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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, Texas7 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.

2018137: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. 2007104:19773-19777.

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. 2020117:2092-2098.

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. 2020126:725-736.

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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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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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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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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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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.