized
[1-13C]pyruvate MRS. By also assessing other hallmarks
of diabetic heart disease, namely impaired energetics
(
31P-MRS), myocardial steatosis (1H-MRS), and diastolic
impairment (echocardiography), we further aimed to
determine the additional information that the hyperpolarized [1-13C]pyruvate technique can provide in the detection of pathological changes in the diabetic heart.
METHODS
The data that support the findings of this study are available
from the corresponding author on reasonable request.
Study Cohort and Study Visit
This research was approved by the National Research Ethics
Committee service (13/SW/0108) and conducted in accordance with the declaration of Helsinki and the Caldicott
principles. All data collection was undertaken at the Oxford
Centre for Clinical Magnetic Resonance at the John Radcliffe
Hospital, Oxford, United Kingdom between March 2016 and
May 2019. Written informed consent was obtained from all
those enrolled. Thirteen people with T2DM, and 12 controls
were recruited from local advertisements. All participants were
aged >18, participants with T2DM were included if they had a
recent glycated hemoglobin between 6 and 9%, no change of
oral medications during the previous 3 months and were not
on insulin therapy. Subjects with T2DM who were taking the
oral antihyperglycemic drug, Metformin, were asked to refrain
from taking their medication for 12 hours before the study to
minimize any potential effect on cardiac redox state.24
All study visits began at 7 am following an overnight fast
lasting at least 9 hours. Diastolic function (echocardiography),
systolic function (CMR), myocardial steatosis (1H-MRS), and
myocardial energetics (31P-MRS) were all assessed in the fasted
state. Additionally, hyperpolarized [1-13C]pyruvate MRS was
undertaken immediately before and 45 minutes after a standardized oral glucose tolerance test consisting of a 75 g glucose
dose (taken in under 5 minutes; Rapilose, Galen Ltd, Craigavon,
United Kingdom). All MR scanning was undertaken at 3T (Tim
Trio MR system, Siemens Healthineers, Erlangen, Germany).
The outline of our study visit is shown in Figure 1, and
additional methodological details are given in the Online Data
Supplement.
Dynamic Nuclear Polarization and Production of
Hyperpolarized [1-13C]Pyruvate
As described in the Online Data Supplement, all starting
materials were prepared in a Grade A sterile environment23
before being loaded into a General Electric SpinLab system
(GE Healthcare, Chicago) for the process of Dynamic Nuclear
Polarization.12 Sufficient polarization levels were achieved
after 2 to 3 hours, after which dissolution was undertaken to
produce the final hyperpolarized [1-13C]pyruvate solution for
injection. Solutions were only released for human injection
if the following criteria were met: pH 6.7 to 8.4, temperature
25.0°C to 37.0°C, polarization ≥15%, (pyruvate) 220 to 280
mmol/L, (electron paramagnetic agent) ≤3.0 μmol/L, appearance: clear, colorless solution with no visible particulate matter.
Administration of the hyperpolarized pyruvate was undertaken
through an 18G venous cannula sited in the left antecubital
fossa at a dose of 0.4 mL/kg and at a rate of 5 mL per second.
Hyperpolarized MR Spectroscopy and Data
Processing
Subjects were scanned supine and hyperpolarized 13C MR spectra were acquired using a 2 channel transmit, 8 channel surfacereceive array (Rapid Biomedical, Rimpar, Germany). Hyperpolarized
data were acquired from a mid-ventricular 10 mm axial slice,
beginning at the start of the injection, using a pulse-acquire
spectroscopy sequence acquired ECG-gated to the R-wave with
a single slice-selective excitation every heartbeat and run for 4
minutes after injection. Total integrated metabolite-to-pyruvate
ratios, known to linearly correlate with first-order chemical kinetic
rate constants, were calculated by summing the first 60 seconds
worth of spectral data acquired following the initial appearance of
the hyperpolarized pyruvate resonance in the acquired spectra.25
Further details are provided in the Online Data Supplement.
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ORIGINAL RESEARCH
Rider et al Hyperpolarized 13C MRI in the Diabetic Human Heart
728 March 13, 2020 Circulation Research. 2020;126:725–736. DOI: 10.1161/CIRCRESAHA.119.316260
Statistical Analysis
All data were analyzed with the operator blinded to the disease
status and metabolic state of the data set. Hyperpolarized
data sets, quantified as described above, were analyzed with
the lme4 and the car packages in R (v3.6.0, R Foundation for
Statistical Computing, Vienna, Austria), with metabolic state
and disease status considered as fixed effects, and subject
ID considered as a random effect, and an ANOVA table computed. Data were subject to a Shapiro-Wilk normality test, and
one outlier corresponding to the [13C]bicarbonate to [1-13C]
pyruvate ratio for an unpaired fasted subject with T2DM with
a Z-score of 9.4 was identified (Grubb test P=0.003, suggesting that point was an outlier). Data derived from this participant were excluded from subsequent analysis. No evidence
of heteroscedasticity was found in the acquired 13C data
(Levene test, P=0.301 for [13C]bicarbonate to [1-13C]pyruvate
ratio, P=0.635 for [1-13C]lactate to [1-13C]pyruvate ratio and
P=0.751 for [1-13C]alanine to [1-13C]pyruvate ratio). This fact
may reflect the comparatively high signal-to-noise ratio of the
acquired spectral data, as it is known that the distribution of
metabolite ratios is approximately normally distributed in the
high signal-to-noise ratio regime.26
Unless otherwise stated, all other analyses were performed
in GraphPad Prism (GraphPad Software, San Diego, CA) via
simple unpaired unequal-variance t tests with the canonical
P<0.05 threshold for statistical significance. All statistical tests
performed are reported in Tables 1 and 2 with the exact P values quoted.
Figure 1. Outline of our typical study visit.
The fasting stipulation in our study restricted our recruitment to what can be considered a fairly mild phenotype of diabetes mellitus—only those
patients receiving oral medication. The total study visit was under 3 hours; however, each hyperpolarized magnetic resonance spectroscopy (MRS)
scan took only a few minutes, meaning its addition to the normal length of routine magnetic resonance protocols would be insignificant. CKD
indicates chronic kidney disease; CMR, cardiac magnetic resonance; eGFR, estimated glomerular filtration rate; and HbA1c, glycated hemoglobin.
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RESULTS
Baseline Population Characterization
Healthy controls (n=12) and people with T2DM
(n=13) were recruited with no difference in age (controls—50.3±11.4 years, people with T2DM—55.2±5.8
years; P=0.190) or sex (controls—8 male/4 female, people
with T2DM—11 male, 2 female). Participants with T2DM
had significantly higher body mass index than controls
(22.6±3.0 versus 29.7±6.8; P=0.003), but baseline myocardial structural characteristics assessed by cine-magnetic resonance imaging including left ventricular ejection
fraction (60±4% versus 57±6%; P=0.228), indexed left
ventricular end-diastolic volume (82±12 versus 79±15
mL/m2; P=0.577) and myocardial mass index (64±10 versus 62±11 g/m2; P=0.658), were not different between
groups (Table 1). Participants with T2DM were confirmed
to be more insulin resistant than the controls (homeostatic
model assessment of insulin resistance, 1.3±0.8 versus
4.3±2.5; P=0.005), with higher fasting blood sugar. Five
controls and 5 people with T2DM from within this cohort
then went on to have fasting [1-13C]pyruvate hyperpolarized MRS, with 5 (2 control, three T2DM) receiving successful repeat [1-13C]pyruvate hyperpolarized MRS 45
minutes after glucose ingestion. Again, this smaller hyperpolarized MRS group was well matched for age and myocardial structural characteristics (Table 1). Example data
acquired from our study population are shown in Figure 2,
demonstrating the breadth of metabolic and structural
parameters acquired in a single scanning session.
Injected Hyperpolarized [1-13C]Pyruvate
Solution Product Specifications
Hyperpolarized [1-13C]pyruvate solution injections were
well tolerated by all subjects with no side effects reported.
Ten participants (5 controls, 5 T2DM) received a total of
15 injections meeting the release criteria. The quality of
these were highly standardized; mean (±SD) pyruvate
concentration was 239±8 mmol/L, residual electron
paramagnetic agent 1.1±0.7 μmol/L, pH 7.7±0.4, temperature 34±1°C, and polarization 34±13%. The mean
polarization time was 150±30 minutes, and dissolution
to injection times were all <90 seconds.
Hyperpolarized 13C MRS
Acquired hyperpolarized spectra were of high quality with
peaks corresponding to [13C]bicarbonate, 13CO2, [1-13C]
lactate and [1-13C]alanine (the downstream metabolites
of [1-13C]pyruvate), clearly visible and appearing 2 to 3
seconds after the ventricular [1-13C]pyruvate resonance.
Example fed and fasted summed spectra from both a
Table 1. Characteristics of Study Population
Study Population Control (n=12) Diabetic (n=13) P Value
General
Age, y 50.3±11.4 55.2±5.8 0.190
Weight, Kg 68.0±13.1 93.7±17.7 <0.001*
BMI, Kg/m2 22.6±3.0 29.7±6.8 0.003*
HbA1c, % 4.9±0.3 6.9±1.0 <0.001*
HOMA IR 1.3±0.8 4.3±2.5 0.005*
Fasting glucose, mmol/L 4.8±0.7 7.9±2.7 0.006*
Medication
ACE-inhibitor 0 6 …
Statin 0 9 …
Metformin 0 11 …
Sulfonylurea 0 5 …
Calcium channel blocker 0 2 …
Thiazide diuretic 0 2 …
Asprin 0 2 …
Liraglutide 0 1 …
Sitagliptin 0 1 …
Echocardiography
E/A 1.3±0.4 1.0±0.3 0.127
E/e′ medial 6.3±2.0 8.1±1.4 0.025*
E/e′ (lateral) 5.1±1.8 6.3±1.8 0.149
E/e′ (mean) 5.7±1.7 7.2±1.4 0.040*
CMR
LVEF, % 60±4 57±6 0.228
LVEDV index, ml/m2 82±12 79±15 0.577
LV mass index, g/m2 64±10 62±11 0.658
RVEF, % 54±4 54±6 0.934
RVEDV index, ml/m2 93±8 84±16 0.095
Spectroscopy
PCr/ATP 1.94±0.21 1.71±0.30 0.042*
Myocardial lipid content,
% of water
1.59±0.88 3.05±1.96 0.026*
13C MRS only group Control (n=5) Diabetic (n=5)
Age, y 49.2±13.1 52±5.2 0.668
Weight, Kg 72.1±7.4 99.2±13.9 0.005*
BMI, Kg/m2 22.6±1.7 31.1±6.1 0.017*
E/e′, mean 4.7±1.1 6.6±1.2 0.030*
PCr/ATP 2.03±0.15 1.75±0.35 0.138
Myocardial lipid content,
% of water
1.29±0.63 3.40±2.26 0.079
*Significance P<0.05.
ACE indicates angiotensin-converting enzyme; BMI, body mass index;
CMR, cardiac magnetic resonance; E/A, early to late diastolic transmitral flow
velocity ratio; E/e', early diastolic transmitral flow velocity to early diastolic
mitral annular tissue velocity ratio; HbA1C, glycated hemoglobin; HOMA
IR, homeostatic model assessment of insulin resistance; LV, left ventricular;
LVEDV, left ventricular end-diastolic volume; LVEF, left ventricular ejection
fraction; MRS, magnetic resonance spectroscopy; PCr, phosphocreatine;
RVEDV, right ventricular end-diastolic volume; and RVEF, right ventricular
ejection fraction.
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control and subject with T2DM are shown in Figure 3,
with typical time courses of substrate and metabolite
signal intensities for a control and a subject with T2DM
also shown. A summary of time-integrated metabolite to
substrate ratios derived from the in vivo hyperpolarized
13C MRS data can be found in Table 2.
The [13C]bicarbonate to [1-13C]pyruvate ratio, shown
previously to linearly correlate with enzymatic flux
through PDH, was significantly reduced by diabetes mellitus (5.3-fold reduction when fasted and 3.5-fold reduction when fed, P=0.013). Conversely, the [1-13C]lactate
to [1-13C]pyruvate ratio, reflecting exchange through
LDH (lactate dehydrogenase), was increased by diabetes mellitus (1.6-fold increase when fasted and 1.8-
fold increase when fed, P<0.001). As a marker of the
balance between glycolytic and oxidative carbohydrate
metabolism,27 the ratio of [13C]bicarbonate and [1-13C]
lactate signals showed a significant reduction in relative
carbohydrate oxidation in the subjects with T2DM (7.5-
fold reduction when fasted and 6-fold reduction when
fed, P<0.001). Transamination of [1-13C]pyruvate to
[1-13C]alanine was not different between subjects with
T2DM and controls (P=0.257). Comparisons of enzymatic flux data as assessed by hyperpolarized MRS are
summarized in Figure 4.
Hyperpolarized MRS also successfully demonstrated
Randle cycle associated increases in PDH flux after
feeding with flux significantly increased 45 minutes after
the oral administration of 75 g of glucose (P<0.001).
Importantly, this increase was discernible not only in
controls (2.2-fold increase) but also in the subjects with
T2DM (3.3-fold increase), in spite of the impaired basal
PDH flux we have demonstrated in this condition. There
were no statistically significant differences in LDH
flux (P=0.072) or the rate of pyruvate transamination
(P=0.077) between the fasted and fed states.
31P and1
H MRS
As expected, within the wider study population, diabetes mellitus significantly impaired cardiac diastolic function (mean E/e′ 5.7±1.7 versus 7.2±1.4; P=0.040),
Table 2. Time-Integrated Metabolite to Substrate Ratios Derived From Hyperpolarized 13C MR Data
Control (n=5) Diabetic (n=5) P Value
Fasted (n=5) Fed (n=2) Fasted (n=5) Fed (n=3) Metabolic State Diseased State Interaction
Bic/Pyr (×10?2) 0.84±0.67 1.84±1.09 0.16±0.14 0.53±0.41 <0.001* 0.013* 0.040*
Lac/Pyr (×10?2) 5.16±1.52 5.94±2.01 8.51±1.38 10.53±1.38 0.072 <0.001* 0.455
Bic/Lac 0.15±0.10 0.30±0.08 0.02±0.02 0.05±0.03 <0.001* <0.001* 0.008*
Ala/Pyr (×10?2) 3.17±1.11 3.70±2.03 3.82±1.05 4.74±0.66 0.077 0.257 0.690
Ala indicates alanine; Bic, bicarbonate; Lac, lactate; MR, smagnetic resonance; and Pyr, pyruvate.
Figure 2. Example data collected during our study from a recruited control (top row) and a subject with type 2 diabetes mellitus
(bottom row).
In characterizing our recruits both structurally (cardiac magnetic resonance [CMR]/Echo) and metabolically (31P magnetic resonance
spectroscopy [MRS], 1H MRS, hyperpolarized 13C MRS), we collate the most comprehensive study of the diabetic cardiac phenotype to date. LV
indicates left ventricular; and RV, right ventricular.
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myocardial energetics (phosphocreatine [PCr]/
ATP 1.94±0.21 versus 1.71±0.30; P=0.042), and
increased myocardial triglyceride content (1.59±0.88
versus 3.05±1.96; P=0.026). The effect sizes for
these differences (E/e′=0.963, PCr/ATP=0.888, myocardial triglyceride content=0.961, G*Power 3.1) were
all lower than the effect sizes calculated for the differences observed between the fasted controls and the
subjects with T2DM from the 13C enzymatic flux data
reported above (bicarbonate/pyruvate=1.405, lactate/
pyruvate=2.308, bicarbonate/lactate=1.803). This
means that, when comparing 2 groups with a simple
Student t test, to observe the differences seen here at
a P value of 0.05 with a power of 90% would require
group sizes of 24, 28, and 24 for E/e′, PCr/ATP and
myocardial triglyceride content respectively versus
group sizes of 12, 6, and 8 for bicarbonate/pyruvate,
lactate/pyruvate, and bicarbonate/lactate, respectively
(G*Power 3.1).
Weak correlations were observed between the PCr/
ATP ratio and the metabolic parameters assessed by
hyperpolarized MRS (ie, positive correlations between
PCr/ATP and the bicarbonate/pyruvate, alanine/pyruvate and bicarbonate/lactate ratios and a negative correlation between PCr/ATP and the lactate/pyruvate
ratio, but these failed to reach statistical significance,
Online Figure I).
Figure 3. Representative examples of hyperpolarized magnetic resonance spectra from both a healthy control and a subject
with type 2 diabetes mellitus in both the fasted and fed states, with 13C containing downstream metabolites labeled.
The [13C]bicarbonate resonance is visibly reduced in the subject with type 2 diabetes mellitus with increases seen during feeding in both controls
and subjects with type 2 diabetes mellitus. Time courses of the normalized signal amplitudes of downstream 13C-labeled metabolic products of
administered [1-13C]pyruvate (shown in blue), in both a control and a subject with type 2 diabetes mellitus are also shown.
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Figure 4. Plots of metabolic flux data for each metabolic product of administered [1-13C]pyruvate.
Flux through PDH (pyruvate dehydrogenase; bicarbonate, A) is reduced in the subjects with type 2 diabetes mellitus (P=0.013), with increases
seen during feeding (P<0.001, E). Levels of [1-13C]lactate were significantly higher in the hearts of people with type 2 diabetes mellitus
(P<0.001, B) with no change observed on feeding (F). The ratio of bicarbonate and lactate was significantly lower in the subjects with type 2
diabetes mellitus (P<0.001, C) and was elevated by feeding (P<0.001, G). No significant differences in [1-13C]alanine were seen across all
injections (D and H). ‘x’ indicates the data point excluded as an outlier. ?P<0.05 in subjects with type 2 diabetes mellitus vs controls and *P<0.05
in fasted subjects vs fed.
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DISCUSSION
In the setting of the rapid global increase in T2DM and
its relationship with heart failure, increasing our understanding of the metabolic changes that occur in diabetes mellitus is becoming increasingly important. Using a
hyperpolarized [1-13C]pyruvate tracer, we have shown that,
following glucose ingestion, the myocardium increases
pyruvate oxidation through PDH (PDH Flux), in line with
the metabolic alterations proposed by the Randle cycle.6
In addition, we have also shown in patients with T2DM
and diastolic dysfunction that PDH flux is reduced, similarly to alterations seen in animal models.7,20 This, therefore, represents the first noninvasive demonstration of
physiological and pathological changes in PDH flux in the
human heart using hyperpolarized MRS. Furthermore, we
have used 31P and1H spectroscopy to confirm that, in the
presence of reduced PDH flux, the diabetic myocardium
has reduced myocardial energetics (PCr/ATP ratio) and
increased myocardial triglyceride content. This is the first
human study to use the multinuclear combination of 1H, 31P, and 13C MR spectroscopy to interrogate myocardial
metabolism and confirms the potential of hyperpolarized
MRS for translation to the clinical quantification of metabolic alterations in cardiac pathology.
Pyruvate Dehydrogenase Flux
Our demonstration that the fasted heart increases PDH
flux after an oral glucose challenge is consistent with
the Randle cycle and confirms previous hyperpolarized
[1-13C]pyruvate experiments in mice,13 rats,14 and pigs.15
While this is an expected result, it is the first demonstration in humans that hyperpolarized [1-13C]pyruvate MR
can detect physiological changes in myocardial metabolism, an important milestone in its clinical translation.
As the post-glucose scan was undertaken |1 hour
after the initial fasted scan, there is the possibility that the
injected pyruvate dose from the first scan may also have
played a part in the increased PDH flux observed. However, it seems unlikely that the |1 g dose of pyruvate given
would have had a significant impact on top of the 75 g
of glucose provided. The variation in PDH flux observed
between the fed and fasted states also illustrates that,
when considering myocardial metabolic readouts, there is
a need to standardize (or at least establish) the prevailing
metabolic conditions under which they are made. To date,
animal models have used glucose loading before hyperpolarized studies to maximize baseline PDH flux, increasing
the power of studies aiming to detect pathological changes.
In contrast to the normal heart, which has metabolic
flexibility, the diabetic heart becomes almost exclusively
reliant on fatty acids as its main catabolic substrate. This
overreliance on fat metabolism is likely underpinned by an
impaired ability to uptake glucose and oxidize the resulting
pyruvate through PDH. Indeed, animal models of diabetes
mellitus have shown PDH inhibition both ex vivo28 and in
vivo.14 In line with this, we have shown here in people with
T2DM, that myocardial PDH flux is reduced compared
with the normal healthy heart. Minimal discernible flux
through PDH was observed in the fasted diabetic state,
with only a small increase demonstrated after glucose
loading, however, our findings show that hyperpolarized
[1-13C]pyruvate studies aimed at measuring alterations in
PDH flux in patients with T2DM are indeed feasible.
Linking Altered Substrate Metabolism to
Altered Function
As diastole is more susceptible to ATP shortage than
systole, alterations in substrate selection may act via
reduced efficiency of ATP production initially as diastolic dysfunction, which is an almost universal finding
in T2DM.29,30 In line with this, we have shown here that
the diabetic myocardium has reduced pyruvate oxidation (reflective of reduced glucose utilization), increased
triglyceride deposition (suggestive of excess fatty acid
uptake), reduced myocardial energetics (with reduced
PCr/ATP), and diastolic dysfunction. As the diabetic
phenotype in this study was not advanced or severe (we
excluded subjects requiring exogenous insulin; average
glycated hemoglobin was 6.9%), this highlights the metabolic inflexibility of the cardiomyocyte in the setting of
lower grades of insulin resistance, and also the ability of
hyperpolarized MR to detect early changes in myocardial
metabolism in diabetes mellitus.
Lactate Dehydrogenase Flux
Incorporation of the 13C label into [1-13C]lactate in our
acquired spectra was significantly higher in subjects with
T2DM in both fasted and fed states suggesting raised
LDH flux in this group. Although it could be assumed that
given [1-13C]pyruvate flux through PDH was lower, that
LDH flux, and therefore the lactate pool size,31 would be
reciprocally increased, this interpretation may be too simplistic. Other factors should be considered, for example, it
has previously been demonstrated that the antihyperglycemic agent, Metformin, has an effect on cardiac redox
state that elevates the observed lactate signal.24 To minimize this effect, the subjects with T2DM studied were
asked to refrain from taking their Metformin on the day
of the study. However, we cannot exclude the possibility
that a chronic effect of their Metformin treatment may
have contributed to the elevated lactate signal observed.
In addition, the myocardial [1-13C]lactate signal following injection of [1-13C]pyruvate has proven much more
diffuse in hyperpolarized short-axis images of the both
the human22 and pig heart32 with a large contribution
from the blood pool. Therefore, [1-13C]lactate generated
in, and effluxed from, the liver may also be contaminating
the cardiac readouts.33 As such, we must be cautious in
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interpreting the exact derivation of the increased lactate
signal from nonlocalized spectra. With metabolite imaging now possible in the human heart,22 this will aid in the
localization of the lactate signal and discern whether or
not its origin is myocardial.
Alanine Aminotransferase Flux
In ex vivo models, the rate of pyruvate transamination
has been shown to increase proportionally as pyruvate
perfusate concentration increases. Labeled alanine is
thus a direct measure of the intracellular availability of
labeled pyruvate, and the alanine signal has, therefore,
been suggested as an alternative normalization standard (as opposed to the pyruvate signal).34 Relative stability of [1-13C]alanine signals in our study, and lack of
difference between groups, suggests cellular bioavailability of administered [1-13C]pyruvate was uniform and
not a potential confounder of the variation of enzymatic
fluxes seen.
Wider Translation to Clinical Practice
The technology of dissolution dynamic nuclear polarization is still in its infancy. The first demonstration of clinical translation was published in 2013 using a prototype
polarizer located inside a cleanroom to prepare sterile
injections for prostate cancer patients.21 The SpinLab is
the clinical-grade second generation of polarizer suitable
for preparing sterile injections outside of a controlled
pharmaceutical facility, and currently, 10 sites worldwide
are injecting hyperpolarized compounds in early-phase
clinical trials. Using this clinical system, we have demonstrated the first step in the clinical translation into
cardiovascular disease characterization through the
observation of metabolic flux changes in the normal and
the diabetic human heart. While technically challenging,
leading in part to our work being performed on a comparatively small number of subjects, the large effect size of
metabolic dysregulation in disease is such that significant
differences in myocardial metabolism, known extensively
to exist from several decades of previous animal experimentation, as well as the effects of novel therapies, can
be conclusively demonstrated in the human heart. Future
studies should build on this proof-of-principle to explore
the impact of other cardiovascular diseases, as well as
the role that possible confounding factors (such as age,
sex, medication use) might have on cardiac metabolism.
As hyperpolarized 13C-imaging allows the in vivo visualization of cardiac metabolism, it has major advantages
over current noninvasive imaging techniques. Hyperpolarized scans are fast (<2 minutes), have no ionizing
radiation, and, due to the ability to simultaneously acquire
standard magnetic resonance imaging acquisitions, have
the potential to directly assess perfusion, ischemia, viability, and altered substrate selection in the same imaging
session. However, the technique does have some limitations. First, the rapid decay of the hyperpolarized signal
(ie, the T1 of hyperpolarized [1-13C]pyruvate in solution
has been measured to be 67.3±2.5 s at 3T35) leads to the
requirement to undertake the hyperpolarization process
adjacent to the magnetic resonance imaging system and
to inject the hyperpolarized tracer immediately after production. While this offers some technical challenges, the
work reported here and by others21–23 demonstrates that
these challenges, as with short-lived positron emission
tomography tracers, can be overcome.
Second, in contrast to positron emission tomography systems, which are capable of measuring picomolar
amounts of radiolabeled molecules, hyperpolarized pyruvate scans require injection of the tracer at millimolar
concentrations. It has previously been suggested that
this supra-physiological dose of pyruvate may impact
the metabolic processes that are being assessed. However, preclinical work in animals has shown that similar doses (|320 mol/kg in previous rat studies versus
the |140 mol/kg used in this work) leads to maximum plasma pyruvate concentrations of |250 μmol/L,
equivalent to physiological pyruvate concentrations
reached during exercise or with dietary interventions.34
In addition, preclinical studies have demonstrated tight
correlations between in vivo hyperpolarized MRS measurements of PDH flux and ex vivo measurements of
PDH enzyme activity.34
While the work described here was undertaken at 3T,
there are advantages and disadvantages to undertaking
hyperpolarized experiments at different field strengths.
Higher field strengths provide increased spectral separation between different metabolites and the subsequent benefits in quantification and selection of different
metabolites for spectral imaging that this brings. Alternatively, the longitudinal relaxation times of hyperpolarized
agents are generally longer at lower field strengths,35
and there is improved B0 homogeneity which will improve
spectral linewidths. As such, 3T seems a reasonable
compromise between these competing factors for such
initial proof-of-concept studies.
In conclusion, this study provides the first demonstration of the ability of hyperpolarized pyruvate to noninvasively assess physiological and pathological changes
in pyruvate dehydrogenase flux in the human heart. In
doing so, we highlight the potential of the technique to
assess metabolic alterations in a range of cardiovascular diseases.
ARTICLE INFORMATION
Received October 28, 2019; revision received January 29, 2020; accepted
February 4, 2020.
Affiliations
From the Oxford Centre for Clinical Magnetic Resonance Research, Radcliffe
Department of Medicine (O.J.R., A.A., J.J.J.J.M., J.Y.C.L., A.J.M.L., M.A.P., C.T., S.N.,
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Circulation Research. 2020;126:725–736. DOI: 10.1161/CIRCRESAHA.119.316260 March 13, 2020 735
D.J.T.), Department of Physiology, Anatomy and Genetics (J.J.J.J.M., J.Y.C.L.,
D.J.T.), and Department of Physics (J.J.J.J.M.), University of Oxford, United Kingdom; School of Life Sciences, Coventry University, United Kingdom (M.S.D.);
Sunnybrook Research Institute, Toronto, Canada (A.Z.L.); and Department of
Radiology (F.A.G., J.T.G.) and Cancer Research UK Cambridge Institute (K.M.B.),
University of Cambridge, United Kingdom.
Sources of Funding
This study was funded by a programme grant from the British Heart Foundation
(RG/11/9/28921). The authors would also like to acknowledge financial support
provided by the British Heart Foundation (BHF) in the form of Clinical Research
Training Fellowships, a BHF Intermediate Clinical Research Fellowship and a
BHF Senior Research Fellowship, respectively (O.J. Rider: FS/14/54/30946,
A. Apps: FS/17/18/32449, A.J.M. Lewis: RE/08/004/23915, M.A. Peterzan:
FS/15/80/31803, and D.J. Tyler: FS/14/17/30634). J.J.J.J. Miller and M.S.
Dodd would like to acknowledge the financial support provided by Novo Nordisk Postdoctoral Fellowships. J.J.J.J. Miller would also like to acknowledge financial support from Engineering and Physical Sciences Research Council. F.A.
Gallagher would like to acknowledge Cancer Research UK (CRUK), the CRUK
Cambridge Centre, the Wellcome Trust and the Cambridge Biomedical Research
Centre. All authors would also like to acknowledge the support provided by the
OXFORD-BHF Centre for Research Excellence (grant RE/13/1/30181) and
the National Institute for Health Research Oxford Biomedical Research Centre
programme.
Acknowledgments
We would like to thank Laura Rodden, Katy Crofts, Katy Briggs, Matthew Wilkins,
and Claire Church and the Clinical Trials Aseptic Service Unit at the Oxford University Hospitals National Health Services Foundation Trust and Anita Chhabra,
Marie-Christine Laurent, Vicky Fernandes, and Matthew Locke from the University of Cambridge for their technical expertise in the preparation of the Sterile
Fluid Pathways (SFPs) used in this study.
Disclosures
F.A. Gallagher has received research support from GE Healthcare. K.M. Brindle
holds patents in the field of hyperpolarized magnetic resonance imaging (MRI)
relating to the use of imaging media comprising lactate and hyperpolarized [13C]
pyruvate, 13C-MR imaging or spectroscopy of cell death, hyperpolarized lactate as
a contrast agent for determination of LDH (lactate dehydrogenase) activity and
imaging of ethanol metabolism. In addition, K.M. Brindle has research agreements
with GE Healthcare which involve the use of hyperpolarized MRI technology. D.J.
Tyler holds a patent relating to the use of hyperpolarized [1-13C]pyruvate for the
assessment of PDH (pyruvate dehydrogenase) flux and has research agreements with GE Healthcare which involve the use of hyperpolarized MRI technology. The other authors report no conflicts.
Supplemental Materials
Expanded Materials & Methods
Supplemental Tables I–II
Supplemental Figure I
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Proof-of-Principle Demonstration of Direct
Metabolic Imaging Following Myocardial Infarction Using Hyperpolarized 13C CMR
研究背景
研究結(jié)果
研究對(duì)象
???????全球?????????????????????????代謝?????????代謝???
??????????????????????磁共振???????????????????????代謝
???
?????代謝??????????PDH??????????LDH?????????????代謝???代
謝??????? PDH ? LDH ?????????????代謝?????研?????極?] 1-13C]pyruvate ??
???????????????????代謝???
??ǖ?? 1?磁共振?????????????????????????????????????????
?極?] 1-13C] ???????????? [
13C] ????? [13C] ???????????????????????
?????代謝???
??ǖ?? 2?磁共振????????????????????????????????????????
?????極?] 1-13C] ???????????????[
13C] ????? [13C] ????????????????
???
2?????????
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研究結(jié)論
應(yīng)用方向
??代謝???????????????????極?] 1-13C]pyruvate ??????????????????
PDH ??????????????????????????]
13C] ????????????????????
??????????研????????????????????????????????代謝????
??????ǘ??????ǘ????????
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p ? 0.015). Patients with baseline [LVGLS] <6.5% had
significantly lower survival rates compared with
those with baseline [LVGLS] $6.5% (p ? 0.017 by
log-rank test) (Figure 1D). From multivariable
analysis, baseline [LVGLS] also showed significant
additive predictive values for all-cause mortality in
addition to the current staging system and response
to chemotherapy (Figure 1E).
In conclusion, LVGLS showed a modest but significant correlation with amyloid load and log NTproBNP. However, despite a modest relationship,
LVGLS showed a significant additive prognostic
value. This finding suggested that impaired LV strain
could be a prognostic factor reflecting more than a
simple amyloid load, encompassing overall impact
from structural damage due to amyloid protein
deposition, cardiotoxicity, and fibrosis. With introduction of new therapeutic options for patients with
AL amyloid, LVGLS could be used as an imaging
biomarker to improve risk stratification.
Darae Kim, MD, PhD
Jin-Oh Choi, MD, PhD
Kihyun Kim, MD, PhD
Seok Jin Kim, MD, PhD
Jung-Sun Kim, MD, PhD
Eun-Seok Jeon, MD, PhD*
*Division of Cardiology
Department of Medicine
Heart Vascular Stroke Institute
Samsung Medical Center
Sungkyunkwan University School of Medicine
81 Irwon-Ro Gangnam-gu
Seoul 06351
Korea
E-mail: eunseok.jeon@samsung.com
https://doi.org/10.1016/j.jcmg.2020.12.009
! 2021 Published by Elsevier on behalf of the American College of Cardiology
Foundation
This research was supported by a fund (code: 2019ER690200) by Research of
Korea Centers for Disease Control and Prevention. The authors have reported
that they have no relationships relevant to the contents of this paper to disclose.
The authors attest they are in compliance with human studies committees and
animal welfare regulations of the authors’ institutions and Food and Drug
Administration guidelines, including patient consent where appropriate. For
more information, visit the Author Center.
REFERENCES
1. Ternacle J, Bodez D, Guellich A, et al. Causes and consequences of longitudinal LV dysfunction assessed by 2D strain echocardiography in cardiac
amyloidosis. J Am Coll Cardiol Img 2016;9:126–38.
2. Buss SJ, Emami M, Mereles D, et al. Longitudinal left ventricular function
for prediction of survival in systemic light-chain amyloidosis: incremental
value compared with clinical and biochemical markers. J Am Coll Cardiol 2012;
60:1067–76.
3. Kumar S, Dispenzieri A, Lacy MQ, et al. Revised prognostic staging system
for light chain amyloidosis incorporating cardiac biomarkers and serum free
light chain measurements. J Clin Oncol 2012;30:989–95.
4. Gertz MA, Comenzo R, Falk RH, et al. Definition of organ involvement and
treatment response in immunoglobulin light chain amyloidosis (AL): a
consensus opinion from the 10th International Symposium on Amyloid
and Amyloidosis, Tours, France, 18-22 April 2004. Am J Hematol 2005;79:
319–28.
5. Palladini G, Dispenzieri A, Gertz MA, et al. New criteria for response to
treatment in immunoglobulin light chain amyloidosis based on free light chain
measurement and cardiac biomarkers: impact on survival outcomes. J Clin
Oncol 2012;30:4541–9.
Proof-of-Principle Demonstration of Direct
Metabolic Imaging Following Myocardial
Infarction Using Hyperpolarized 13C CMR
Although ischemic heart disease is a major contributor to global disease burden, there remains scope to
improve diagnosis, risk stratification, and management of myocardial ischemia. The recent ISCHEMIA
(International Study of Comparative Health Effectiveness With Medical and Invasive Approaches) trial
showed that after an average follow-up of 3.2 years,
invasive therapy did not reduce major adverse cardiac events compared with optimal medical therapy
in patients with stable ischemic heart disease (1). The
presence of ischemia invariably leads to alterations in
the balance between aerobic and anaerobic
metabolism, and therefore, noninvasive detection of
these metabolic alterations may lead to
improvements in patient care pathways. Although
current cardiac magnetic resonance (CMR)
techniques are able to assess altered perfusion and
scar burden, they cannot directly measure metabolic
alterations. In addition, whereas positron emission
tomography with 18F-fluorodeoxyglucose allows
assessment of glucose uptake, it is unable to report
on the metabolic fate of glucose beyond its initial
phosphorylation by hexokinase, and so a new
approach is required.
The fate of glucose metabolism after glycolysis
depends on the prevailing metabolic conditions and
thus has the potential to be used diagnostically, with
the equilibrium between pyruvate dehydrogenase
(PDH) activity and lactate dehydrogenase (LDH) activity indicating the balance between aerobic and
anaerobic metabolism (2). The recently demonstrated
technique of hyperpolarized cardiac magnetic
resonance (hp-CMR) offers the ability to
noninvasively monitor PDH and LDH activity (3),
and may provide the potential for direct imaging of
metabolism in the ischemic heart (Figure 1A).
Whereas this potential has been established in
animal models (4,5), we present here the first hpCMR images of pathological human myocardial
metabolism in ischemic heart disease.
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Studies were approved by the National Research
Ethics Committee (17/WM/0200). Hyperpolarized
[1-13C]pyruvate was prepared in a GE SPINlab hyperpolarizer (GE Healthcare, Chicago, Illinois) and
administered intravenously (0.1 mmol/kg) (3).
Hyperpolarized 13C images were acquired on a
Siemens 3T Tim Trio scanner (Siemens Healthineers,
Erlangen, Germany) using a cardiac-gated sequence
consisting of interleaved spectral-spatial excitations
of pyruvate, lactate, and bicarbonate resonances
followed by a hybrid-shot spiral (HSS) readout (6).
A 2-dimensional implementation of HSS was used in
case 1 and encoded 3 short-axis slices (basal, mid,
apical) of 20-mm thickness per heartbeat with
nominal 10-mm in-plane resolution (flip angles:
pyruvate 12!; lactate/bicarbonate 60!). Imaging was
performed over an end-expiration breath-hold
started 22 s after injection; all 3 slices were encoded
each heartbeat for 1 metabolite, and the 3
metabolites were acquired over 3 subsequent
heartbeats in the order pyruvate, bicarbonate, and
lactate. Three interleaves were used to acquire the
presented data, requiring 9 heartbeats in total. For
case 2, a 3-dimensional implementation of HSS was
used and encoded a 384 " 384 " 120 mm3 volume
with nominal 6-mm in-plane resolution and 3
excitations per heartbeat (flip angles: pyruvate 6!;
lactate/bicarbonate 30!) and 12 excitations per
volume. As for the 2-dimensional case, 3 interleaves
were used to acquire the presented data, requiring
36 heartbeats in total.
Case 1: A 67-year-old man with type 2 diabetes
presented with chest pain, non–ST-segment elevation
myocardial infarction (cardiac troponin I 44 ng/l), and
electrocardiographic evidence of anterolateral territory ischemia. Coronary angiography revealed disease of the distal left main and proximal left anterior
descending coronary arteries with angiographic
appearances consistent with a chronic total occlusion
of the right coronary artery, which was dominant.
CMR and late gadolinium enhancement imaging were
undertaken to assess viability and inform revascularization. This demonstrated 2 separate areas of
infarction: subendocardial infarction (25% to 50%,
intermediate viability, presumed acute) of the midand apical anterior and anterolateral walls (4 of 17
segments), and transmural (75% to 100%, nonviable,
presumed old) infarction of the inferior septum (2
of 17 segments) (Figure 1B). Hyperpolarized [1-13C]
pyruvate imaging (Figure 1C) was undertaken 5 days
following the onset of chest pain and showed an
absence of 13C-bicarbonate and [1-13C]lactate signals
in the nonviable inferior septum, but 13Cbicarbonate and [1-13C]lactate signals were seen in
the anterior wall in the region of the subendocardial
infarction, demonstrating ongoing oxidative
metabolism in the recently infarcted anterior wall.
Case 2: A 76-year-old woman presented 24 h after a
severe episode of chest pain to a regional hospital on
the island of Jersey. By this time, anterior Q waves
were seen on the electrocardiogram; however, pain
persisted and ST-segment elevation was still apparent,
so the patient was treated with intravenous thrombolytic therapy and flown to our center with the capability for primary coronary intervention for ongoing
management. On arrival, the patient was stable
without symptoms; echocardiography revealed an
akinetic anterior wall. On day 4 following the first
onset of pain, CMR was undertaken to assess anterior
wall viability before invasive angiography. Hyperpolarized [1-13C]pyruvate imaging was also undertaken at this time. Late gadolinium enhancement
imaging revealed transmural (75% to 100%, nonviable)
infarction in the mid- and apical anterior walls,
alongside the mid-anterolateral and mid-apical lateral
walls (5 of 17 segments), with significant microvascular
obstruction typical of acute infarction (Figure 1D).
Hyperpolarized [1-13C]pyruvate imaging (Figure 1E)
showed absent 13C-bicarbonate and [1-13C]lactate
signals in the transmural infarction, but both 13Cbicarbonate and [1-13C]lactate signals were observed
in the inferior lateral walls. Management options
were discussed with the patient, and a conservative
course of action was pursued in the first instance,
with invasive angiography reserved for any
recurrence of symptoms.
This is the first report to our knowledge of in vivo
imaging of pathological metabolism in the human
heart using hp-MRI. These 2 cases show that, whereas
nonviable segments with transmural infarction show
reduced PDH-mediated aerobic conversion to 13C-bicarbonate, viable segments following subendocardial
infarction have preserved 13C-bicarbonate signal. This
shows the difference in ongoing oxidative metabolism (the hallmark of viability) that exists between
viable (hibernating) and nonviable myocardium.
Further studies are now needed to investigate
whether such a biomarker could be useful in stratifying those that would benefit from revascularization. Despite the presence of reduced PDH flux in the
diabetic heart, previous work has demonstrated the
potential for this technique to be applied in the diabetic heart (7). Because the subject in case 1 also had
type 2 diabetes, this work further emphasizes the
ability of hp-MRI to image metabolism in the
diabetic heart.
In addition, we have shown that following hyperpolarized [1-13C]pyruvate injection, [1-13C]lactate
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FIGURE 1 Proof-of-Principle Metabolic Images Acquired From the Ischemic Heart Using Hyperpolarized 13C CMR
Label Exchange of [1-13C]Pyruvate Metabolism
In!ammation
& Ischaemia
Aerobic
Respiration
Rapid
Equilibrium O
O
O
O O
O
O
O
OH O
OH
[1-13C]Lactate [1-13C]Pyruvate 13CO2
13C 13C
13C 13C
13C-Bicarbonate
LDH PDH
Subject 1: Non ST segment elevation MI (2-dimensional HSS)
Anatomical (1H) Pyruvate (13C) Bicarbonate (13C) Lactate (13C)
Anatomical (1
H) Pyruvate (13C) Bicarbonate (13 Lactate ( C) 13C)
10cm
10cm
6cm
3cm
Heart
Spleen
B
C
Pyruvate Bicarbonate Lactate
Pyruvate Lactate Bicarbonate
1.5 T Late
Gadolinium
Late Gadolinium
Color scales represent
Signal-to-Noise Ratio
Color scales represent
Signal-to-Noise Ratio
Subject 2: ST segment MI (3-dimensional HSS)
12 18 24 6 12 18 20 30 40
10 80 2 16 12
D E
A
(A) Schematic representation of metabolic pathways observable following injection of hyperpolarized [1-13C]pyruvate. (B and C) Representative late gadolinium/metabolic images acquired from Subject #1. (D and E) Representative late gadolinium/metabolic images acquired from
Subject #2. CMR ? cardiac magnetic resonance; HSS ? hybrid-shot spiral; LDH ? lactate dehydrogenase; PDH ? pyruvate dehydrogenase.
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signals were absent in the nonviable sections, but
were seen in the viable recently infarcted segment in
case 1 and in the remote myocardium in case 2.
Although this possibly represents residual ischemia
in the infarcted segment in case 1, this may also be
explained by inflammatory changes. The origin of
these [1-13C]lactate signals requires further
clarification.
These results demonstrate the emerging potential
for hyperpolarized imaging in ischemic heart disease.
The detection of downstream conversion to either
bicarbonate or lactate after hyperpolarized [1-13C]pyruvate injection has the potential to characterize
the metabolic state of viable myocardium noninvasively. Because this can be achieved in a single
90-s scan, and in the absence of ionizing radiation,
this is an exciting prospect for future cardiovascular
research.
Andrew Apps, MDy
Justin Y.C. Lau, PhDy
Jack J.J.J. Miller, DPhil
Andrew Tyler, MChem
Liam A.J. Young, MChem
Andrew J.M. Lewis, DPhil
Gareth Barnes, MD
Claire Trumper, BSc
Stefan Neubauer, MD
Oliver J. Rider, DPhilz
Damian J. Tyler, PhDz*
*Oxford Centre for Clinical Magnetic Resonance Research
Division of Cardiovascular Medicine
Radcliffe Department of Medicine
University of Oxford
Oxford OX3 9DU
United Kingdom
E-mail: damian.tyler@dpag.ox.ac.uk
https://doi.org/10.1016/j.jcmg.2020.12.023
? 2021 The Authors. Published by Elsevier on behalf of the American College of
Cardiology Foundation. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
yDrs. Apps and Lau contributed equally to this work as joint first authors. zDrs.
Rider and Tyler contributed equally to this work as senior authors. This work
was funded by the British Heart Foundation (BHF) grants RG/11/9/28921, FS/17/
18/32449 (Dr. Apps), RE/08/004/23915 (Dr. Lewis), FS/14/54/30946 (Dr. Rider),
FS/14/17/30634 (Prof. Tyler), and FS/19/18/34252), as well as the National Institute for Health Research Oxford Biomedical Research Centre (Dr. Lau), a Novo
Nordisk Postdoctoral Fellowship (Dr. Miller), the Engineering and Physical Sciences Research Council grant EP/L016052/1 (Dr. Tyler), and the Medical
Research Council (Dr. Young). All authors would also like to acknowledge the
support provided by the OXFORD-BHF Centre for Research Excellence (grant
RE/13/1/30181). All other authors have reported that they have no relationships
relevant to the contents of this paper to disclose. The authors thank Katy Briggs,
Katy Crofts, Paloma Delgado, Matt Wilkins, Claire Church, Laura Rodden, and
the Clinical Trials Aseptic Services Unit.
The authors attest they are in compliance with human studies committees and
animal welfare regulations of the authors’ institutions and Food and Drug
Administration guidelines, including patient consent where appropriate. For
more information, visit the Author Center.
REFERENCES
1. Maron DJ, Hochman JS, Reynolds HR, et al. Initial invasive or
conservative strategy for stable coronary disease. N Engl J Med 2020;382:
1395–407.
2. Apps A, Lau J, Peterzan M, Neubauer S, Tyler D, Rider O. Hyperpolarised
magnetic resonance for in vivo real-time metabolic imaging. Heart 2018;104:
1484–91.
3. Cunningham CH, Lau JYC, Chen AP, et al. Hyperpolarized 13C metabolic MRI
of the human heart: initial experience. Circ Res 2016;119:1177–82.
4. Ball DR, Cruickshank R, Carr CA, et al. Metabolic imaging of acute and
chronic infarction in the perfused rat heart using hyperpolarised [1-13C]pyruvate. NMR Biomed 2013;26:1441–50.
5. Yoshihara HAI, Bastiaansen JAM, Berthonneche C, Comment A, Schwitter J.
An intact small animal model of myocardial ischemia-reperfusion: characterization of metabolic changes by hyperpolarized13C MR spectroscopy. Am J
Physiol Heart Circ Physiol 2015;309:H2058–66.
6. Tyler A, Lau JYC, Ball V, et al. A 3D hybrid-shot spiral sequence for
hyperpolarized 13C imaging. Magn Reson Med 2021;85:790–801.
7. Rider OJ, Apps A, Miller JJJJ, et al. Non-invasive in vivo assessment of
cardiac metabolism in the healthy and diabetic human heart using hyperpolarized 13 C MRI. Circ Res 2020;126:725–36.
LETTERS TO THE EDITOR
Hemodynamic Assessment in the
Cardiac Intensive Care Unit
May Echocardiography Solve the Conundrum?
Jentzer et al. (1) performed a retrospective study to
investigate the association among bidimensional (2D)
echocardiography-derived hemodynamic
parameters, Society for Cardiovascular Angiography
and Interventions shock stages, and in-hospital
mortality in patients admitted to the cardiac
intensive care unit (CICU). Interestingly, a 2Dechocardiographic assessment of the hemodynamic
status at admission showed to be significantly
predictive of in-hospital outcomes. Specifically, after
correcting for potential confounders, a reduced
stroke volume index (<35 ml/m2
) and increased left
ventricular filling pressures (mitral E/e0 ratio >15)
were independently associated with 2-fold and 50%
increased risk of in-hospital mortality, respectively (1).
Hemodynamic data can be determinant in characterizing the type of shock and guiding patient
management in critically ill patients (2). On the
contrary, previous observational studies and
randomized controlled trials in shock patients did
not only fail to show any benefit of invasive
hemodynamic assessment, but found in most cases
an increased risk of in-hospital mortality. This
finding was likely related to the complications of
an invasive hemodynamic assessment, which
Letters to the Editor JACC: CARDIOVASCULAR IMAGING, VOL. 14, NO. 6, 2021
JUNE 2021:1271 – 9 2
1288
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神經(jīng)篇
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First Hyperpolarized [2-13C] Pyruvate MR
Studies of Human Brain Metabolism
研究背景
研究結(jié)果
研究對(duì)象
應(yīng)用方向
研究結(jié)論
?極??Hyperpolarized, HP?????? [1-13C]pyruvate ????????????????代謝? [
13C]CO2 ?] 13C]
lactate ?????????????????全?????????代謝?????研???????????
?? 13C ???????? [2-13C]pyruvate ?代謝??? 13C ?????????????代謝???????glutamate???????citrate????????????? [1-13C]pyruvate ?????? ?? IDH ???????
研?????????????代謝????? HP[2-13C]pyruvate ????????
?研?????? GMP ????????] 2-13C]pyruvate ?????????????????????????
??極????研????????代謝?????
?????????[2-13C]pyruvate代謝???
?A?1H T2FLAIR
?B?[2-13C]lactate代謝???
?C?[5-13C]glutamte代謝???
4??????ǖ???????代謝
??代謝????ǘ??????????????????研?
4??????????????]2-13C]pyruvate??代謝??[2-13C]lactate?]5-13C]glutamte?????????
???????-???????????代謝??[1-13C]pyruvate???????????HP[2-13C]pyruvate???
??????????
Neoplasia. 2019 Jan;21(1):1-16. doi: 10.1016/j.neo.2018.09.006.
A B C
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Journal Pre-proofs
First Hyperpolarized [2-13C]Pyruvate MR Studies of Human Brain Metabolism
Brian T Chung, Hsin-Yu Chen, Jeremy Gordon, Daniele Mammoli, Renuka
Sriram, Adam W Autry, Lydia M Le Page, Myriam Chaumeil, Peter Shin,
James Slater, Chou T Tan, Chris Suszczynski, Susan Chang, Yan Li, Robert
A Bok, Sabrina M Ronen, Peder EZ Larson, John Kurhanewicz, Daniel B
Vigneron
PII: S1090-7807(19)30256-3
DOI: https://doi.org/10.1016/j.jmr.2019.106617
Reference: YJMRE 106617
To appear in: Journal of Magnetic Resonance
Received Date: 16 July 2019
Revised Date: 4 October 2019
Accepted Date: 6 October 2019
Please cite this article as: B.T. Chung, H-Y. Chen, J. Gordon, D. Mammoli, R. Sriram, A.W. Autry, L.M. Le
Page, M. Chaumeil, P. Shin, J. Slater, C.T. Tan, C. Suszczynski, S. Chang, Y. Li, R.A. Bok, S.M. Ronen, P. EZ
Larson, J. Kurhanewicz, D.B. Vigneron, First Hyperpolarized [2-13C]Pyruvate MR Studies of Human Brain
Metabolism, Journal of Magnetic Resonance (2019), doi: https://doi.org/10.1016/j.jmr.2019.106617
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
? 2019 Published by Elsevier Inc.
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First Hyperpolarized [2-13C]Pyruvate MR Studies of
Human Brain Metabolism
Revision
Brian T Chung1,2, Hsin-Yu Chen1, Jeremy Gordon1, Daniele Mammoli1, Renuka
Sriram1, Adam W Autry1, Lydia M Le Page1,3, Myriam Chaumeil1,3, Peter Shin1, James
Slater1, Chou T Tan4, Chris Suszczynski4, Susan Chang5, Yan Li1, Robert A Bok1,
Sabrina M Ronen1, Peder EZ Larson1, John Kurhanewicz1, Daniel B Vigneron1
1Department of Radiology and Biomedical Imaging, University of California, San
Francisco, CA 94158, USA
2UCSF – UC Berkeley Graduate Program in Bioengineering, University of California
3Department of Physical Therapy and Rehabilitation Science, University of
California, San Francisco, CA 94158, USA
4ISOTEC Stable Isotope Division, MilliporeSigma, Merck KGaA, Miamisburg, OH
45342, USA
5Department of Medicine, University of California, San Francisco, CA 94158, USA
Corresponding author:
Brian Thomas Chung
Department of Radiology and Biomedical Imaging
University of California, San Francisco
1700 Fourth Street
Byers Hall Suite 102
San Francisco, CA 94158
Email: Brian.Chung@ucsf.edu
Phone: 415-514-4802, Fax: 415-514-4451
Keywords: Hyperpolarized C13, Metabolic Imaging, Brain Metabolism
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Abstract
We developed methods for the preparation of hyperpolarized (HP) sterile [2-
13C]pyruvate to test its feasibility in first-ever human NMR studies following FDA-IND
& IRB approval. Spectral results using this MR stable-isotope imaging approach
demonstrated the feasibility of investigating human cerebral energy metabolism by
measuring the dynamic conversion of HP [2-13C]pyruvate to [2-13C]lactate and [5-
13C]glutamate in the brain of four healthy volunteers. Metabolite kinetics, signal-tonoise (SNR) and area-under-curve (AUC) ratios, and calculated [2-13C]pyruvate to [2-
13C]lactate conversion rates (kPL) were measured and showed similar but not
identical inter-subject values. The kPL measurements were equivalent with prior
human HP [1-13C]pyruvate measurements.
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Introduction
Dissolution Dynamic Nuclear Polarization (dDNP) provides over 10,000 fold signal
enhancement for hyperpolarized carbon-13 (HP-13C) MRI, enabling a novel stableisotope molecular imaging approach for preclinical and recently clinical research
studies demonstrating both safety and translational potential for human HP-13C
molecular imaging1-4. HP [1-13C]pyruvate MR metabolic imaging has been applied to
identify tumor metabolism5, assess aggressiveness6, evaluate treatment response7,
and probe organ function4,8.
MR detection of the conversion of HP [1-13C]pyruvate to [1-13C]lactate catalyzed by
lactate dehydrogenase (LDH) has shown research value and clinical potential in
Phase I trials of cancer patients reflecting the Warburg Effect3 with greatly
upregulated LDH activity9,10. In approaching the tricarboxylic acid (TCA) cycle, [1-
13C]pyruvate is enzymatically metabolized via pyruvate dehydrogenase (PDH) and
converted to 13CO2, thereby preventing direct detection of downstream TCA cycle
metabolites. Prior animal studies using HP pyruvate with the 13C isotope enriched in
the 2-position ([2-13C]pyruvate) have successfully shown direct detection as the HP
13C labeled atoms are carried over into acetyl-CoA, a precursor to the TCA cycle, and
on to [5-13C]glutamate, acetyl-carnitine and other metabolites as shown in Figure
111,12. Therefore, HP [2-13C]pyruvate provides novel metabolic information different
from HP [1-13C]pyruvate due to its unique positioning atop multiple anaplerotic and
cataplerotic metabolic cascades in the TCA cycle with known fast conversions13.
Prior preclinical studies have shown differences in [2-13C]pyruvate to [5-
13C]glutamate metabolism with isocitrate dehydrogenase (IDH) mutations in brain
tumor models that are not detected by HP [1-13C]pyruvate MR14.
The goal of this study was to develop methods for the hyperpolarization and
preparation of sterile [2-13C]pyruvate with FDA-IND and IRB approval for first-ever
human studies. We sought to investigate HP [2-13C]pyruvate conversion to [2-
13C]lactate and [5-13C]glutamate in the normal brain in four volunteers,
demonstrating a significant first step for HP metabolic imaging to diagnose
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neurological disorders potentially at an early stage and monitor treatment response.
Unlike animal studies, these human experiments were performed without
anesthesia that significantly reduces brain pyruvate metabolism15 and therefore are
more relevant to future patient studies.
Figure 1: Diagram showing [2-13C]pyruvate metabolism investigated in this
hyperpolarized NMR spectroscopy study of the human brain.
Methods
[2-13C]Pyruvate: FDA-IND, IRB, Human Volunteers
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[2-13C]pyruvate was produced by MilliporeSigma Isotec Stable Isotopes
(Miamisburg, OH) following Good Manufacturing Practices (GMP) for first-ever use
in human HP MR studies. All human studies followed an IRB and FDA IND-approved
protocol with informed consent. Proton T2-FLAIR anatomical reference imaging
scans showed volunteers had no acute abnormalities.
Preclinical Quality Control: T1, Polarization, Purity, Animal Studies
T1 relaxation times and liquid-state polarization levels of [2-13C]pyruvate were
measured with independent characterization experiments in solution and murine
models. [2-13C]pyruvate T1 measurements of 47 sec and polarization levels of
15.61% reaffirmed literature values16. Pyruvic acid solution NMR testing was
performed using a Varian VNMRS 500 MHz (Varian Medical Systems, Palo Alto, CA)
to confirm the absence of impurities.
In-vivo spectroscopic animal studies were performed on a 3T GE MR scanner
following IACUC approval, prior to human volunteer studies to test in vivo
performance. Non-localized dynamic 13C NMR spectra were acquired with hardpulsed excitation (TR/TE = 3 sec/35 msec) in Sprague-Dawley rats for detection of
[5-13C]glutamate, [2-13C]lactate and other metabolite resonances such as
acetylcarnitine and acetoacetate17.
Clinical Preparation: Hyperpolarization, SPINlab
A 1.46 g sample of 14 M 99% enriched [2-13C]-labeled pyruvic acid (Millipore-Sigma,
Miamisburg, OH) mixed with 15 mM trityl radical (GE Healthcare, Oslo, Norway)
was pre-filled in a single-use, pharma-kit polymer fluid pathway and polarized for
over 2 hours in a SPINlab polarizer (General Electric, Niskayuna, NY) operating at 5
Tesla and 0.77 Kelvin, with microwave irradiation frequency in the 94.0 - 94.1 GHz
band. Following the protocol approved by the University of California San Francisco
IRB and the FDA IND, and after dissolution and meeting all quality control
specifications and pharmacist approval, 0.43 mL/kg of the hyperpolarized pyruvate
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solution (250 mM) was injected intravenously at a rate of 5 mL/s using a power
injector (Medrad Inc., Warrendale, PA) followed by 20 mL of sterile saline.
MR Protocol
Volunteers were measured using a 3T MR scanner (MR750, 50 mT/m gradient
amplitude, 200 T/m/s slew rate; GE Healthcare, Waukesha, WI) and scanned with a
volume excitation and 32-channel receive 13C array coil for brain studies18. A 400
sec hard pulse excitation provided an approximately 2.5 kHz excitation bandwidth,
with a nominal flip angle of 40° at the center frequency of 141 ppm calibrated using
a built-in urea phantom. The [2-13C]pyruvate, [5-13C]glutamate, and [2-13C]lactate
doublet resonances were excited with 7°, 30°, 5° and 2.1° flip angles respectively.
The acquisition used temporal and spectral resolutions of 2 sec and 2.4 Hz across 30
timepoints for a total scan time of 2 minutes.
Data Analysis
Dynamic spectroscopic data yielding kinetic rates and curves was reconstructed
after zero-filling free induction decays. The 32-channel data was combined with a
phase-sensitive summation followed by line broadening of 5 Hz19.
For the pyruvate-to-lactate conversion (kPL) kinetic model, the measured pyruvate
magnetization functioned as the input for fitting the lactate magnetization. The
MATLAB model was solved based on minimization of a constrained least-squares
error computed across measured and estimated lactate using a trust-regionreflective algorithm. The input-less fitting was chosen over integral ratios due to
improved accuracy by accounting for variability in delivery times20. The analytical
tools used are available from the Hyperpolarized MRI Toolbox via the
Hyperpolarized Technology Resource Center:
https://doi.org/10.5281/zenodo.1198915.
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Quantitative data processing and display were performed using MATLAB (The
MathWorks Inc., Natick, MA) and MestReNova (Mestrelab, Santiago de Compostela,
Spain). Zero- and first-order phase corrections were performed, and baseline was
subtracted by fitting a spline to signal-free regions of the smoothed spectrum.
Metabolites of interest were quantified following prior assignments by selecting and
integrating across peak boundaries17. Single timepoint data 16 seconds following
injection was further analyzed and interpreted following singular value
decomposition techniques20-22.
Results
Volunteer Spectra
HP [2-13C]pyruvate, [2-13C]lactate, [5-13C]glutamate and other metabolites were
successfully observed and quantitatively measured for the first time in four
volunteers. Figure 2 shows a representative summed spectra over the total 2 min
scan time for a healthy volunteer using a pulse and acquire scheme with the RF
profile shown in Figure 3. Figures 4 and 5 depict spectra and kinetics of measured
metabolite resonances for each of the four volunteers.
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Figure 2: Representative Carbon-13 NMR summed spectrum from the brain of a
healthy volunteer acquired with a 32-channel head coil following an injection of 1.43
mL/kg of 250mM [2-13C]pyruvate. Peak identification was assigned following those
by Park et al. from studies of HP [2-13C]pyruvate in the murine brain: A) [2-
13C]pyruvate, B) [5-13C]glutamate, C) [1-13C]citrate and/or [5-13C]glutamine, D) [1-
13C]pyruvate (natural abundance doublet), E) [2-13C]pyruvate-hydrate, F) [2-
13C]lactate doublet17.
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Figure 3: Flip angle plot of the RF excitation pulse sequence with the parameters used
for this study. Note the decreased excitation of the upfield [2-13C]lactate resonance
versus the downfield by approximately one half.
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Figure 4: Spectra for all four volunteers at a single timepoint 16 seconds postinjection. Similar levels of [5-13C]glutamate and [2-13C]lactate reflect the underlying
biochemistry of the healthy human brain of similar rates of conversion of [2-
13C]pyruvate to [2-13C]lactate catalyzed by LDH as [2-13C]pyruvate to [5-
13C]glutamate catalyzed by PDH.
Figure 5: Dynamic plots of metabolite kinetics for each of the four volunteers. Results
were consistent noting minor differences in intensity scale. As shown in the
corresponding Figure 4, the rates of conversion of [2-13C]pyruvate to [2-13C]lactate
and [5-13C]glutamate are similar in the normal human brain.
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Volunteer [2-13C]Pyruvate [5-13C]Glutamate [2-13C]PyruvateHydrate[2-13C]Lactate
(Left Peak)
[2-13C]Lactate
(Right Peak)
1 885.93 91.18 169.68 24.63 9.55
2 1278.34 68.36 265.33 42.58 18.55
3 2114.06 83.03 428.07 72.67 32.95
4 964.09 57.43 219.63 50.93 22.05
Mean 1310.61 486.57 75.00 13.03 270.68 96.96 47.70 17.27 20.77 8.38
Table 1: SNR for each volunteer from a single timepoint 16 seconds post-injection
with calculated mean and standard error.
Volunteer Lac / Pyr Glu / Pyr Pyr-Hyd / Pyr Glu / Lac
1 0.024 0.027 0.163 1.125
2 0.030 0.045 0.178 1.500
3 0.028 0.030 0.180 1.071
4 0.038 0.037 0.191 0.974
Mean 0.030 0.005 0.035 0.007 0.178 0.010 1.168 0.199
Table 2: AUC metabolite ratios for each volunteer summed across all timepoints
with calculated mean and standard error.
SNR & Metabolite Ratios
Tables 1 and 2 summarize measured SNR from the single timepoint data and AUC
metabolite ratios summed across all timepoints for 4 volunteers. Measured values
and calculated mean and standard error across volunteers were consistent within
expected ranges16. The observed variations in SNR can be attributed to multiple
factors including brain volumes, polarization values, and delivery times from the
polarizer to the subject. These demonstrated however minimal effects on the ratios
and the kinetic values that showed tight agreement between volunteers. The third
volunteer dataset showed the highest SNR with AUC ratios near median and was
hence selected as the representative spectrum for peak identification in Figure 2.
The [2-13C]lactate (left and right peaks) correspond to the left and right resonances
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of the [2-13C]lactate doublet in the 13C MRS spectra. The left (downfield) resonance
is about two-fold higher due to the excitation profile shown in Figure 3.
Figure 6: Plots showing kPL analysis demonstrated similar results between a
previously acquired [1-13C]pyruvate dataset from a volunteer (left) and [2-
13C]pyruvate volunteer dataset acquired in this study with the mean + standard error
for all 4 volunteers (right).
[2-13C]Pyruvate kPL Model:
Figure 6 shows a MATLAB plot of a measured [2-13C]pyruvate kPL value from the
volunteer studies with calculated mean and standard error of 0.011 ± 0.002 sec-1.
The values were consistent with prior [1-13C]pyruvate kPL values of 0.012 sec-1
acquired using a similar setup and non-selective pulse-acquire strategy17,20.
Identical results of pyruvate to lactate kinetics across a previously processed [1-
13C]pyruvate dataset and newly acquired [2-13C]pyruvate datasets from volunteers
lends verification to the robustness and consistency of approach.
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Figure 7: Images acquired using a metabolite-specific flip angle schedule and echo
planar imaging (EPI) readout. From left to right 1H proton reference image, overlaid
[2-13C]pyruvate, and [5-13C]glutamate images of a volunteer’s brain are shown. The
single-shot HP 13C EPI images were acquired with: resolution = 2.5 x 2.5 cm2, slice
thickness = 5 cm, bandwidth = 6 kHz, TR = 3s, TE = 2.8s, and flip angles θPyr = 10°, θGlu
= 60°. Average SNR of the pyruvate signal = 682 and glutamate signal = 31.1.
Initial Volunteer EPI Studies
Figure 7 shows initial data and feasibility of HP 13C imaging of the [2-13C]pyrvuate
conversion to [5-13C]glutamate using a specialized 13C 32-channel head coil. As
shown in Figure 2 not only was the uptake of HP [2-13C]pyruvate in the human brain
observed, but also its metabolic conversion to [2-13C]lactate, [5-13C]glutamate, and
other metabolites, similar to prior animal study results17.
Discussion and Conclusion
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In this study we worked with the ISOTEC Stable Isotope Division of MilliporeSigma,
Merck KGaA to develop GMP grade 99% enriched [2-13C]pyruvate meeting the
purity specifications established for [1-13C]pyruvate used in numerous human
studies following FDA-IND and IRB approved protocols. Prior to human studies with
HP [2-13C]pyruvate, we first tested the purity and polarization through in vitro NMR
analysis and performed a process qualification for testing and demonstrating the
sterility of the polarized solution. The NMR spectra in Figure 4 and quantitative
values in Tables 1 and 2 demonstrated excellent data repeatability affirming the
consistency of the preparation and processing methods. Metabolite ratios and
dynamic plots in these initial studies directly reflected the excitation profile of the
RF pulse that was optimized to capture the bandwidth encompassing metabolic
byproducts and provided normative values for future human brain HP [2-
13C]pyruvate NMR studies. Lastly pyruvate to lactate kinetic modeling from these [2-
13C]pyruvate studies yielded kPL values that were consistent with results from a
prior HP [1-13C]pyruvate dataset in healthy human brain.
Future Directions
This study demonstrated feasibility and initial normative values for HP [2-
13C]pyruvate NMR and thus serves as the groundwork for designing new studies of
neurological disorders. These future studies would clearly benefit from an imaging
approach to investigate HP [2-13C]pyruvate MRI variations associated with anatomy
and pathology and examine differences using centrality metrics and connectomic
analytical methods with HP [1-13C]pyruvate MRI22. HP metabolic information can
also be linked with modalities such as functional and diffusion MRI to build
increasingly comprehensive representations of neural function, structure and
metabolism23. Centrality metrics processing higher-order descriptors of multivalued metabolite kinetics with advances in machine learning may further elucidate
new methods for detecting early stages of neurological disorders.
Acknowledgements
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This work was supported by NIH grants P41EB0135898, U01EB026412,
R01CA197254, R01CA172845, R01NS102156 and the UCSF NICO project. The
authors would additionally like to thank Romelyn Delos Santos, Kimberly Okamoto,
Mary McPolin, and Hope Williams for their help with volunteer studies.
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Highlights
Human brain TCA cycle metabolism was investigated using HP [2-13C]
pyruvate.
A rapid metabolic MR study with a RF bandwidth of 2.5 kHz for 2
minutes was performed.
Similar but not identical glutamate and lactate levels were recorded
across volunteers.
kPL values were calculated & found to agree with prior [1-13C]pyruvate
data.
Future studies may investigate early predictors for cancer metabolic
reprogramming and neurological disorders.
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Graphical abstract
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Declaration of interests
? The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
?The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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Kinetic Modeling of Hyperpolarized Carbon-13 Pyruvate Metabolism in the Human
Brain
研究背景
研究結(jié)果
研究對(duì)象
研究結(jié)論
?極??Hyperpolarized, HP?[
13C] ????????????????????集??????????????
???????????????????集????????代謝??????代謝????????????
???????
?????????????? [1-13C]pyruvate ????代謝?? ?kPL ???????代謝?? ?kPB?kPL ? kPB
???代謝?????????????????????????????????????????研???
??????????????????????
?研??????????????????????????? kPL ? kPB 代謝????????????????
??????????????????代謝?????
????????????????????2 ??????????
????代謝?????
?A????代謝?? ?kPL?FLAIR ?????????????ǘ
??代謝???B?????
10?????????ǖ??代謝???????????????
?研??????極?]1-13C]pyruvate?????研?????????????代謝?kPL?kPB?????????
?代謝???????????????????????????????????????????????
????
?????kPL????????????????????研?????????????
A
B
IEEE Trans Med Imaging. 2020 Feb;39(2):320-327.
doi: 10.1109/TMI.2019.2926437.
