DISS. ETH NO. 20945

Quantitative, SNR-enhanced and Localized 13C

Magnetic Resonance Spectroscopy A dissertation submitted to the ETH ZURICH for the degree of Doctor of Sciences

presented by Xing Chen Master Biomed. Eng., Zhejiang University born June 13th, 1981 citizen of China

accepted on the recommendation of Prof. Dr. Peter Boesiger, examiner Dr. Anke Henning, co-examiner Prof. Dr. Markus Rudin, co-examiner 2012

献给我的父母 —— 陈宝岩，李丹红 Dedicated to my parents – Baoyan Chen, Danhong Li

Contents ZUSAMMENFASSUNG ........................................................................................... 7 SUMMARY ................................................................................................................. 11 CHAPTER 1: INTRODUCTION .......................................................................... 15 CHAPTER 2: OPTICALLY TRANSMITTED AND INDUCTIVELY COUPLED ERETIC FOR QUANTITATIVE PROTON-DECOUPLED

13

C

MRS .............................................................................................................................. 25 CHAPTER 3: J-REFOCUSED 1H PRESS DEPT FOR LOCALIZED

13

C

MR SPECTROSCOPY ........................................................................................... 43 CHAPTER 4: QUANTIFICATION OF FATTY ACIDS IN HUMAN CALF ADIPOSE TISSUE AND

MUSCLE BY 13C MRS USING

J-

REFOCUSED PRESS DEPT AND ERETIC ................................................... 73 CHAPTER 5: CHOLESTEROL DETECTION IN ADIPOSE TISSUE BY NATURAL ABUNDANCE IN VIVO 13C MRS AT 7T .................................... 93 CHAPTER 6: CONCLUSION AND OUTLOOK ............................................. 99 APPENDIX ............................................................................................................... 103 REFERENCES........................................................................................................ 109 ACKNOWLEDGEMENTS ................................................................................... 123 CURRICULUM VITAE .......................................................................................... 127 LIST OF PUBLICATIONS ................................................................................... 129

Zusammenfassung

7

Zusammenfassung Die Magnetresonanzspektroskopie (MRS) hat sich zu einem standardmäßig in den biologischen Wissenschaften

verwendeten analytischen Werkzeug

entwickelt. Eine große Vielfalt von verschiedenen Kernen und Isotopen wird verwendet, da jeder Kern seine eigenen Vor- und Nachteile hat. Kohlenstoff-13 (13C)-MRS macht

einzigartige Informationen über komplexe biologische

Systeme, vom Molekül bis zu ganzen Organismen, zugänglich. Ein sehr allgemeines Merkmal der

13

C-Spektren ist die hohe spektrale Dispersion,

welche die Auflösung der Resonanzlinien einer großen Anzahl von Substanzen ermöglicht und damit neue Perspektiven zur Untersuchung von Metaboliten eröffnet, die mittels Protonen (1H)-MRS schwer zugänglich sind. Außerdem ist Kohlenstoff außer Protonen der einzige Kern, der praktisch ubiquitär in organischen Verbindungen vorkommt und damit mit 1H-MRS vergleichbare Möglichkeiten bezüglich Metabolismusforschung bietet. Darüber hinaus sind die T2 Relaxationszeiten in der 13C-MRS in der Regel länger als die in der 1HMRS; damit ermöglicht nur 13C-MRS die in vivo Quantifizierung von großen Molekülen wie Glykogen. Obwohl

13

Kombination mit der Infusion von

C-MRS eine grosse Bedeutung in 13

C-angereicherten Substraten und

13

hyperpolarisierten C-basierten Kontrastmitteln erlangt hat, enthalten auch 13CMRS-Spektren, die auf dem natürlichen Vorkommen des 13C-Isotops beruhen, wertvolle Informationen.

Zusammenfassung

8

Einer der größten Nachteile von 13C-MRS für die Anwendung am Menschen ist die geringe Empfindlichkeit, die eine Folge der geringen natürlichen Häufigkeit des 13C-Isotops und der niedrigen gyromagnetischen Konstante des C-Kerns ist. Außerdem sind an die meisten Kohlenstoffe Protonen gebunden, wodurch Multiplettstrukturen auftreten, welche für eine weitere Abnahme der Empfindlichkeit verantwortlich sind. In Bezug auf eine wesentliche Voraussetzung der in vivo MRS, die räumliche Lokalisation, ist die Anwendbarkeit von Lokalisationsverfahren, die in der 1H oder Phosphor (31P) MRS gebräuchlich sind, in der

13

C-MRS beschränkt. Das ist aufgrund des

großen Frequenzbereiches, in dem

13

C Resonanzlinien vorkommen, und der

geringen natürlichen Häufigkeit der

13

C-Kerne. Darüber hinaus haben

verschiedene Stoffwechselprodukte unterschiedliche Eigenschaften bezüglich ihres T1-und T2-Relaxationsverhalten, die unterschiedliche Methoden zur Lokalisierung und Quantifizierung erfordern oder ermöglichen. Schließlich stellt die Bestimmung von molaren Konzentrationen der Stoffwechselprodukte im Gewebe ein zentrales Thema in der klinischen und physiologischen Forschung dar; dies erfordert einen zuverlässigen und stabilen ReferenzStandard mit hohem Signal-Rausch-Verhältnis (SNR), der generell in der 13CMRS fehlt. Das Ziel dieser Arbeit war es, Methoden zu entwickeln, welche die zuverlässige Anwendung von quantitativer, SNR-verbesserter und lokalisierter in vivo

13

C-MRS welche auf natürlich vorkommenden

13

C basiert, in

physiologischen Studien ermöglicht. Drei methodische Aspekte wurden dabei und im Hinblick auf spezifische Metaboliten untersucht: (1) stabiler Referenzstandard für die quantitative Analyse, (2) reproduzierbare SNRVerbesserung und (3) die räumliche Lokalisation. Mit Bezug auf den Referenzstandard für die quantitative Analyse wurde ein optisch übertragenes

Zusammenfassung

9

und induktiv gekoppeltes elektronisches Referenzsignal für die Bestimmung von In-vivo-Konzentrationen (ERETIC)-benutzt. Die Implementation hat die gleichzeitige

Protonenentkopplung

und

ERETIC-basierte

Metabolitenquantifizierung ermöglicht, und somit die Anwendbarkeit der ERETIC Methode auf NOE-verbesserte und Protonen-entkoppelte in vivo 13CMRS erweitert. Die ERETIC Signalstabilität unter dem Einfluß der gleichzeitigen Protonenentkopplung wurde untersucht. Die vorgeschlagene Quantifizierungsmethode wurde gegen konventionelle interne und externe Referenzstandards für die Metabolitenquantifizierung in der menschliche Skelettmuskulatur

cross-validiert.

Bezüglich

SNR-Verbesserung

und

räumlicher Lokalisierung wurde eine J-refokussierte Protonen PRESS lokalisierte DEPT Sequenz präsentiert, die gleichzeitig eine vier-fache SNRVerbesserung und konsistente räumliche Lokalisierung von Signalen einer Vielzahl von Metaboliten mittels in vivo

13

C-MRS ermöglicht. Die

Unterdrückung der J-Modulation während PRESS und die dadurch hervorgerufene

Wiederherstellung

der

theoretisch

zu

erwartenden

Signalverstärkung durch PRESS lokalisiertes DEPT wurden mittels ProduktOperator-Formalismus, numerisch durch Spin-Dichtematrix-Simulationen für unterschiedliche skalare Kopplungsbedingungen und experimentell mit einem Glutamat Phantom für verschiedenen Echozeiten demonstriert. Die Anwendung der Sequenz für den Nachweis von gesättigten und ungesättigten Fettsäuren im adipösen Gewebe des Knochenmarkes und im Skelettmuskel in der Wade von gesunden Probanden ergab hohe Signalverbesserungen, wodurch gleichzeitig alle Fettkomponenten messbar wurden. Schließlich wurde die J-refokusierte PRESS-lokalisierte DEPT Sequenz mit der ERETIC-Referenzmethode kombiniert, um die Fettsäurezusammensetzung in

menschlichem adipösen

Gewebe im Knochenmark und im Skelettmuskel in drei verschiedenen Gruppen

Zusammenfassung

10

mit unterschiedlichem Ernährungsverhalten (Allesesser, Vegetarier und Veganer) zu quantifizieren. Es wurden relative und absolute molare Fettsäurekonzentrationen ermittelt. Die Analyse ergab, dass das Fettsäure-Profil im Knochenmark stabil ist und die langfristige Diät wiederspiegelt, während die Fettsäuren im Muskel eher mit kurzfristigen Stoffwechselaktivitäten in Zusammenhang stehen. Die Ergebnisse zeigen die Anwendbarkeit der quantitativen, SNR-verbesserten und lokalisierten

13

C-MRS für umfangreiche

nicht-invasive Untersuchungen der Auswirkungen von Nahrungsaufnahme, körperlicher Bewegung oder Pathologien des Fettsäurestoffwechsels und deren potentielle Anwendbarkeit für klinische Diagnostik.

Zusammenfassung

11

Summary Nuclear magnetic resonance spectroscopy has become a standard analytical tool in biological sciences. A large variety of different nuclei and isotopes have been used with each nucleus having its own advantages and disadvantages. Carbon-13 (13C) MRS provides unique information concerning complex biological systems, from molecules to whole organisms. A very general feature of 13C spectra is their high spectral dispersion, which allows the resolution of resonances of a large number of substances, and thereby the study of metabolites which are difficult to resolve in proton (1H) MRS. Furthermore carbon is the only nucleus besides protons that is practically ubiquitous in organic compounds, which makes it comparable to 1H with respect to metabolism research. In addition, T2 relaxation times in 13C MRS are generally longer than those in 1H MRS and thus only 13C MRS enables in vivo detection of large molecules such as glycogen. Although developed in combination with infusion of hyperpolarized

13

C agents, natural abundance

13

C MRS has been greatly

13

C-enriched substrates and

13

C MRS spectra still hold

valuable information. One of the major drawbacks of

13

C MRS applied to humans is its low

sensitivity, which is a consequence of the low natural abundance of only 1% and the low gyromagnetic constant of the carbon nucleus. Furthermore, most carbons have attached protons, resulting in multiplet structures which further

12

Summary

decrease sensitivity. With respect to the essential prerequisite of in vivo MRS, the spatial localization, the large chemical-shift range and low natural abundance of the 13C nuclei make most commonly used localization schemes in 1

H or phosphorus (31P) MRS not easily transferrable to 13C MRS. In addition,

different target metabolites have different properties regarding their relaxation behavior, such as T2 relaxation times, which lead to differences in the required methodology. Finally as the final aim and key issue in clinical and physiological studies, knowing the metabolite molar concentrations in tissues requires a reliable and stable reference standard with high signal-to-noise ratio (SNR), which is missing in 13C MRS. The objective of this thesis was the methodological development for quantitative, SNR enhanced and localized natural abundance in vivo 13C MRS, which allows for reliable applications in physiological studies. Three methodological aspects including a stable reference standard for quantification, reproducible SNR enhancement and proper volume localization were targeted and investigated with regard to specific metabolites. With respect to the reference standard, we introduced an optically transmitted and inductively coupled ERETIC (Electric Reference To access In vivo Concentrations) implementation, which enables simultaneous proton decoupling and ERETIC based metabolite quantification, and hence extends the applicability of the ERETIC method to NOE enhanced and proton decoupled in vivo

13

C MRS.

ERETIC signal stability under the influence of simultaneous proton decoupling was investigated. The proposed quantification method was cross-validated against internal and external reference standards on human skeletal muscle. With respect to SNR enhancement and localization, a J-refocused proton PRESS localized DEPT sequence was presented and implemented for obtaining simultaneously SNR enhanced and localized signals from a large number of

Zusammenfassung

13

metabolites by in vivo 13C MR spectroscopy. The suppression of J-modulation during PRESS and the substantial recovery of signal enhancement by Jrefocused PRESS localized DEPT were demonstrated theoretically by product operator formalism, numerically by the spin density matrix simulations for different scalar coupling conditions, and experimentally with a glutamate phantom at various echo times. Applying the sequence for localized detection of saturated and unsaturated fatty acids in the calf bone marrow and skeletal muscle of healthy subjects yielded high signal enhancements simultaneously obtained for all components. Finally the J-refocused PRESS localized DEPT sequence was combined with the ERETIC reference standard and used to assess fatty acid concentrations from human calf tibial bone marrow and skeletal muscle among omnivores, vegetarians and vegans. Both relative and absolute molar metabolite concentrations are evaluated and analyzed. Seen from the concentration values and statistic analysis, the fatty acid profile in the bone marrow is stable and reflects the long-term diet, while the fatty acids in the muscle are related to short-term metabolic activity. The results demonstrate the applicability of quantitative, SNR enhanced and localized

13

C MRS for large

scale noninvasive investigation of the impact of dietary intake, physical exercise or pathology on fatty acid metabolism and related diagnosis.

14

Summary

Chapter 1: Introduction

Chapter 1:

Introduction

15

Chapter 1: Introduction

16

1.1 Introduction Carbon-13 (13C) magnetic resonance spectroscopy (MRS) in vivo accesses metabolic information of humans not easily obtained by other non-invasive methods. Since almost all metabolically relevant compounds contain carbon, 13

C MRS is in principle able to detect many metabolites and can thus offer

complementary information to that obtained with proton (1H) and/or phosphorus-31 (31P) MRS. The large chemical shift range of

13

C MR spectra

allows the resolution of resonances of a large number of substances, and thereby the study of some metabolites which are difficult to resolve in 1H MRS. In addition, T2 relaxation times in 13C MRS are in generally longer than those in 1H MRS and thus only

13

C MRS enables the in vivo detection of large

molecules such as glycogen. Last but not least infusion studies based on specifically

13

C labeled metabolites such as glucose enable insight into

metabolic turnover rates such as TCA cycling and glutamate and glutamine cycling. One of the major drawbacks of

13

C MRS applied to humans is its low

sensitivity, which is a consequence of the low natural abundance (only 1%) and the low gyromagnetic constant of the carbon nucleus (only 1/4 of the proton nucleus). Furthermore, most carbons have attached protons, resulting in multiplet structures which further decrease sensitivity. An essential prerequisite of in vivo MRS is the spatial localization of the spectra. Although many localization techniques are successful in 1H and 31P MRS, the large chemicalshift range and low natural abundance of the 13C nuclei make their direct application in 13C MRS difficult. In addition, different target metabolites have different properties regarding their relaxation behavior, such as short T2 relaxation times, which lead to differences in the required methodology. Finally

Chapter 1: Introduction

17

knowing the metabolite molar concentrations in tissues is the key issue in clinical and physiological studies in order to assess the diseases and physiological processes by MRS. To sum up, reproducible SNR (Signal-toNoise Ratio) enhancement, proper volume definition and a stable reference standard for metabolite quantification are necessary to achieve quantitative 13C MRS.

1.2 Objectives The aim of this dissertation is to overcome some of these technical challenges in

13

C MRS in order to realize the reliable application of quantitative, SNR

enhanced and localized MRS in human physiological studies. Specific aims were the development of a reliable reference standard for metabolite quantification, robust SNR enhancement and an accurate localization scheme. With the proposed solutions to the above aspects, a noninvasive study of fatty acids among different dietary groups was performed and analyzed.

1.3 Theoretical Background Quantification Absolute concentrations of metabolites expressed in mmol / L can be in principle calculated from the peak areas since the measurable signal is directly proportional to the number of spins, which is proportional to the molar concentration. However, many physical parameters as well as tissue specific

Chapter 1: Introduction

18

properties are not known exactly. Different quantification strategies and corresponding calibrations have been developed that allow for the quantification of in vivo metabolite concentrations by comparing metabolite peak areas to the area of a reference standard with known concentration. The internal water reference has been commonly used in 1H spectra due to its high SNR and relatively stable content for a specific tissue type. Similarly the concentration of other stable metabolites in the tissue, such as creatine, can be assigned an average literature value and used as internal reference standard for 13

C spectra. But the concentrations of internal references might change and be

unreliable especially in disorders. Furthermore the signal intensity of creatine is very low in

13

C MRS and if using water as reference, different transmit and

receive B1 field patterns between 1H and 13C should be considered, all of which reduce the quantification precision. In addition to the above internal references, external reference standards i.e. a phantom solution with known metabolite concentration can be placed next to the subject and measured in a separate scan each time. But strictly identical experimental conditions are required or influence factors such as receiver gain, coil loading and processing procedures should be corrected for. Reciprocity principle has also been widely applied by using the voltage that is needed in order to obtain the maximal signal response for calibration and reference. Recently, the ERETIC (Electric REference To assess In vivo Concentrations) method based on the injection of an artificial synthetic reference signal has been developed, which allows for the acquisition of reference signal and metabolite signals in a single scan. The ERETIC method can be seen as a further development of the phantom based quantification strategy which allows simultaneous acquisition of both metabolite and reference signals after a single calibration against a high precision phantom. It has been further improved by

Chapter 1: Introduction

19

exploring in optical transmission and inductive coupling concerning the signal stability and reproducibility for in vivo 1H MRS. While differences in coil load and receiver gain are directly considered by the ERETIC method, only volume definition, B1+, T1, T2 as well as temperature have to be taken into account as additional parameters to correct for. In our work, the ERETIC method will be implemented on a 3T human MR scanner and combined with a dual-tuned 1H/13C volume calf coil for 13C MRS. The artificial peak as the crucial reference standard will be investigated under the specific application for multinuclear MRS namely simultaneous proton decoupling during acquisition and ERETIC injection and cross-validated regarding to other reference methods in order to ensure the accuracy and reliability for different target metabolites.

SNR enhancement 13

C spectra can be improved and simplified by proton decoupling due to the

collapse of C-H couplings, but SNR enhancement is only effective for in-phase multiplets. Based on dipolar interaction, nuclear Overhauser enhancement (NOE) enhances sensitivity of when

1

13

C through a rearrangement of populations

H is saturated. Nonetheless only a small fraction of theoretical

enhancement factor can be obtained in vivo, indicating the presence of other competitive relaxation mechanisms. Based on the J-coupling effect, polarization transfer techniques are used in multinuclear MRS by transferring polarization from highly polarized nuclei to less sensitive spin systems. A theoretically higher enhancement factor can be achieved by polarization transfer than by NOE. In our work two commonly used polarization transfer methods DEPT (Distortionless Enhancement by Polarization Transfer) and INEPT (Insensitive

Chapter 1: Introduction

20

Nuclei Enhanced by Polarization Transfer) will be implemented on the 3T scanner. The extremely short echo time needed for polarization transfer, due to the large J coupling constants between carbon and attached protons, puts constrained requirements on the radiofrequency pulse and sequence design. Most pulses are too long to realize effective enhancement, while block pulse might result in not fully enhanced signals if inhomogeneous transmit fields (B1) exists. The DEPT and INEPT sequences require simultaneous excitation of proton and carbon nuclei and need to be adapted to clinical MRI systems which exhibit a significant switching time for the start of radiofrequency transmission on two transmit channels. . For the metabolites with different MR properties, different combination of the techniques will be necessary. Therefore according to our target metabolites in the calf tissue including glycogen and fatty acids, different SNR enhancement techniques will be discussed, compared and combined.

Localization Restricting signal detection to a well-defined region of interest is crucial for meaningful in vivo MRS. Resulting from the intrinsic low sensitivity of

13

C

MRS, the use of surface coils or half-volume coils without localization scheme is most commonly used in order to achieve a high SNR and to circumvent the chemical shift displacement artifact resulting from the large frequency dispersion if conventional localization schemes are applied to

13

C MRS.

However, only a non-sharply defined sensitivity area is produced by the inhomogeneous B1 field obtained by such coils, which is inaccurate and insufficient for subsequent Millimolar metabolite quantification. Based on eight scans to achieve full 3D localization, ISIS (Image Selected In Vivo

Chapter 1: Introduction

21

Spectroscopy) is the most popular localization technique in multinuclear MRS especially for the observation of short T2 species. Even though ISIS is typically executed with adiabatic pulses for inversion and excitation, it still suffers from signal loss and contamination caused by so-called ‘T1 smearing’ as well as transverse T2 relaxation during inversion pulses. STEAM (Stimulated Echo Acquisition Mode) is a localization technique capable of 3D localization in a single acquisition, but only half of the magnetization is obtained while the other half is completely dephased, which makes it improper for the low sensitive

13

C MRS. The PRESS (Point RESolved Spectroscopy)

localization method is a so-called double spin-echo method, which is the most commonly used localization scheme in 1H MRS. Compared to 1H spectra, however, the full chemical shift range of most biologically interesting 13C resonances is approximately 160 - 200 ppm wide. Therefore chemical shift displacement error problems are important factors to consider when localized

13

C MRS is required. Hence localization can be

conducted using the proton channel and combined with polarization transfer enhancements, which avoids the large chemical shift displacement if localization is conducted directly using the carbon channel. However direct carbon localization is also applicable in some cases. The extremely short T2 of some metabolites like glycogen makes echo-base localization methods and the use of polarization transfer unfeasible, and thus only ISIS could be possibly employed. Outer Volume Suppression (OVS) will be combined in 13C MRS in order to reduce signal contamination from outside. Targeting at different metabolites, a proper volume should be defined through different localization schemes for the successive quantification.

Chapter 1: Introduction

22 Applications

The combination of the above described quantification reference standard, SNR enhancement methods and localization schemes are going to improve the applicability of 13C MR spectroscopy for clinical use and physiological studies. Metabolites of specific interest include glycogen and fatty acids, which are important sources of energy for muscular contraction and general metabolism. The degree of exercise and nutrition induced changes of glycogen and fatty acid has been of intense interest in the exercise and nutrition science community as well as among clinicians, and skeletal muscle glycogen and fatty acid metabolism has been found in association with insulin resistance I diabetes patients. Metabolically distinct tissue types such as adipose tissue and skeletal muscle should be separately investigated to get a meaningful physiological interpretation due to the different metabolic activities and roles.

1.4 Outline The main focus of this dissertation is natural abundance 13C MRS methodology and corresponding applications. A reliable reference standard with respect to absolute quantification, reproducible SNR-enhancement methods and accurate localization schemes will be introduced, investigated and demonstrated. Specifically, the second chapter of this thesis describes the optically transmitted and inductively coupled ERETIC reference standard being implemented, which enables simultaneous proton decoupling and ERETIC based metabolite quantification by in vivo 13C MRS. Glycogen as an important compound in human metabolism can only be detected noninvasively by in vivo

Chapter 1: Introduction

23

13

C MRS. Since it separates itself from other metabolites regarding to the

extremely short T1 and T2 relaxation times, a SNR-enhanced, quantitative and localized glycogen detection protocoll is also investigated in this chapter. In the third chapter a J-refocused proton PRESS localized DEPT sequence is introduced and presented for obtaining simultaneously enhanced and localized signals from a large number of metabolites. The suppression of homonuclear Jmodulation during PRESS and the substantial recovery of signal enhancement by J-refocused PRESS localized DEPT are demonstrated theoretically by product operator formalism, numerically by the spin density matrix simulations for different scalar coupling conditions, and experimentally with a glutamate phantom at various echo times. The applicability of the proposed sequence for localized detection of fatty acids is described in the third chapter. In the forth chapter assessment of fatty acids from human calf tibial bone marrow and skeletal muscle among omnivores, vegetarians and vegans by the J-refocused PRESS localized DEPT sequence with optically transmitted and inductively coupled ERETIC as the quantification reference is presented. Both relative and absolute molar metabolite concentrations are evaluated and analyzed. The fifth chapter describes the application of the J-refocused PRESS localized DEPT sequence at 7T on adipose tissue.

24

Chapter 1: Introduction

Chapter 2: ERETIC

25

Chapter 2:

Optically transmitted and inductively coupled ERETIC for quantitative protondecoupled 13C MRS

Published in: Magnetic Resonance in Medicine 2012; 67:1-7.

Chapter 2: ERETIC

26

2.1 Introduction Millimolar (mM) concentrations of metabolites in specific tissues are of fundamental interest to the understanding of metabolism in clinical diagnostics and physiological studies. Magnetic Resonance Spectroscopy (MRS) is a powerful technique for this purpose because of the direct proportionality of signal intensities in spectra and the amount of resonating nuclei (1,2). While the internal water reference method has become standard for the quantification of metabolite concentrations in mM (absolute quantification) by 1H MRS (3), absolute quantification by heteronuclear MRS is a particularly difficult but important task. The goal of this study was to establish a reliable and stable method that enables absolute quantification of metabolite concentrations by in vivo 13C MRS in physiological and pathological conditions. Unlike in 1H MRS, internal reference standards are not easily available for in vivo

13

C MRS. The internal water reference method is unreliable, since the

transmit and receive B1 field patterns in the related proton measurement differs from the B1 fields in the heteronuclear measurement. This problem is augmented at high field strengths, and a theoretical or experimental B1 correction is not straightforward (3). Alternatively, creatine is frequently used as internal reference for quantification since it can be detected by

13

C MRS

directly (4-7). However the signal intensity of the respective resonance line is very low and the quantification precision is further reduced by the baseline distortion stemming from lipid resonances. With external reference methods, strictly identical experimental conditions are required or correction factors need always to be considered in each paired measurement (3). Some conditions to take into account in this context such as receiver gain stability, temperature of

Chapter 2: ERETIC

27

the phantom, coil loading conditions or receive B1 need considerable effort to be well controlled. The total measurement time is also usually prolonged due to the need of an extra spectral measurement of the external reference phantom solution each time. As an alternative, the Electric Reference To access In vivo Concentrations (ERETIC) method can be used for quantification. An additional synthetic NMR-like reference signal is injected during the acquisition of the spectrum independent of the real metabolite signal (2,8-12). The ERETIC signal has previously been implemented via radiation through a broad band antenna in vitro for solution state NMR (2), ex vivo using 13C magic angle spinning NMR spectroscopy (8), in vivo

31

P (9) and 1H MRS (13). In order to make the

quantification more accurate, an inductively coupled reference signal injection was introduced to make the calibration factor immune to changes in coil loading conditions (10). This modification has been investigated by in vitro 1H and 31P MRS (10) and 19F imaging quantification (11), and recently by in vivo 31

P MRS on skeletal muscle(14) using animal MR systems. However, the

reference signal in these setups is sent through the second RF channel, which makes the simultaneous use of proton decoupling and ERETIC quantification for multinuclear spectroscopy (31P, 13C) impossible. Although in heteronuclear MRS proton decoupling is commonly used to achieve sufficient signal intensities and reduction of spectral overlap, neither an implementation that allows for simultaneous injection of ERETIC and proton decoupling has been shown so far, nor has the compatibility of these two methods ever been investigated. In 1H MRS Heinzer-Schweizer et al have furthermore proposed optic fiber transmission instead of electrical transmission of the calibration signal to eliminate parasitic coupling between transmit and ERETIC cables. This substantially improved quantification accuracy and reproducibility for in

Chapter 2: ERETIC

28

vivo studies on a clinical MR platform(12), where repeated repositioning of RF coils and the regarding transmit and ERETIC cables is required.

Here, we propose the ERETIC method implemented via an optical transmission line, inductive coupling and compatible with additional proton decoupling as a reliable reference standard for absolute quantification of multinuclear MRS in vivo. In addition, we demonstrate the compatibility of the ERETIC method and simultaneous proton decoupling. The synthetic reference signal is injected into a

13

C/1H dual-tuned calf volume coil on a clinical 3T scanner. Quantification

results are cross-validated with internal and external reference standards in phantoms and for glycogen and unsaturated fatty acids (UFA) detection in human skeletal muscle on healthy subjects with inter- and intra-individual experiments.

2.2 Methods MRI Instruments All experiments were performed on a Philips Achieva 3T human MRI scanner (Philips Healthcare, Best, Netherlands). A shielded dual-tuned

13

C/1H

transmit/receive calf volume coil (RAPID Biomedical GmbH) was used for 13C excitation and detection as well as for 1H based imaging, preparation phases, nuclear Overhauser enhancement (NOE) and proton decoupling. ERETIC Layout and Setting The adapted ERETIC technique using inductive coupling and optical transmission (an equivalent in vivo 1H MRS setup is described in detail and

Chapter 2: ERETIC

29

validated in ref. (15)) was implemented by modifying the above described commercial dual-tune calf volume coil. The ERETIC loop was placed as close as possible (~1.0 cm) to the

13

C coil in order to maximize the inductive

coupling between the ERETIC loop and the receive coil during acquisition. The loop size was minimized (~1 cm) to avoid direct interaction with the patient load. The ERETIC signal was generated by a low power auxiliary broadband channel of the 3T spectrometer, which in conventional MRI operation delivers a control signal for tuning and detuning of receive-only coils. This implementation, which required extensive software modifications, left the proton channel free to be used for simultaneous proton decoupling. It also enabled precise synchronization of ERETIC transmission and signal acquisition and hence a very high phase stability of the artificial reference peak. The signal generated by the auxiliary channel was tuned to the same frequency as the resonance frequency of

13

C and it was used to modulate the bias current of a

high-frequency compatible light emitting diode (LED); in this way it is possible to convert an electrical signal into an optical signal. The light generated from the LED travelled into the scanner room through an optic fiber (10 meters in length) (Fig.1 top left) and the electric reference signal was recovered by a photodiode which is directly connected to the ERETIC loop via a matching network inside the case of the dual-tune calf volume coil, as shown in Fig.1 (bottom left). The ERETIC pulse shape was set to Lorentzian; the amplitude, frequency, phase and decay time constant were also adjusted with respect to the signals in the 13C NMR skeletal muscle spectra from the scanner console.

30

Chapter 2: ERETIC

Figure 1 The optical fiber (as circled) transmitting the ERETIC signal (top left); The ERETIC setup attached to the 13C coil (bottom left): (a) photodiode with matching network, (b) T-bias network, (c) ERETIC loop; Acquisition sequence with ISIS localization, NOE and proton decoupling for SNR enhancement and ERETIC reference (top right); Volume of interest chosen in calf muscle and the position of the external reference phantom (bottom right).

Spectroscopy Sequence Volume selection was achieved by using a three-dimensional ISIS localization scheme, involving three hyperbolic secant pulses (duration 4.5 ms, bandwidth 2788 Hz) and one block excitation pulse (duration 1ms, bandwidth 5040 Hz), as shown in Fig.1 (top right). The carrier frequencies of all pulses were positioned at the frequency of the respective target metabolites (glycogen, UFA, creatine or dimethyl sulfoxide (DMSO)) in order to reduce the effect of imperfect slice profiles and chemical shift displacement. Localization also provides exact volume sizes and hence regarding correction factors, which is a

Chapter 2: ERETIC

31

prerequisite for ERETIC based quantification of tissue concentrations in the unit of mmol / liter and was also used in the frame of the presented crossvalidation with internal and external standards in this study. A block pulse (duration 1.1 ms, bandwidth 903 Hz) NOE (proton channel) was applied 1 ms before the first selective inversion pulse of the ISIS localization scheme (carbon channel), saturating the 1H channel at 5.4 ppm. Continuous wave proton decoupling was used during acquisition with a bandwidth of 33 Hz covering the scalar coupled 1H range of glycogen and UFA. The decoupling power delivered to the coil was 64 W ensuring fully decoupled peaks. To keep the repetition time as short as possible, the spectra for glycogen quantification were acquired with 512 sample points and 6000 Hz bandwidth while all the other spectra were acquired with 1024 sample points and 8000 Hz bandwidth. ERETIC Signal Stability during Proton Decoupling The stability of the ERETIC signal during SNR enhancement (NOE enhanced and proton decoupling) and especially proton decoupling was assessed so that interference between these simultaneously transmitted signals could be excluded. Inter-individual experiments were performed on six subjects (two males and four females, age = 27 ± 4 years old, weight = 75 ± 15 kg). Eleven intra-individual experiments were carried out on one male subject (30 years old, 90 kg). All the 17 experiments together have been performed in a time period of 3 months. In each experiment, two protocols were applied, one with and the other one without proton decoupling and NOE enhancement. The receiver gain was always set to the same value in each experiment. 15 of the 17 experiments were set to the same ERETIC signal scaling, while half and one-eighth of the above ERETIC amplitude settings were performed in the other 2 measurements.

32

Chapter 2: ERETIC

In vivo Cross-validation The cross-validation measurements were performed in five experiments. For all subjects a voxel was chosen as large as possible that included calf muscle tissue and excluded subcutaneous lipids and bones (Fig.1 bottom right). The glycogen and UFA concentrations were calculated based on the ERETIC reference signal intensity, which had been calibrated against a DMSO phantom with known concentrations before. The repetition times of the two related scans were 550 ms with 4096 averages for glycogen and 3000 ms with 296 averages for UFA, leading to measurement time of 40 min and 15 min respectively. For crossvalidation against an internal reference-standard method, additional spectra centered on the creatine resonance frequency were acquired from the same volume as glycogen and UFA in each volunteer; creatine was assigned a concentration of 30 mmol / kg wet weight as indicated in literature (16). The repetition time for the internal creatine reference measurements was 8000 ms with 496 averages, resulting in a total measurement time of 60 min. For crossvalidation against an external reference standard a 50 ml phantom tube containing 105.6 mM DMSO was placed above the lower part of the calf for each volunteer, as shown in Fig.1 (bottom right). The measurement time for the external DMSO reference measurement was 17 min from a 15000 ms repetition time and 64 averages. Spectral Processing and Fitting All post-processing and visualization of spectra and fitting results were performed using the IDL based custom-built software package “Spectator”. Obtained 13C NMR spectra were zero filled to 1024 sample points. Zero order manual phase adjustment was applied to each spectrum and no filtering or baseline correction was used. All spectra were fitted in TDFDfit (17) using

Chapter 2: ERETIC

33

Voigt shapes for all peaks. The fitting stability regarding to the SNR level with and without NOE enhancement and proton decoupling was compared. Absolute Quantification The following equation [2.1] was used to calculate metabolite concentrations using the ERETIC reference standard after the one-time in vitro calibration Cm = C phan ,cal *

AERETIC ,cal Aphan ,cal * f s ,cal * f c ,cal * f o ,cal

*

Am * f s * f c * f o , AERETIC

(2.1)

and equation [2.2] was used for respective metabolite quantifications based on internal creatine and external DMSO reference standards, Cm = Cref *

Am * f s * f c * f o , Aref

(2.2)

where C stands for concentration, A for measured peak area and f for correction factor. The subscript cal indicates the in vitro calibration session, and s, c, o are the corrections with respect to sequence, coil and other parameters. With regard to sequence correction factors, average NOE enhancement factors of the target metabolites were experimentally determined to be 1.42 ± 0.32 for glycogen and 1.11 ± 0.35 for UFA. Volume sizes were considered in the calculation of tissue absolute concentrations in the case of ERETIC and external references. T1 relaxation times of glycogen, UFA, creatine and DMSO were all measured with the inversion recovery series method. The obtained values which were 73 ms for glycogen (corresponding to literature value (18,19)), 360.9 ms for UFA peak at 131.26 ppm, 535.5 ms for UFA peak at 129.61 ppm, 2820 ms for creatine and 5457 ms for DMSO were used for the T1 corrections that are needed to correct for repetition time induced saturation effects. The ISIS localization loss for glycogen (20) resulting from short T1 and T2 (8 ms (19,21)) relaxation times was assessed experimentally (~66.6 %) in vivo. The coil

Chapter 2: ERETIC

34

correction factor can be considered as 1 since coil loading is accounted for in the ERETIC method as well as for the internal and external reference standards (simultaneous phantom acquisition); B1 is rather homogenous for

13

C MRS

using the calf volume coil and receiver gains were fixed to zero attenuation. The other correction parameter consists of a temperature correction factor 0.9613 (22) for ERETIC and external reference method and the number of contributing spins per molecule. The UFA peak at 129.61 ppm corresponds to the polyunsaturated fatty acids (PUFA), while the other one at 131.26 ppm corresponds to the sum of the polyand monounsaturated fatty acid (MUFA). The pure MUFA concentration is calculated through the subtraction of the two peaks.

2.3 Results Fitting stability The fitting results of spectra with versus without SNR enhancement are plotted in Fig.2 (a) and (b). A zoomed view of the spectral range from 125 ppm to 135 ppm that covers UFA peaks is shown in Fig.2 (c) and (d), and from 90 ppm to 120 ppm that covers glycogen and ERETIC peaks in Fig.2 (e) and (f). The excellent fit quality is reflected by flat residues. The fitting errors, expressed as CRLB (Cramer-Rao Lower Bound) (23) were analyzed and used as acceptance criteria. A CRLB < 20 % is normally taken as an acceptance criterion in recent

Chapter 2: ERETIC

35

Figure 2 Calf muscle 13C spectra (blue), fitting (red) and residue (green): (a) ISIS localization; (b) ISIS localization with NOE and proton decoupling; (c) (d) zoomed to 125-135 ppm: unsaturated fatty acids; (e) (f) zoomed to 90-120 ppm: glycogen and ERETIC. (a) and (b), (c) and (d) as well as (e) and (f) are scaled equally, respectively.

literature (24) and was also used here for DMSO, UFA and the ERETIC signals. No cutoff CRLB was set for creatine and a CRLB limit < 30 % was used for glycogen in order to include a sufficient amount of data. The average CRLBs were 4.16 ± 0.15 % and 4.69 ± 1.58 % for the ERETIC signal intensities with

36

Chapter 2: ERETIC

and without SNR enhancement respectively and 19.11 ± 4.70 % and 10.18 ± 5.48 % for the glycogen signal intensities before and after NOE enhancement and proton decoupling for the in vivo measurements. For the two UFA peaks, CRLBs of 6.22 ± 2.11 % (131.26 ppm) and 18.43 ± 6.61 % (129.61 ppm) respectively were achieved with proton decoupling and NOE enhancement. The average fitting error for creatine was 42.54 ± 8.02 % due to the very low signal intensities, even though, the residuals were still good and the values were usable for quantification. Interference of ERETIC signal injection and proton decoupling Effect of the ERETIC signal on proton decoupling Neither significant increase in the spectral noise nor pre-amplifier spiking was observed after the installation of the ERETIC loop even during simultaneous injection of ERETIC signal and proton decoupling in any of the measurements conducted in this study and the decoupling performance remained unchanged. Stability of the ERETIC signal during proton decoupling Fig. 3 displays the results of stability tests with regard to ERETIC signal intensities with and without proton decoupling from the 17 reproducibility tests among 7 healthy subjects performed over 3 months. Peak areas were determined by TDFDfit and assigned to an arbitrary institutional unit, as plotted along the y axis in Fig.3 (left). In the 15 experiments with the same ERETIC signal scaling, the resulting ERETIC peak areas were 5.94 ± 0.26 with and 6.01 ± 0.31 without NOE and proton decoupling enhancement, respectively (n = 15, p = 0.32). The slopes of lines connecting the two areas in the original and enhanced spectra indicate the change of ERETIC intensities between the two measurements on the same subject (same loading). The mean ratio of ERETIC intensities between the two sets of experiments is 1.01 ± 0.04 (100.65 ±

Chapter 2: ERETIC

37

4.18 %). The inter-individual ratio was 1.00 ± 0.04 (99.7 ± 3.88 %) (n = 6), and the intra-individual ratio was 1.02 ± 0.05 (101.64 ± 4.56%) (n = 11), as plotted in Fig.3 (right). Thus ERETIC signal intensities can be considered to be equal among different subjects, at different times or days over a period of 3 months, including experiments with and without SNR enhancement.

Figure 3 Stability of ERETIC signal. The left plot shows the ERETIC signal intensities in an institutional unit for spectra without (left) and with (right) SNR enhancement (proton decoupling and NOE) from 17 in vivo experiments. Fitting errors (CRLBs) are indicated as error bars. The right plot shows the ratios of the ERETIC signal intensities between the two spectra acquired with and without SNR enhancement, while the solid line indicates the average and the dashed lines the 95 % confidence interval. Closed circles stand for intra-individual experiments and open circles for inter-individual experiments.

Absolute concentrations and cross-validation The cross-validation results are plotted in Fig. 4. The average glycogen concentration was 85.41 ± 13.14 mmol / liter, and the average MUFA and PUFA concentrations were 138.73 ± 54.55 mmol / liter and 108.48 ± 32.79 mmol / liter according to the ERETIC method. The values obtained from the

38

Chapter 2: ERETIC

Figure 4 Glycogen (A), MUFA (B) and PUFA (C) concentrations as estimated from three types of experiments throughout 5 in vivo scan sessions: ERETIC-based, internal creatine reference based and external DMSO reference based quantification.

Chapter 2: ERETIC

39

ERETIC-based and the creatine-based methods correspond well with each other, while the external reference shows larger deviations from the results of the other two methods in two experiments. Taking the creatine-based method as the most established standard, the difference between ERETIC-based and creatinebased quantification was 1.85 ± 1.21 % for glycogen and 1.84 ± 1.00 % for UFA while the deviation between external reference standard and creatinebased quantification was 6.95 ± 9.52 % and 3.19 ± 2.60 % respectively.

Table 1 Comparison of glycogen and UFA quantification deviation by using ERETIC and external reference methods with internal creatine-based method as standard. ER, Cr and Ex stand for ERETIC, Creatine-base and external reference methods.

Glycogen UFA

¦ER-Cr¦/Cr

¦Ex-Cr¦/Cr

1.85 ± 1.21 % 1.84 ± 1.00 %

6.95 ± 9.52 % 3.19 ± 2.60 %

2.4 Discussion An implementation of the ERETIC reference standard is presented that allows for simultaneous proton decoupling for quantitative, SNR enhanced

13

C MRS

on a clinical platform. The ERETIC signal is stable and reliable in the presence of SNR enhancement and the additional ERETIC loop does neither hamper the efficiency of proton decoupling or NOE enhancement nor introduce additional noise or artifacts by mitigation of coupling between carbon and proton channel. The reproducibility measurements spread over 3 months and were performed at different times during the day, and all ratios between ERETIC signals acquired with and without SNR enhancement fall into the 95 % confidence interval centered at 1.01 including inter- and intra-individual values. The ERETIC

Chapter 2: ERETIC

40

signal stability is also independent of amplitude settings, which allows for proper adjustment for different metabolites. These results demonstrate that the ERETIC signal can be used as a reference standard for quantification of metabolite tissue concentrations in SNR enhanced, multi-nuclear MRS with no SNR or performance penalty. Additional validation measurements with regard to short-term stability, interscan variability, scaling with changes in coil load and receive gain of the proposed ERETIC implementation based on optical signal transmission and inductive coupling of the ERETIC signal into a volume coil were performed in an earlier 1H MRS study (15). The related results apply for the present implementation equally. The influence of different leg sizes on the impedance inside the receive coil, which is commonly referred to as coil loading, depends on the resonance frequency, and is lower in

13

C MRS compared to 1H MRS.

This is reflected by the low standard deviation of the ERETIC signal intensities in our reproducibility tests, which also implies a high stability of the ERETIC signal over 3 months without the need of recalibration.

From the cross-validation study, it is clear that the results from the ERETIC method are comparable to those gained from the internal creatine reference method, so far the most established standard for in vivo metabolite quantification by

13

C MRS, with respect to accuracy and reproducibility.

Moreover, after a one-time calibration, the ERETIC quantification method can be used without additional measurements or scan time prolongation as is true for the standard non-localized internal creatine reference method as reported for glycogen quantification. However, the latter approach is not suitable for quantification of fatty acids due to the enormous difference in signal amplitudes between them and creatine. A significant scan time prolongation would be

Chapter 2: ERETIC

41

required to reach sufficient signal-to-noise ratios for creatine, which is avoided by using the ERETIC reference. The still very low signal intensities of creatine might lead to further fitting errors and therefore affect the final quantification precision. Signal amplitudes of the ERETIC reference can be freely chosen, and additionally, the ERETIC reference can be applied to all conditions including pathological cases, while the concentration of creatine may change in muscle disorders and literature values for muscular creatine content in healthy subjects also vary between 27.2 and 36.2 mmol / kgww (25). In comparison to the external reference standard, the ERETIC method has proven to be more reliable (Table 1). The glycogen concentration value determined with the external reference method deviated in two cases significantly from those results stemming from the other two reference methods. This could result from positioning of the phantom close to coil elements, a region with inhomogeneous effective magnetic field (B1), prone to flip angle miscalibration due to the lack of iterative power optimization methods suitable to low signal amplitudes present in 13C MRS and where warming up of the external reference phantom during proton decoupling might occur. The cross-validation experiments finally also prove that the optically transmitted and inductively coupled ERETIC method can be easily applied to the quantification of

13

C

MRS visible metabolites with very different properties with regard to signal intensity, relaxation behavior and resonance frequency. Localization is preferred for ERETIC-based quantification of tissue metabolite levels since it enables the application of accurate volume correction factors. This has the positive side-effect of spatially specific metabolic information (for instance fatty acids in muscle versus subcutaneous tissue and bone marrow) and an improvement of the fitting quality by minimizing baseline distortions stemming from subcutaneous and bone marrow lipids which exhibit very high

Chapter 2: ERETIC

42

concentrations. However, it might also be combined with an image segmentation based volume determination which makes it more applicable to glycogen quantification without localization induced signal loss.

2.5 Conclusion An implementation of the optically transmitted and inductively coupled ERETIC method that is compatible with simultaneous proton decoupling is described and validated as a reliable reference standard for quantification of NOE enhanced and proton decoupled multinuclear MRS in vivo. The ERETIC reference signal exhibits long-term phase and amplitude stability in presence and absence of proton decoupling, while frequency, amplitude, phase and line width can be freely adjusted; the method is independent of metabolic changes induced by disorders and requires no additional measurement time except a one-time calibration. Hence it is easy to use for reliable quantification of glycogen, UFA and potentially any other desired

13

C or

31

P detectable

metabolites.

2.6 Acknowledgment S. Heinzer-Schweizer and M. Pavan contributed equally to this work. We thank Johannes Slotboom and Roland Kreis for providing TDFDfit; Zurich Center for Integrative Human Physiology (ZIHP) for funding and RAPID Biomedical as well as Philips Healthcare for technical support.

Chapter 3: J-refocused PRESS DEPT

43

Chapter 3:

J-refocused 1H PRESS DEPT for Localized 13C

MR Spectroscopy

Published in: NMR in Biomed 2013, Feb 25. [Epub ahead of print]

Chapter 3: J-refocused PRESS DEPT

44

3.1 Introduction Localized

13

C magnetic resonance spectroscopy (MRS) provides a wealth of

highly specific information for the study of metabolism in vivo, such as determining metabolic turnover rates in the brain by assessment of incorporation during infusion of

13

C label

13

C labeled substrates (1,2), and lipid

composition in adipose tissue (3,4) where fatty acids are analyzed and quantified. While 13C MRS has its own unique insight and advantages over 1H MRS, it is, however, methodologically more challenging than 1H or 31P MRS. Two major issues that must be overcome to achieve acceptable spectral quality are low sensitivity, and localization in presence of a large chemical shift dispersion. Polarization transfer methods such as Distortionless Enhanced Polarization Transfer (DEPT) (5) can be used to enhance sensitivity in multinuclear MRS by transferring polarization from highly polarized nuclei to less sensitive spins, namely from 1H to

13

C when applied to

13

C MRS. The main advantage of

polarization transfer over direct excitation methods combined with nuclear Overhauser enhancement (NOE) is the consistent signal-to-noise ratio (SNR) increase. Compared to NOE, whose enhancement factors are sensitive to pathological conditions since it is based on dipolar relaxation (2,6), the signal enhancement by polarization transfer depends only on the echo time relative to the scalar coupling constants (7-9), and can thus be quantitatively calculated or empirically determined. Together with relaxation effects, the SNR can be enhanced by up to 3-4 fold using polarization transfer where the theoretical value is 4 (=γH/γC, the ratio of proton and carbon gyromagnetic ratios).

Chapter 3: J-refocused PRESS DEPT

45

The combination of DEPT with proton localization methods (1) avoids the large chemical shift displacement that occurs for direct

13

C localization and

enables tissue specific investigation of target metabolites. ISIS (Image Selected in Vivo Spectroscopy (10)) has been implemented on the proton resonance before polarization is transferred from proton to carbon (1) and the resulting 3D ISIS DEPT sequence has been used to eliminate the scalp lipid and detect metabolites from rat and human brain (1,6) for tricarboxylic acid cycling (TCA) and neurotransmitter cycling rate measurements. However, ISIS suffers from significant signal contamination from regions outside the volume of interest (VOI) for metabolites with long T1 relaxation times (11-13). This effect is referred to as T1 smearing and depends on the volume size (11), on the order of the eight encoding steps (12,13), and on the ratio of TR / T1 (13). Furthermore, signal loss from the VOI and signal contributions from outside can be caused by slice profile degradation for metabolites with very short T2 relaxation time (14). In addition, ISIS as a method that requires eight encoding steps for 3D localization is very sensitive to physiological and patient motion, which obscures the subtraction scheme. Finally, strong lipid contamination artifacts due to magnetization transfer (MT) and NOE have been recently reported as consequence of a single ISIS inversion pulse used in the SPECIAL (SPin-ECho full Intensity Acquired Localized) sequence for 1H MRS localization at high field strength (15), querying the applicability of ISIS DEPT to fatty acid quantification by

13

C MRS. Unlike spin-echo based localization schemes, the

above described signal losses and/or contamination of uncertain origin from outside the VOI occurring for ISIS due to T1 and T2 relaxation effects, motion, magnetization transfer and NOE cannot be straightforwardly corrected, which makes ISIS DEPT unreliable for metabolite quantification.

Chapter 3: J-refocused PRESS DEPT

46

By comparison, PRESS (Point RESolved Spectroscopy (16)), as the most widely used method for localized in vivo 1H spectroscopy, offers several advantages. As a single shot 3D localization method it shows a low sensitivity to physiological or patient motion, allows for high temporal resolution in functional spectroscopy and shot-by-shot frequency and phase correction (17). In contrast to ISIS, PRESS also facilitates high localization efficiency with a low amount of signal contribution from outside the VOI for a large range of T1 and T2 relaxation times. PRESS is thus widely applicable, robust and except for metabolites with extremely short T2 relaxation time like glycogen, SNR penalties due to T2 relaxation can be minimized by the use of short echo times (TE). In addition, the signal loss due to T1 and T2 relaxation effects can be easily corrected for making PRESS a reliable method for metabolites quantification. Hence the combination of PRESS and DEPT (18) for robust in vivo 13C MRS localization is highly promising. However, the homonuclear proton scalar couplings ubiquitous in most metabolites modify the proton spin coherence distribution during the protonPRESS localization as well as the subsequent DEPT enhancement. It has been observed that the signal amplitudes of 1H multiplet resonances vary with the pulse sequence timing when echo-driven volume selective methods such as PRESS are applied (19-21). Besides the spatial variation in the evolution due to the chemical shift displacement effect (19), which could be overcome or reduced by Outer Volume Saturation (OVS) (22,23) or Inner Volume Saturation (24,25), another crucial source of signal loss and deleterious coherence distribution are resulting from J-coupling modulation (20,21,26). The latter effect occurs during PRESS localization and ultimately leads to a substantial reduction in the

13

C signal enhancement during the subsequent

DEPT period. The resulting DEPT enhancement has been described as a

Chapter 3: J-refocused PRESS DEPT

47

metabolite specific nonmonotonic function of the PRESS echo times (TE) (18). The related study represents the only report of PRESS localized DEPT, and does only show

13

C intensity simulation results for hard and soft pulses and

experimental data acquired by hard pulse PRESS localized DEPT from a spherical (d = 2 cm) glutamate phantom to support this finding (18). A homonuclear J-refocused spin-echo sequence has been presented theoretically and experimentally to completely remove the weak scalar modulation (26) and partially suppress the strong

scalar modulation (21)

during double echo pulse schemes. It has been applied on citrate (21), glutamate and glutamine in phantoms (20) in 1H MRS and to minimize Jmodulation of amino acid neurotransmitters in spectroscopic imaging (27) in 1

H MRSI. Complete recovery of the signal from the weakly J-coupled nuclei

for two spin systems could be achieved (26) and a substantial suppression for systems with multiple coupled spins was predicted and verified in vitro (20,21,26). In the present study, J-refocused soft pulse 1H PRESS localized DEPT with robust coherence transfer pathway selection by a 32-step phase cycle, optimized gradient spoiling and OVS (Outer Volume Suppression) is introduced for robust localization and simultaneous signal enhancement of a large range of spin systems in 13C MR spectroscopy. The suppression of homonuclear proton J-modulation as well as an improvement in SNR enhancement by J-refocused PRESS-DEPT was investigated theoretically by product operator formalism for weakly coupled spin systems and numerically by spin density matrix simulations for strongly coupled spin systems as well as experimentally in a glutamate phantom. The stability of SNR enhancement, and thus the feasibility of reliable metabolite quantification, with the proposed method were demonstrated by adopting glutamate phantoms with different

Chapter 3: J-refocused PRESS DEPT

48

known concentrations. Finally in vivo application of the PRESS localized DEPT sequence is demonstrated for the first time. More specifically the in vivo performance of J-refocused 1H PRESS localized DEPT with regard to SNR enhancement and localization efficiency is investigated by detecting fatty acids in the calf bone marrow and skeletal muscle of five healthy subjects.

3.2 Methods J-refocused 1H PRESS DEPT sequence The entire sequence is shown in Fig. 1. A 90o RF pulse orthogonal to the first excitation pulse is inserted in the middle of the double echo PRESS localization part, which reverses the direction of homo-nuclear proton J-coupling evolution at this point. Complete or partial refocusing of scalar coupling terms will be established after the second half of the double echo sequence, while the chemical shifts are refocused by each echo pulse. While the J-refocused 1H PRESS substitutes the first 90o pulse of a conventional DEPT experiment, the residual part of the DEPT sequence remains unchanged and is implemented subsequently. The pulses from proton and carbon channels are applied simultaneously regarding the timing of the reference point (in this case the middle) of the pulse. To verify the refocusing effect during PRESS localization and the signal recovery during DEPT enhancement, theoretical evaluation by product operator formalism, numerical spin density matrix simulation and experimental measurements were performed.

Chapter 3: J-refocused PRESS DEPT

49

Figure 1 J-refocused PRESS DEPT 13C-[1H] sequence.

Theoretical and Numerical verification A detailed description of the J-refocusing theory on the example of weakly coupled homo-nuclear spin system I1I2 by vector formalism (19) and I1I2,n (n = 1, 2, 3; number of magnetically equivalent spins) by product operators (20,26), as well as on the example of a strongly coupled homo-nuclear AB spin system by density matrix calculation (21) can be found in previous reports. However, the actual scalar coupling conditions in most metabolites are more complex. First, I1I2S (weak homo-nuclear and hetero-nuclear J-coupling) or ABX (strong homo-nuclear and weak hetero-nuclear J-coupling) three-spin systems (namely CH2) often constitute a basic unit in bio-molecules investigated by heteronuclear experiments like 1H localized DEPT enhanced 13C MRS. Hence the weak hetero-nuclear J-coupling should also be taken into account together with the homo-nuclear J-couplings for a related analysis of the entire PRESS

50

Chapter 3: J-refocused PRESS DEPT

DEPT sequence. Second, hetero-nuclear J-coupling counts as weak coupling (the chemical shifts difference ν is 10 times larger than the J-coupling constant), while homo-nuclear J-coupling is often considered as strong coupling. But the actual homo-nuclear J-coupling constants cover a large range in comparison with the chemical shift difference, and the homo-nuclear J-couplings also exist among protons attached to different i.e. adjacent carbons which may build up additional weak homo-nuclear J-couplings. The resulting complex spin systems include weak hetero-nuclear J-couplings, strong homo-nuclear J-couplings and weak homo-nuclear J-couplings with a range of different coupling constants. Therefore a theoretical analysis that takes all the J-coupling conditions into account is necessary. Product operator analysis for I1I2S spin system In contrast to previous reports, all scalar couplings involved, including heteronuclear JIS couplings between I1 and S, between I2 and S, as well as homonuclear JII coupling between I1 and I2, are taken into account to analyze the entire J-refocused PRESS DEPT sequence for a weakly coupled I1I2S spin system by the product operator formalism. Spin density matrix simulations for ABX spin system and glutamate Computer simulations were performed by using the GAMMA C++ library (28). A three-spin system is assumed where A and B spins have similar chemical shift offsets with a different frequency ν of 10Hz, separated by 96 MHz from another spin X (simulation of proton and carbon at 3T). The weak heteronuclear coupling JCH between AX and BX has a fixed constant of 135Hz, and the homo-nuclear coupling constant J between A and B varies from 0.1ν (weakly coupled) to 1.5ν (strongly coupled). Different PRESS echo times were investigated corresponding to the J constants while the DEPT echo time was set to 3.7 ms according to 1/2JCH. The spectra of the A and B spins after PRESS

Chapter 3: J-refocused PRESS DEPT

51

and the spectra of the X spin after PRESS DEPT were compared with and without the J-refocusing pulse. Spectra of glutamate as an example of a metabolite of interest with real Jcoupling behavior were also simulated with and without J-refocusing pulse; PRESS TE was set to 32 ms (within 1/2JHH) and DEPT TE was set to 3.7 ms (1/2JCH). Glutamate has two CH2 (C3 and C4) and one CH (C2) spin systems adjacently with J-coupling constants in a range of 5 to 16 Hz for H-H coupling (29), and 135 and 155 Hz for C-H couplings and chemical shift offsets 2.06 ppm, 2.14 ppm, 2.36 ppm, 2.37 ppm and 3.78 ppm in proton frequency and 27.8 ppm, 34.2 ppm and 55.6 ppm in carbon frequency (18). This represents a typical coupling condition in a large variety of metabolites. Experimental verification in glutamate and oil phantoms Owing to the high solubility property of monosodium glutamate, it was used for experimental verification in 13C MRS. The effectiveness of the suppression of J-modulation and recovery of SNR enhancement by the proposed J-refocused 1

H PRESS DEPT sequence was investigated with a 250 mL phantom of 887

mM monosodium glutamate. Two groups of spectra were obtained: one using PRESS localized DEPT, and the other one using J-refocused PRESS localized DEPT. At short TEs (26 ms, 32 ms, 40 ms, 48 ms, 56 ms) no proton decoupling was applied in order to observe the phase evolution of each coupled peak. To show the signal intensity trend in a large PRESS echo time range, spectra were acquired with proton decoupling as TEs increase from 32 ms to 350 ms. Repetition time was 15000 ms with 128 averages. The VOI covered the whole phantom area. The polarization transfer echo time τ was 3.7 ms with the flip angle of the last pulse set to 45o which was optimal for the CH2 groups. The carrier frequency of 1H was centered at 2.5 ppm and the carrier frequency of

52

Chapter 3: J-refocused PRESS DEPT

13

C was centered at 30 ppm. C2 (CH group), C3 and C4 (CH2 groups) signal

intensities of glutamate were measured. To validate the applicability of the proposed sequence for quantification, the glutamate peak intensities were also measured and analyzed in phantoms with different glutamate concentrations (from 254 to 1270 mM/L) using the same sequence parameters as described above. The J-refocused 1H PRESS DEPT and direct

13

C PRESS sequences were

applied on 1L colza oil with the sequence parameters set according to Jcoupling condition and constants from general lipid (same as in vivo application). In vivo Application and SNR enhancement factors Spectra from fatty acids were acquired by the proposed J-refocused PRESS DEPT sequence from the calf bone marrow and calf skeletal muscle of five healthy subjects. Fatty acids contain long chains of adjacent CH, CH2 and CH3 spin systems with homo-nuclear J-coupling constants in a range of 6-18 Hz (30,31) and hetero-nuclear J-coupling constants in a range of 120-160 Hz (32), which is a highly similar J-coupling regime as glutamate. Written informed consent was obtained from all volunteers in accordance to local ethics guidelines. T1 weighted turbo spin echo imaging was executed before acquiring localized spectra. The volume sizes from the calf bone marrow and muscle were around 20*21*250 mm3 and 42*42*162 mm3 respectively, with minor adjustments to different subjects. The PRESS echo time was 32 ms (roughly within 1/2JHH for homo-nuclear J-coupling constants of 6-18 Hz), while the polarization transfer echo time τ was 3.7 ms for saturated fatty acids (roughly 1/2JCH for hetero-nuclear J-coupling constants of around 135 Hz) detection and 3.25 ms for unsaturated fatty acids (hetero-nuclear J-coupling constant of 154

Chapter 3: J-refocused PRESS DEPT

53

Hz) detection. The carrier frequency of 1H radiofrequency pulses, in PRESS localization, DEPT and proton decoupling, was centered at 1.65 ppm for saturated and 5.31 ppm for unsaturated fatty acids. The carrier frequency of 13C radio frequency pulses was centered at 30 ppm for saturated and 130 ppm for unsaturated fatty acids in 13C spectra. Waltz16 decoupling was used with 17 μT B1 resulting in a bandwidth of 964 Hz. Optimized selective pulses were used in J-refocused PRESS localization with 4419 Hz bandwidth for the excitation and J-refocusing pulses, and 2808 Hz bandwidth for the chemical shift refocusing pulses. Block pulses with a minimum bandwidth of 1270 Hz were adopted in DEPT enhancement in order to achieve a short polarization transfer echo time corresponding to the relatively large heteronuclear J-coupling constants. The flip angle of the last pulse β was set to 45o for the saturated fatty acids detection and 90o for the unsaturated fatty acids detection, leading to a theoretical enhancement factor of 1.0*(γH/γC) for a CH2 spin system (45o), 1.06*(γH/γC) for a CH3 spin system (35o) and 1.0*(γH/γC) for a CH spin system (90o) (33). The acquisition bandwidth was 8000 Hz and 1024 sample points were acquired. Repetition time was 6 s and 256 acquisitions were averaged. Second order FASTERMAP B0 shimming was used on the same volume of interest. In order to avoid unwanted coherence terms particularly in presence of B1 inhomogeneity, three coherence pathway selection methods were combined together: OVS (23); strong and unequal spoiling gradients; and a 32-step phase cycle. Six 90o OVS pulses with 13459 Hz bandwidth (25) were applied in all three directions on the 1H frequency as a complementary localization scheme. Unequal spoiling gradient strengths were used simultaneously in orthogonal directions (Fig. 1) to avoid refocusing of previously spoiled unwanted coherences. The spoiling gradient strengths were set empirically as 34 mT/m and 28 mT/m respectively to realize a strength ratio of around 1.2. The 16-step

Chapter 3: J-refocused PRESS DEPT

54

phase cycle of PRESS was combined with a 2-step phase cycle of DEPT on the last β pulse, which leads to a final 32-step phase cycle (listed in appendix 1). In order to analyze the SNR enhancement factors on the fatty acid components, spectra with direct 13C PRESS localization were also obtained using the same pulse shapes and timing parameters. Chemical Shift Displacement (CSD) of different fatty acid components from the direct

13

C PRESS localization was

significant and analyzed according to the following equation (34) ∆X ∆ω = VX BW

(3.1)

where ∆X is the spatial displacement of a localized volume,

VX

is a given voxel

size in the x direction, ∆ω represents the difference in Larmor frequency and BW is the spectral bandwidth of the RF pulse. Since the signals from outside

the nominal volume of interest (VOI) were saturated by broadband OVS, a CSD correction factor was defined as the ratio between the part of the resonance line specific excitation volume that overlapped with the nominal volume and the size of the nominal volume. T1 and T2 relaxation times were also taken into account by using literature values for proton resonances (T1 as 365 ms, T2 as 133 ms) (35) and measured values for carbon resonances. T1 relaxation times for carbon resonances were measured by inversion recovery sequence (34) and T2 relaxation times were measured by spin echo series (34) from the whole calf without localization volume. The final SNR enhancement factors were corrected for CSD, T1 and T2 effects. MRI Instruments All experiments were performed on a Philips Achieva 3T human MRI scanner (Philips Healthcare, Best, Netherlands). A dual-tuned

13

C/1H human calf

volume coil (RAPID Biomedical GmbH, Rimpar, Germany) was used for

Chapter 3: J-refocused PRESS DEPT

55

transmission and reception. Imaging, preparation phases such as center frequency determination, B0 shimming and proton decoupling were executed on the proton frequency. The maximum B1 of the proton channel is 30 μT and of the carbon channel is 120 μT. Post processing and Quantification All spectra were processed with Spectator, a custom written IDL (IDL Workbench Version 7.1.0, Exelis VIS, Boulder, Colorado, USA) graphical user interface. Zero order phase correction was performed manually and no Gaussian or exponential filtering was applied. The spectra were fitted by an embedded tool TDFDfit (36) using Voigt line shape models.

3.3 Results Theoretical and Numerical verification Product operator analysis for I1I2S spin system After J-refocused PRESS in the case of TE1 = TE2, all weak J-couplings including both hetero-nuclear and homo-nuclear J-couplings are refocused −( I1 y + I 2 y ) cos(π J II TE1 ) cos(π J II TE2 ) + 2( I1x I 2 z + I1z I 2 x ) cos(π J II TE1 ) sin(π J II TE2 ) −( I 2 y + I1 y ) sin(π J II TE1 ) sin(π J II TE2 ) − 2( I1z I 2 x + I1x I 2 z ) sin(π J II TE1 ) cos(π J II TE2 )

(3.2)

= −( I1 y + I 2 y )(cos(π J II TE1 ) cos(π J II TE2 ) + sin(π J II TE1 ) sin(π J II TE2 )) = −( I1 y + I 2 y )

After DEPT, the polarization is transferred to the S spin from each of the coupled I spins and detectable S spin coherences are

Chapter 3: J-refocused PRESS DEPT

56

→ −2( I1z + I 2 z ) S y sin β cos(π J II 2τ ) cos3 (π J ISτ ) sin(π J ISτ ) +8 I1z I 2 z S x sin β cos(π J II 2τ ) cos 2 (π J ISτ ) sin 2 (π J ISτ ) +2 S x sin β cos(π J II 2τ ) cos 2 (π J ISτ ) sin 2 (π J ISτ )

(3.3)

+2( I1z + I 2 z ) S y sin β cos(π J II 2τ ) cos(π J ISτ ) sin 3 (π J ISτ ) −8 I1z I 2 z S x cos β sin β cos(π J II 2τ ) cos 2 (π J ISτ ) sin 2 (π J ISτ ) −2( I1z + I 2 z ) S y cos β sin β cos(π J II 2τ ) cos(π J ISτ ) sin 3 (π J ISτ ) −2( I1z + I 2 z ) S y cos β sin β cos(π J II 2τ ) cos(π J ISτ ) sin 3 (π J ISτ ) +2 S x cos β sin β cos(π J II 2τ ) sin 4 (π J ISτ )

When τ equals to 1 / 2JIS, it leads to 1 2 J IS cos(π J ISτ ) = 0 sin(π J ISτ ) =1

τ=

 → = 2 S x cos β sin β cos(π J II / J IS )

(3.4)

To reach the highest enhancement, the optimal flip angle β is 45o for I1I2S spin systems. Similarly it can be deduced that the result for I1I2I3S spin system is 3cos2βsinβ when the optimal β is 35o, and for IS spin system is sinβ when the optimal β is 90o. The final S spin intensity also depends on the homo-nuclear and the hetero-nuclear J-coupling constants, which can usually be neglected for in vivo 13C MRS since the C-H couplings constants in most metabolites are in the range of 125-200 Hz (34) and H-H couplings constants are in the range of 0.5-18 Hz (29) resulting in cos(πJII/JIS) ->1. By alternating the phase of the last β pulse on the I spin and subtraction the resulting signal, which is termed as the DEPT phase cycle, the signal from additionally directly excited S spin will be cancelled out, and therefore these terms can be omitted. The fully detailed product operator calculation is provided as supplementary online material. Spin density matrix simulations for ABX spin system and glutamate Fig. 2 shows a series of PRESS spectra on A and B (namely H in CH2) with homo-nuclear J-coupling constants varying from weak coupling (0.1ν) to very strong coupling (1.5ν), and the corresponding spectra under J-refocused PRESS.

Chapter 3: J-refocused PRESS DEPT

57

For PRESS TE starting from 1/4JHH (not shown) up to 1/2JHH (Fig. 2a), the Jrefocusing effect is pronounced for weakly coupled spins and reduces as the Jcoupling constant gets higher. At very strong J-coupling the J-refocusing pulse has almost no effect on the appearance or SNR of the multiplet for TE < 1/2JHH. In contrast for TE of 1/JHH (Fig. 2b), in-phase terms are created and SNR is increased for weak and really strong couplings, but for intermediate couplings there is obvious signal loss induced by the J-refocusing pulse.

Figure 2 Simulated AB real spectra obtained by PRESS (left) and J-refocused PRESS (right) with the homonuclear J-coupling constant varying from 0.1 to 1.5 times of the chemical shifts difference for TE of PRESS at 1/2JHH (a) and 1/JHH (b) respectively.

58

Chapter 3: J-refocused PRESS DEPT

Figure 3 Simulated X real spectra obtained by PRESS DEPT (left) and J-refocused PRESS DEPT (right) with the homonuclear J-coupling constant varying from 0.1 to 1.5 times of the chemical shifts difference for TE of PRESS at 1/2JHH (a) and 1/JHH (b) respectively.

The related X spin spectra (namely C in CH2) obtained by PRESS DEPT with and without J-refocusing pulse are shown in Fig. 3. For PRESS TE up to 1/2JHH (Fig. 3a), the recovery of signal enhancement by DEPT is achieved by the refocusing pulse in case of weak and intermediate J-coupling constants, while the full signal enhancement is achieved for strong coupling constants with and without J-refocusing pulse. For TE at 1/JHH (Fig. 3b), in-phase magnetization is reached for all J-coupling constants if the J-refocusing pulse is applied, while

Chapter 3: J-refocused PRESS DEPT

59

the full signal intensity in only achieved for weak J-coupling, partial signal recovery is observed for very strong J-coupling and signal decrease in comparison to PRESS DEPT without J-refocusing appears for intermediate Jcoupling. The signal intensities of the X spin after DEPT are in agreement with the in-phase signal intensities of AB spins after PRESS, since only the polarization of in-phase terms can be transferred to the X spins during DEPT enhancement. In

13

C MRS, the adopted PRESS TE for in vivo measurements is usually not

longer than 1/2JHH in order to minimize T2 relaxation losses (see discussion). In this case J-refocused PRESS DEPT results in full signal enhancement for all Jcoupling constants. Fig. 4 shows simulated glutamate spectra obtained by proton-detected PRESS (Fig. 4a) and carbon-detected PRESS DEPT (Fig. 4b) with (right column) and without (left column) the J-refocusing pulse. At TE of 32 ms (within 1/2JHH), the enhanced carbon signals after DEPT are restored as expected, with more inphase proton terms built up after PRESS localization.

Figure 4 Simulated glutamate real spectra obtained by 1H PRESS (with hetero-nuclear C-H couplings taken into account which should be considered for the following DEPT) (a) and 13C PRESS DEPT (b) with (right) and without (left) J-refocusing pulse for a TE of 32ms.

Chapter 3: J-refocused PRESS DEPT

60

Experimental verification in glutamate and oil phantoms Fig. 5 shows the

13

C acquired unfiltered PRESS TE series obtained without

proton decoupling by PRESS DEPT and J-refocused PRESS DEPT from the glutamate phantom, respectively. The negative impact of homo-nuclear Jcoupling can be observed from the rapid loss of signal intensity with increasing TE, as seen in Fig. 5a. For comparison, in Fig. 5b, the suppression of J modulation was clearly demonstrated by the signal enhancement at various TEs. In both CH (C2 in glutamate) and CH2 (C3 and C4 in glutamate) spin systems, the obvious recovery of signal enhancement was detected.

Figure 5 Measured glutamate 13C real spectra versus TEs (1/2JHH: 31.5-107.1 ms): (a) PRESS localized DEPT and (b) J-refocused PRESS localized DEPT.

Chapter 3: J-refocused PRESS DEPT

61

Fig. 6 shows the measured 13C intensities (normalized to the maximum value) versus TE obtained by proton-decoupled PRESS DEPT with and without Jrefocusing pulse (blue and red lines, respectively). The 13C intensities without J-refocusing pulse (blue line) follow nonmonotonical functions with increasing TE. At around 50-70 ms there is the first local minimum of signal intensity appearing, which corresponds to the time point 62.5 ms of 1/2JHH for the average of homo-nuclear J-coupling constants in glutamate between protons attached to adjacent carbons, which is around 8 Hz. The recovery of the C4 intensity at around 120 ms without J-refocusing pulse was broader and higher than that of C3 (blue line), which is due to the absence of the additional 1H homonuclear J-coupling between C2 and C3 (18). With the J-refocusing pulse inserted, there is an obvious signal enhancement for TE values less than approximately 90 ms (between 1/2JHH and 1/JHH of the smallest J-coupling constant 6.41 Hz between C3 and C4) for C3 and C4 resonances, and 110 ms (corresponding to 1/2JHH of the smallest J-coupling constant 4.65 Hz between C2 and adjacent C3) for C2 resonance (red line). With the suppression of Jcoupling modulation and the accompanying elimination of signal recovery resulting from J-coupling effects, the signal decreased quasi monotonically with similar transverse relaxation time for all three resonance lines (red line). From Fig. 7 can be seen that the relationship between glutamate peak intensities and the glutamate phantom concentrations is linear for all three groups of resonance lines (C2, C3, C4). The regression lines indicate that the signal enhancement with J-refocused PRESS DEPT is stable for different concentrations and the sequence is therefore applicable for metabolite quantifications if the actual enhancement factors and relaxation times are taken into consideration.

62

Chapter 3: J-refocused PRESS DEPT

Figure 6 Measured normalized glutamate 13C intensities (C2: CH; C3: CH2; C4: CH2) as a function of the PRESS echo times (1/2JHH: 31.5-107.1 ms) when TE1 = TE2, with PRESS localized DEPT (blue plot) and J-refocused PRESS localized DEPT (red plot), respectively.

Chapter 3: J-refocused PRESS DEPT

63

Figure 7 Measured glutamate 13C intensities acquired with the J-refocused PRESS DEPT 13C-[1H] sequence, versus glutamate phantom concentrations.

Fig. 8 (top) shows the 13C spectra from the colza oil phantom acquired with Jrefocused 1H PRESS DEPT and direct

13

C PRESS sequences respectively.

Obvious signal enhancement was observed for all the visible peaks. Proton decoupling was average forward power limited and not fully efficient in the oil phantom that poorly loads the 1H coil. In combination with strong signal intensities, slight baseline distortions result. In vivo Application and SNR enhancement factors The spectra acquired with the J-refocused 1H PRESS DEPT and direct

13

C

3

PRESS from the calf bone marrow (20.28*21.46*249.51 mm = 109 mL) and muscle (41.73*42.18*162.14 mm3 = 285 mL) are shown in Fig. 8. All the spectra were equally scaled and show the regions of saturated (10-40 ppm) and unsaturated (125-135 ppm) fatty acids. All the recognizable peaks obtained with J-refocused 1H PRESS DEPT were assigned (Table 1) in agreement with literature (3). In the bone marrow, which contains mainly adipose tissue, higher signal intensities of the fatty acids were observed from a smaller voxel compared to the skeletal muscle. Peaks 3 and 6 (Table 1) could be clearly seen

Chapter 3: J-refocused PRESS DEPT

64

from the J-refocused PRESS DEPT bone marrow spectra, while they were not detectable in the muscle or with direct

13

C MRS acquisition. In general the

13

comparison of direct C PRESS and J-refocused PRESS DEPT spectra reflects a clear SNR enhancement for all resonance lines in the latter case. According to the above theoretical and phantom results an effective suppression of J modulation is achieved for the entire range of H-H J-coupling constants (6-18 Hz) for the selected PRESS TE of 32 ms (1/2JHH: 28-83 ms). Coherence transfer pathway selection is robust as demonstrated by in-phase peaks at the expected resonances frequencies, correct peak amplitude dependence on the voxel size in the same tissue type and SNR enhancement factors in agreement with theoretical expectations at both voxel positions. For determination of the exact in vivo SNR enhancement factors CSD correction factors for direct

13

C PRESS were calculated as listed in Table 1.

Except for the obvious signal reduction of the CH3 group (peak 9) by CDS, peak 8 has the biggest frequency difference from the 13C center frequency. The chemical shift displacement for peak 8 was 5% with the optimized excitation pulse and 8% with the refocusing pulse. For a complete 3D volume, the resonance of peak 8 had 80% in common with the nominal voxel. The measured T1 and T2 relaxation times were listed in Table 1. Since the T2 relaxation time of proton lipid is shorter than the T2 relaxation times of most carbon resonances, there was relatively more signal loss in proton PRESS localized acquisition compared to carbon PRESS. The repetition time (6s) was longer than 5*T1 of all protons and most carbons except for peak 8 and 9. The correction for relaxations was hence mainly due to transverse relaxation (Table 1).

Chapter 3: J-refocused PRESS DEPT

65

Figure 8 The fatty acids spectra acquired from colza oil (top) and from calf bone marrow (middle) and muscle (bottom) with the J-refocused PRESS DEPT 13C-[1H] sequence (top spectra) and direct 13C PRESS sequence (bottom spectra). The numbers correspond to the assignments in Table 1.

Chapter 3: J-refocused PRESS DEPT

66

Table 1 SNR enhancement factors of the J-refocused PRESS DEPT sequence for different fat components as determined from five subjects. Peak

Fat component

(1) (2) (3) (4) (5) (6) (7) (8) (9)

COO-CH2-CH2-R =CH-CH2-CH3 -CH2-CH2-CH2-CH3 (CH2)n envelope =CH-CH2=CH-CH2-CH= COO-CH2-CH2-CH2-CH2-CH3 -CH2-CH3 -HC=CH-CH2-CH=CH-HC=CH-HC=CH-CH2-CH=CH-

(10) (11) a

Chemical shift 33.83 32.18 31.74 29.80 27.37 25.78 25.02 22.86 14.11

CSDa

T1 (ms)

T2 (ms)

Cor.b

Enh. factorc

89% 94% 95% 99% 92% 88% 86% 80% 59%

199.2 1048.5 428.5 419.55 270.75 1658 2890

94.16 210.3 168.2 156.6 104 196.6 227.7

0.95 1.06 1.04 1.03 0.95 1.03 -

1.93±0.84 3.09±0.59 4.02±0.40 3.71±0.72 3.32±0.30 3.40±0.20 -

131.26

96%

541.3

290.3

1.08

3.59±0.57

129.61

99%

919.7

270.4

1.04

indicated as how much in common with the chosen voxel for direct localization b correction for relaxations c corrected enhancement factor red 'C' indicates the resonance carbon atom from the lipid chains

3.85±0.49 13

C PRESS

In vivo SNR enhancement factors for different fatty acid resonances were obtained by comparing the spectra from the bone marrow using J-refocused 1H PRESS localized DEPT and direct

13

C PRESS localization (Table 1). High

signal enhancements were achieved for all the fatty acid components. No SNR enhancement factors could be calculated for peak 3 and 6 since they could not be quantified using direct 13C detection. No SNR enhancement factor could be measured for the CH3 group (peak 9) in our experiment either, because it was missing or barely seen in the spectra with direct 13C localization due to the big chemical shift displacement that shifted the related excitation volume out of the calf. The signal intensities of the spectra from the muscle using direct

13

C

PRESS localization were so low that most of the components could be hardly observed or fitted (Fig. 8).

Chapter 3: J-refocused PRESS DEPT

67

3.4 Discussion As illustrated from theoretical calculation by product operators and spin density matrix simulations, and experimentally demonstrated by glutamate phantom measurements and in vivo detection of saturated and unsaturated fatty acids, the J-refocused 1H PRESS DEPT sequence effectively suppresses the J-modulation for all CH, CH2 and CH3 spin systems in case of short proton PRESS TE. A substantial and robust in vivo SNR enhancement of a broad range of 13C MRS signals and J-coupling constants was achieved from a 1H PRESS localized volume avoiding the big chemical shift displacements occurring in the case of direct 13C acquisition. The combination of a 32-step phase cycling scheme, OVS and unequal spoiling gradients ensured a robust and reproducible coherence transfer pathway selection. Especially when many coherence terms were produced by both homo-nuclear and hetero-nuclear scalar coupling in high order spin systems, deleterious phase distortion and visibility of unwanted peaks were observed without the effective phase cycling or spoiling gradients. Phase distortion problems were already reported in the only previous PRESS DEPT study (18) and might have been caused by an insufficient phase cycle in combination with poor localization profiles of the utilized hard pulses. Without proper OVS, the signals from outside the volume might still interfere or contribute to the acquired signal intensities, leading to unreliable quantification results. The numerical simulations for an ABX spin system clearly show the refocusing effect and signal recovery at TE up to 1/2JHH for J-coupling constants smaller than 0.5ν, while at stronger J-coupling the full SNR enhancement is achieved with or without the J-refocusing pulse. Hence J-refocused PRESS DEPT results in full signal enhancement for all J-coupling constants when TE is chosen to be

68

Chapter 3: J-refocused PRESS DEPT

smaller or equal to 1/2JHH. In reality, the H-H coupling constants in most metabolites have a range of 0.5-18 Hz (29,30), so the corresponding echo time range with regard to 1/2JHH is 28-1000 ms. In line with the requirements to fall into the range of 1/2JHH for all homo-nuclear J-coupling constants occurring in the investigated metabolites, in vivo PRESS localized DEPT measurements should be performed at the shortest possible PRESS echo times to minimize signal loss due to transverse relaxation. Considering common hardware setups and RF pulses used for in vivo MRS minimum echo times are usually in the range of 15-35 ms, which corresponds well to 1/2JHH requirement for almost all metabolites and does not reach the shortest 1/JHH of 56 ms. PRESS TEs in the order of 1/JHH should be generally avoided due to the signal loss for intermediate coupling unless only relatively weak (J < 0.25ν) and/or very strong (J > 1.25ν) J-coupling occurs. However, if a closer look is taken at the H-H coupling constants of common metabolites, it is easy to find that the Jcoupling constants do not evenly cover the range from 0.5 to 18 Hz, but mainly focus in two regions instead. The protons attached to the same carbon have obviously strong J-coupling constants, around 1.5ν in the example of glutamate, while the protons attached to different carbons have relatively weak J-coupling constants, around 0.03ν to 0.3ν in glutamate. Therefore as predicted by the simulation and proven by the glutamate phantom experiments, the suppression of J modulation and recovery of signal intensities can be obtained for most metabolites even for longer TEs towards 1/JHH. For PRESS DEPT without J-refocusing pulses, the normalized glutamate

13

C

intensities as a function of the PRESS echo times show a nonmonotonical behavior, as reported by Yahya et al (18). This effect is caused by the loss of in-phase coherence terms inherited from the PRESS localization. With the Jrefocusing pulse inserted, an effective suppression of this so called J-

Chapter 3: J-refocused PRESS DEPT

69

modulation effect was achieved up to a PRESS TE of 90 ms, which is between the average TE value for 1/2JHH and 1/JHH. At longer PRESS TEs for example 130 ms, the original recovery of the signals through J-coupling evolution was eliminated. The minor fluctuation of final 13C intensities with varying TEs can be traced analytically to glutamate being a higher order spin system with various J-coupling constants in the range from 4.67 Hz to 15.89 Hz (18). More specifically these experimental results are in agreement with previous theoretical analyses of weakly coupled homo-nuclear I1I2,n (n=1,2,3) spin systems. In these higher order spin systems J-coupling modulation is substantially but not entirely suppressed by the J-refocusing pulse at a PRESS TE up to 1/2JHH (20,26) in comparison to non-refocused PRESS. In the validation experiments using phantoms with various glutamate concentrations, the stable signal enhancement with the proposed sequence, as predicted from the linear trend lines (R-squared value (37) close to 1) ensures an accurate quantification if the same timing parameters are used. The slight deviation of the C2 trend line from the C3 and C4 trend lines most likely originates from the different theoretical DEPT enhancement factors of 0.707*(γH/γC) for C2 from a CH spin system and 1.0*(γH/γC) for the C3 and C4 carbons from CH2 spin systems by using 45o flip angle for the last 1H DEPT pulse. Under the restriction of short DEPT echo times, only block pulses can be implemented which limits the bandwidth of DEPT pulses and requires more than one measurement if applied on a large chemical shift range of metabolites, such as required for simultaneous assessment of saturated and unsaturated fatty acids. The signal intensities of enhanced multiplets without proton decoupling such as glutamate C3 and C4 in Fig. 5 are not perfect 1:2:1 triplets due to the

70

Chapter 3: J-refocused PRESS DEPT

combination of the above bandwidth limitation of block pulses and slight center frequency shifts. All the fatty acid components from a J-refocused PRESS (TE = 32 ms) localized volume were simultaneously and effectively SNR enhanced by the subsequent DEPT at a polarization transfer echo time τ of 3.7 ms for saturated and 3.25 ms for unsaturated fatty acids respectively, despite of the variety of CH coupling constants. The slight baseline distortion visible in J-refocused PRESS DEPT data acquired from the bone marrow might result from incomplete proton decoupling in combination with the large lipid signal intensity especially after DEPT enhancement. The signal enhancement factors depend mainly on the polarization transfer echo time parameter corresponding to the 1H-13C J-coupling constants. In our experiment, 3.7 ms τ was used to achieve full enhancement for a J-coupling constant of 135 Hz which is the literature value for CH2 groups (34), while the actual J-coupling constants of the saturated fatty acids range from 125 Hz to 145 Hz. The J-coupling constant of the unsaturated fatty acids was measured to be 154 Hz in vivo. Most SNR enhancement factors approach the theoretical value of 4. Besides the different C-H J-coupling constants and imprecision in spectral fitting especially in the case of direct 13C PRESS, another reason for the variation of SNR enhancement factors might result from errors with regard to relaxation corrections. For all proton resonances of fatty acids, only one T1 and one T2 value were found in the literature and adopted in the correction. But in reality the proton resonances may relax differently in various chemical environments. While due to the long repetition time T1 correction could be omitted, T2 correction might introduce additional variation of SNR enhancement factors. For example peak 1, which seems to have a significantly lower SNR enhancement, might in reality have a faster proton T2 relaxation since the related proton nucleus is adjacent to a

Chapter 3: J-refocused PRESS DEPT

71

COO- structure. At higher field strengths or for the simultaneous assessment of saturated and unsaturated fatty acids in one measurement, overprescribed PRESS can be combined to further reduce the chemical shift displacement. Altogether J-refocused PRESS DEPT represents a robust 1H localized SNR enhancement method for in vivo fatty acid quantification by

13

C MRS. In

contrast, ISIS was reported to suffer from MT and NOE induced localization errors if applied in the presence of lipid rich tissue (15), which puts question marks behind the applicability of ISIS DEPT for this purpose.

3.5 Conclusion The J-refocused

1

H PRESS localized DEPT sequence was introduced,

implemented and investigated for natural abundance

13

C MRS. Theoretical

analysis, numerical simulation results, glutamate phantom and in vivo fatty acids experiments show its ability to suppress homo-nuclear proton Jmodulation for the investigated spin systems and achieve substantial SNR enhancement for a large range of J-coupling constants. In addition, a novel phase cycling scheme in combination with OVS and unequal spoiling gradients was shown to effectively suppress unwanted coherence contamination for in vivo application. The potential use of the sequence is to obtain reliable signal gain for 13C MRS and to assess concentrations of any metabolites of interest in specific locations simultaneously.

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72

3.6 Acknowledgements We thank the HARTMANN MÜLLER foundation for medical research (grant 1433) and the ZIHP (Zurich Center for Integrative Human Physiology) for fundings, and Erin MacMillan for proof reading the manuscript.

Chapter 4: Fatty acids at 3T

73

Chapter 4:

Quantification of Fatty Acids in Human Calf Adipose Tissue and Muscle by 13C MRS using

J-refocused

PRESS

ERETIC

Modified based a manuscript (to be submitted).

DEPT

and

Chapter 4: Fatty acids at 3T

74

4.1 Introduction Fatty acids serve as an important source of energy for muscular contraction and general metabolism (1-5). Skeletal muscle fatty acid metabolism has been found in association with insulin resistance (6-8). Compared to proton (1H) MRS, which enables distinction of intra-myocellular (IMCL) and extramyocellular (EMCL) lipids (6,7), in vivo carbon 13 (13C) MRS offers a better separation of saturated and unsaturated fatty acid components and might allow for additional detection of omega-3, omega-6 (9) and trans-fatty acids (10), cholesterol (11) and di-glycerides (12). The analysis of fatty acids composition of human muscle has been reported ex vivo by 13C NMR spectroscopy and gas chromatography on surgical muscle samples (8,13). In addition, the in vivo 13C MRS profile of saturated and unsaturated fatty acids has been shown mainly from subcutaneous adipose tissue using surface coil (2,4,5,14) or half-volume coil (1,3) excitation and reception without any additional localization scheme, which was the option of choice due to the intrinsic low sensitivity and large frequency dispersion hampering direct localization in 13C MRS. However, muscle as the metabolically active tissue has been barely studied by in vivo

13

C MRS. To our knowledge only C. Wary (14) has shown a lipid

spectrum from calf muscle combining surface coil localization with surface spoiling gradients to suppress the signals from subcutaneous fat. However contamination from the bone marrow and the insufficient suppression of subcutaneous fat after certain depth was not considered and resulted in a spectrum from mixed adipose tissue and muscle origin. To enhance the SNR (signal-to-noise ratio) in

13

C MRS, DEPT (Distortionless Enhanced

Polarization Transfer) (15) as a well established method has been previously used and combined with different proton localization schemes to overcome the

Chapter 4: Fatty acids at 3T severe chemical shift displacement problem inherent to direct

75 13

C localization

(16-18). ISIS (Image Selected in Vivo Spectroscopy) (19) can in principle be used for metabolites with short T1 and T2 relaxation times such as lipids, but is sensitive to physiological motion and transmit field inhomogeneity due to its 8 inversion based encoding steps, as well as suffers from magnetization transfer and Nuclear Overhauser Effect induced localization errors occur if applied to lipid rich tissue (20). Due to its high localization efficiency PRESS (Point RESolved Spectroscopy) (21) is the most widely used 1H MRS localization scheme and applicable to lipid quantification but suffers from the homonuclear scalar coupling modulation, which exacerbates the DEPT enhancement (22). A J-refocused proton PRESS localized DEPT sequence for in vivo SNR enhanced and localized

13

C MRS has been recently introduced, implemented and

validated (23). Related phantom and in vivo experiments prove its capability to suppress the homonuclear scalar coupling evolution during PRESS and restore the DEPT enhancement for all relevant resonance lines in lipids. While the relative concentrations of saturated and unsaturated fatty acids have been prevalently used for dietary (1,3,4) or disease (5,13) studies, absolute molar concentrations of fatty acids have been reported only once from two subjects using endogenous water and fat as a reference and a localization approach of limited efficiency as described above (14). In general internal reference standards can hardly be applied to pathological studies since concentrations of the corresponding metabolites such as water or creatine may be altered. In 13C MRS additional problems arise due to the low SNR of the 13C creatine resonance and in case of internal water referencing due to the different transmit field homogeneity between carbon and proton frequencies. In recent years, ERETIC (Electric Reference To access In vivo Concentration) (24) as a synthetic reference signal for quantification of molar metabolite concentrations,

Chapter 4: Fatty acids at 3T

76

which is independent of physiological conditions, has undergone major improvement with respect to the stability and reproducibility (25) and has been enabled and validated for

13

C MRS applications (26).

By combining recent methodology of J-refocused proton PRESS for localization, DEPT for SNR enhancement, and optically transmitted and inductively coupled ERETIC as quantification reference, the specific aims of this work were: 1, to establish fatty acids quantification from the metabolically distinct tissue types adipose tissue and skeletal muscle by 13C MRS for the first time; 2, to evaluate relative compound proportions and absolute molar concentrations of all the fatty acid subgroups in both tissue types; 3, to determine the sensitivity of the proposed method to detect different lipid profiles among omnivores, vegetarians and vegans.

4.2 Methods MRS acquisition MRI instruments All experiments were performed on a 3T Philips Achieva human MRI scanner (Philips Healthcare, Netherlands). A commercial dual-tuned 13C/1H human calf volume coil (RAPID Biomedical GmbH, Germany) was used for both transmission and reception and included the previously reported custom modification to implement the ERETIC reference standard for quantitative 13C MRS (26). Imaging and preparation phases such as F0 determination and B0 shimming were executed on the proton frequency. The maximum B1 of the proton channel is 30 μT and of the carbon channel is 120 μT.

Chapter 4: Fatty acids at 3T

77

Spectroscopy sequence As shown in Figure 1, J-refocused PRESS and OVS (Outer volume suppression) localization was performed on the proton frequency. Optimized selective pulses were used with 4419 Hz bandwidth for the PRESS excitation and J-refocusing, and 2808 Hz bandwidth for the chemical shift refocusing as well as 13459 Hz bandwidth for OVS. The subsequent DEPT enhancement radiofrequency pulses were placed on both proton and carbon frequencies and transferred the polarization from the J-refocused and localized protons to the coupled carbons, using block pulses with a minimum bandwidth of 1270 Hz. The standard Philips setup allowed for simultaneous transmission on the proton and the carbon frequency with reference points of related DEPT pulses being aligned. Waltz-16 cycle proton decoupling was used with 17μT B1 resulting in a bandwidth of 964 Hz centered at the corresponding proton spectra. The sequence performance was described in detail elsewhere (23). The synthetic ERETIC reference signal was injected simultaneously with proton decoupling and carbon signal acquisition. The frequency of the ERETIC reference signal was set to avoid overlapping with any metabolites, and the amplitude was fixed in all the measurements. A detailed description of the ERETIC implementation and related quantification reliability tests can be found in Chen et al [19]. The volumes of interest were localized in the calf tibial bone marrow and skeletal muscle with volume sizes around 105 mL (20*21*250 mm3) and 286 mL (42*42*162 mm3) respectively. Minor adjustment was made to the volume position and size for different subjects. At each location, one spectrum targeting at saturated fatty acids (14-34 ppm) and one spectrum targeting at unsaturated fatty acids (128-132 ppm) were acquired, with the flip angle β of the last pulse in DEPT set as 45o and 90o respectively. The PRESS TE was 32 ms. The DEPT τ was 3.7 ms for saturated fatty acids detection and 3.25 ms for unsaturated

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fatty acids detection. The repetition time was 6 s and 4 s with 256 averages. The spectra were acquired with 1024 sample points and 8000 Hz acquisition bandwidth.

Figure 1 Localized volumes from calf tibial bone marrow and skeletal muscle (left) and J-refocused proton PRESS localized DEPT sequence, combined with ERETIC reference during the acquisition (right). The volume sizes were around 105 mL and 286 mL in the bone marrow and muscle, respectively. PRESS echo time TE was 32 ms and DEPT echo time τ was 3.7ms and 3.25 ms for saturated and unsaturated fatty acids detection with the flip angle β set as 45o and 90o respectively.

Parameter measurement Carbon T1 and T2 relaxation times of each peak component were measured with the inversion recovery series and echo time series (27) on two subjects without localization scheme. Proton T1 and T2 relaxation times were taken from literature as 365 ms and 133 ms in the bone marrow and 1420 and 32 ms in the muscle (28). In order to calculate the DEPT enhancement factor for each resonance line, the spectra with direct 13C PRESS localization and the spectra with the adopted sequence were obtained and compared using the same pulse shapes and timing parameters on a large volume from another two subjects. For

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this purpose three groups of spectra were acquired focusing on the unsaturated fatty acids, methylene group and methyl group of saturated fatty acids respectively in order to minimize the chemical shift displacement in the direct 13

C spectra. Chemical Shift Displacement (CSD) of each carbon resonance in

the direct

13

C localization experiment was assessed by the ratio between the

part of the resonance line specific excitation volume that overlapped with the nominal volume and the size of the nominal volume, which was calculated as the difference in Larmor frequency divided by the bandwidth of each RF pulse. The final DEPT enhancement factors were corrected for proton T1 and T2 relaxation during proton PRESS localization and carbon T1 and T2 relaxation during direct 13C PRESS localization as well as CSD.

Subjects Written informed consent was obtained from all the subjects in accordance to local ethics guidelines. Twenty subjects in total were studied and divided into three age- and sex-matched groups, as listed in Table 1. Among the seven vegans, one male subject who has been vegan for 11 years has regularly taken omega-3 supplement for 2 years. All the other subjects did not use special nutrition supplements. Table 1 Subjects information (Mean ± SD) Dietary type Omnivore (n = 6) Vegetarian (n = 7) Vegan a (n = 7) a

Number and Gender 3 female, 3 male 4 female, 3 male 3 female, 4 male

Age b (years) 34.5±9.9 27.3±5.3 28.9±6.4

BMI c, d (kg/m2) 24.0±1.9 22.3±1.6 22.1±1.8

Except for one (11 years), the other subjects have been vegans for an average of 2 years (2.0 ± 1.6). b no significant difference c Body mass index d no significant difference

Chapter 4: Fatty acids at 3T

80 MRS data analysis

All the spectra were post-processed with Spectator, a custom written IDL (IDL Workbench Version 7.1.0) graphical user interface, and fitted by an embedded tool TDFDfit (29), without Gaussian or exponential filtering. Zero order phase correction was performed manually before fitting. Since the peak around 128 ppm originates only from PUFA (polyunsaturated fatty acids) and the peak around 130 ppm originates from the sum of MUFA (monounsaturated fatty acids) and PUFA, IPUFA represents the peak intensity at 128 ppm alone and IMUFA is the subtraction of the peak intensity at 130 ppm minus the peak area at 128 ppm. All the methylene and methyl carbons are from the saturated part of fatty acids, so the sum intensity of six predominant peaks around 30 ppm and the methyl peak around 14 ppm is defined as ISaturated. Relative concentrations: The percentage of each carbon component: % Each component = IEach component / (ISaturated + IMUFA + IPUFA)

(4.1)

The percentage of saturated carbons: % Saturated = ISaturated / (ISaturated + IMUFA + IPUFA)

(4.2)

The percentage of MUFA carbons: % MUFA = IMUFA / (ISaturated + IMUFA + IPUFA)

(4.3)

The percentage of PUFA carbons: % PUFA = IPUFA / (ISaturated + IMUFA + IPUFA)

(4.4)

The ratio of MUFA/PUFA was analyzed since it was reported to reflect abnormalities in cystic fibrosis (5) and diet conditions (1): MUFA / PUFA = IMUFA / IPUFA

(4.5)

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81

The ratio of middle chain methylene to methyl representing the mean chain length / degree of saturation of fatty acids (1) was also calculated: Middle chain methylene / methyl = I(CH2)envelop / ICH3

(4.6)

Absolute concentrations: According to the following equation (26): 𝑪𝑚 = 𝑪𝑝ℎ𝑎𝑛,𝑐𝑎𝑙 ∗

𝑨𝐸𝑅𝐸𝑇𝐼𝐶,𝑐𝑎𝑙

𝑨𝑝ℎ𝑎𝑛,𝑐𝑎𝑙 ∗𝒇𝑠,𝑐𝑎𝑙 ∗𝒇𝑐,𝑐𝑎𝑙 ∗𝒇𝑜,𝑐𝑎𝑙

∗

𝑨𝑚 ∗𝒇𝑠 ∗𝒇𝑐 ∗𝒇𝑜 𝑨𝐸𝑅𝐸𝑇𝐼𝐶

(4.7)

the absolute concentration in the unit of mM / L of each fatty acid component was accessed with the previously calibrated ERETIC reference signal (26). C stands for molar concentration, A for measured peak area and f for correction factor. The subscript cal indicates the in vitro ERETIC reference calibration session against a DMSO (dimethyl sulfoxide) phantom with known concentration. Coil load and the influence of receive chain (coil / hardware correction factors fc) were explicitly corrected by ERETIC, while T1 and T2 relaxation times during proton PRESS localization, corrected DEPT enhancement factors, volume sizes (sequence correction factors fs) and the temperature (other correction factor fo) were considered. The details of absolute quantification with respect to the ERETIC reference standard were described before (26). Statistic analysis: The results were expressed as mean ± SD. Statistical analysis was done by Student’s unpaired, two-tailed t-test. When the P-value was less than 0.05 (30), the comparison was considered as significantly different. Other more significant P-values (0.005 and 0.001) are also observed and marked.

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4.3 Results 13

C spectra of fatty acids

Figure 2 shows the

13

C spectra of both saturated and unsaturated fatty acids

from calf tibial bone marrow and skeletal muscle. Good SNR and a flat base line were obtained from both tissue types. The different carbon resonances from the saturated fatty acids around 30 ppm can be clearly distinguished and fitted. The peak assignments are listed in Table 2. In Figure 2, spectra on the left are from a male omnivore and spectra on the right are from an age- and sex-matched vegan who has taken omega-3 supplement for two years and is vegan for 11 years. It can be seen that the signal intensity of PUFA (peak 2) obtained from the vegan was obviously higher than the intensity from the omnivore in both bone marrow and muscle, which reflects the omega-3 supplement. The ratio of MUFA to PUFA was 0.72 in the calf tibial bone marrow and 1.35 in the calf muscle from the vegan, while 2.56 in the calf tibial bone marrow and 1.42 in the calf muscle from the omnivore. The percentage of the saturated fatty acids in the whole fatty acids pool was 85.94% and 87.46% from the vegan and 92.20% and 93.11% from the omnivore in the bone marrow and muscle respectively. The ERETIC reference signal was stable during all the measurements on each subject with a variance less than 2%.

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Figure 2 13C Spectra from calf tibial bone marrow (top) and skeletal muscle (bottom) from a male omnivore (left) and an age- and sex-matched vegan who has taken omega-3 supplement for 2 years (right). The ERETIC peak is shown in all the spectra and the peak assignments of fatty acids are listed in Table 2. Table 2 Peak assignments in the 13C spectra as well as the measured T1, T2 relaxation times in carbon frequency and DEPT enhancement factors after corrected for CSD, T1 and T2 relaxations of each carbon resonance. Peak no. 1 2 3 4 5 6 7 8 9

Chemical shift (ppm) 131.26 129.61 33.83 32.18 29.80 27.37 25.02 22.86 14.11

lipid components

CSD* 96%

T1 relaxation time (ms) 541.3

T2 relaxation time (ms) 290.3

Corrected DEPT factor 3.59±0.57

-HC=CH-CH2-CH=CH-HC=CH-HC=CH-CH2-CH= CHCOO-CH2-CH2-R (C2) =CH-CH2-CH3 (ω3) (CH2)n envelope =CH-CH2- (allylic α=cis) COO-CH2-CH2- (C3) -CH2-CH2-CH3 (ω2) -CH2-CH3 (CH3)

99% 89% 94% 99% 92% 86% 80% 99%**

919.7 199.2 1048.5 428.5 419.5 270.8 1658 2890

207.4 94.16 210.3 168.2 156.6 104 196.6 227.7

3.85±0.49 1.93±0.84 3.09±0.59 4.02±0.40 3.71±0.72 3.32±0.30 3.40±0.20 3.50±0.51

* indicated as how much in common with the chosen voxel for direct localization ** from a separate measurement

13

C PRESS

Relative concentrations Figure 3 plots the relative concentrations of all the fatty acid components in calf tibial bone marrow and skeletal muscle among the three dietary groups. Similar

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Chapter 4: Fatty acids at 3T

proportions were found in both tissue types from all the groups. Small standard deviation bars indicate the stable proportions of the fatty acid subgroups especially with regard to bone marrow results. In calf tibial bone marrow (Figure 3 left), there are obvious differences between dietary groups for peak 5 (middle methylene group), peak 1 – peak 2 (MUFA) and peak 2 (PUFA), while not observed in skeletal muscle (Figure 3 right). The relative concentration of the middle methylene group (peak 5) is lower in the muscle than in the bone marrow especially for vegetarians (39.74±6.34% and 47.78±6.17%; p < 0.05) and vegans (39.64±3.73% and 44.96±3.85%; p < 0.005). The relative concentrations of unsaturated fatty acids including both MUFA (peak 1 – peak 2) and PUFA (peak 2) in the muscle are higher compared to the bone marrow (Table 3) in these two dietary groups. It was also particularly noticeable that peak 3 had higher relative concentrations in the muscle in comparison to bone marrow of vegetarians (7.44±1.98% and 3.70±1.38% with p < 0.001) and vegans (9.09±3.18% and 4.26±1.50% with p < 0.005), but not significant in omnivores. Statistically significant differences for relative concentrations of peak 3 between omnivores and vegetarians (p < 0.05) and between omnivores and vegans (p < 0.05) were found, but not between vegetarians and vegans.

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85

Figure 3 Relative concentrations of each fatty acid component from the calf tibial bone marrow (left) and muscle (right) among omnivores, vegetarians and vegans. The mean value and the standard deviation were plotted in each dietary group.

Table 3 lists the percentages of saturated fatty acids, MUFA and PUFA, as well as the ratios between the two unsaturated fatty acid types and between two major saturated peaks indicating average chain length. In the muscle less saturated fatty acids were found compared to the bone marrow for all the dietary groups indicated by the smaller %Saturated values. In the bone marrow, %Saturated from vegans is significantly different from that in omnivores and vegetarians, but no significant difference was found between omnivores and vegetarians. A similar correlation could not be found in the metabolically active muscle where the lipid profile reflects the short-term dietary intake and muscular activity. It was interesting to find out that for vegetarians all the listed values except for MUFA/PUFA in the bone marrow were significantly different from those in the muscle. For omnivores %PUFA in the bone marrow and in the muscle were also significantly different. No

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86

obvious difference was found between females and males among all the three groups. Table 3 Relative concentrations and ratios of the fatty acids (mean ± SD) a

Bone marrow Muscle

h

Omnivore * g Vegetarian * Vegan Omnivore Vegetarian Vegan

% Saturated 92.97±1.84 92.54±2.70 f 88.17±4.97 * 88.13±6.63 87.10±3.86 86.32±7.26

% MUFA 5.04±1.09 5.04±1.96 7.32±2.98 8.05±5.27 8.24±2.82 8.78±4.57

b

c

% PUFA 1.99±1.05 2.42±1.40 4.50±2.66 3.82±1.50 4.65±1.26 4.90±2.83

MUFA / PUFA 3.00±1.50 2.40±0.82 2.17±0.77 2.03±0.77 1.79±0.45 1.79±0.69

d

(CH2) envelop / CH3 5.14±0.90 5.20±0.60 4.98±0.95 5.98±3.39 4.12±0.72 4.48±0.73

e

a the sum of the peak area of 3 to 9 divided by the sum of the peak area of 1 to 9 b the subtraction of peak area of 1 and 2 divided by the sum of the peak area of 1 to 9 c the peak area of 2 divided by the sum of the peak area of 1 to 9 d (peak 1 – peak 2) / peak 2 e peak 5 / peak 9 *f p < 0.05 difference between vegans and omnivores, p < 0.05 difference between vegans and vegetarians, no significant difference between omnivores and vegetarians in the bone marrow *g p < 0.05 difference in vegetarians between bone marrow and muscle for all the values except for MUFA / PUFA with P < 0.1 *h p < 0.05 difference in omnivores between bone marrow and muscle for % PUFA

Absolute concentrations The measured T1 and T2 relaxation times at 3T as well as the T1-, T2- and CSDcorrected DEPT enhancement factors of each fatty acid component are listed in Table 2. The absolute concentrations of fatty acid compounds in the unit of mM / L obtained via the ERETIC reference standard are plotted in Figure 4. The molar concentrations of fatty acids in the muscle are about 10 times lower than in the bone marrow (Figure 4, Table 4). In agreement with the larger standard deviation of relative concentrations, in skeletal muscle compared to bone marrow, the absolute molar concentrations of fatty acid components in the muscle show also a large dynamic range.

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87

Figure 4 Absolute concentrations in mM / L of each fatty acid component from the calf tibial bone marrow (left) and muscle (right) among omnivores, vegetarians and vegans. The mean value and the standard deviation were plotted in each dietary group.

The sum of the concentrations of all saturated, all unsaturated and all fatty acids are listed in Table 4 and plotted in Figure 5. Significantly higher molar concentrations of unsaturated fatty acids in the bone marrow were observed for vegans compared to omnivores and vegetarians, while no significant difference was found between omnivores and vegetarians. It was interesting to find that there is a significant difference between females and males for omnivores for the unsaturated fatty acids concentrations in the bone marrow (Figure 5). In addition the molar concentrations of saturated, unsaturated and all fatty acids from the vegan subject who has taken Omega-3 for two years were 3404.34 mM/L, 505.32 mM/L, 3909.66 mM/L in the bone marrow and 782.78 mM/L, 97.37 mM/L, 880.15 mM/L in the muscle, respectively. Compared to the average concentrations among vegans (in Table 4), this subject had obvious lower fatty acid level in the bone marrow but higher level in the muscle.

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Table 4 Absolute concentrations of saturated, unsaturated and total fatty acids (mean ± SD) Bone marrow b Muscle

Omnivore c Vegetarian Vegan Omnivore Vegetarian Vegan

Saturated (mM / L) 4527.56±1377.95 5871.63±1781.91 5693.55±3006.21 504.10±223.73 466.34±187.05 595.55±249.57

Unsaturated (mM / L) 321.20±151.26 419.60±156.52 599.44±123.86 a 56.29±21.52 58.44±17.78 64.02±17.13

Total (mM / L) 4848.76±1495.14 6291.23±1830.91 6292.99±3088.67 560.39±220.68 524.78±200.55 629.57±261.64

a

p < 0.05 difference between vegans and omnivore, no significant difference between omnivore and vegetarian in the bone marrow b p < 0.001 difference between bone marrow and muscle for all groups and for all concentration values c p < 0.05 difference in omnivore between female and male for unsaturated and total concentration values in bone marrow

Figure 5 Gender-marked absolute concentrations in mM / L of saturated, unsaturated and total fatty acids from the calf tibial bone marrow (left) and muscle (right) among omnivores, vegetarians and vegans.

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4.4 Discussion The proposed combination of J-refocused PRESS DEPT and ERETIC for quantitative, localized and SNR enhanced

13

C MRS was capable of detecting

the relative and absolute concentrations of all the fatty acids subgroups from two metabolically distinct tissue types: adipose tissue and skeletal muscle. Localized in vivo 13C MR spectra could be acquired from calf bone marrow and skeletal muscle, without obvious contamination from improper coherence pathway selections or signal loss due to homonuclear scalar couplings (23). With the ERETIC reference, the absolute molar concentrations of all the fatty acid components could be reliably obtained and analyzed among three dietary groups. The fatty acids profile obtained from the calf tibial bone marrow was in agreement with the previous report from the calf subcutaneous adipose tissue (1-5,14). Both percentages and molar concentrations are relatively stable and reflect long-term diet. It has been reported that triglyceride composition was not found differently between calf subcutaneous adipose tissue and tibial bone marrow for a given subject by 1H MRS at 7T (31). Our results provide the same conclusion by 13C MRS. The half-life of fatty acids in the adipose tissue were reported around two years (32). In our study, the vegan subjects had been vegans for an average of two years and the most vegetarian subjects were born to be vegetarians or vegetarians longer than at least two years. The difference in the fatty acids profile could be clearly distinguished especially regarding to the percentages and absolute molar concentrations of unsaturated fatty acids, indicating that the half-life of fatty acids in the bone marrow could be similar or shorter than 2 years. Since the lipid profile of bone marrow is related to a longterm dietary intake, the result confirmed a previous dietary assessment (3) that

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the vegan population has a significant higher intake of unsaturated fatty acids, particularly polyunsaturated fatty acids while no significant differences in dietary fat content and composition are found between omnivores and vegetarians. While the lipid profile from the bone marrow corresponds to the general diet of the subject, in the muscle there was no significant difference being found related to diet among different dietary groups. In addition the variation of molar concentrations is large, which reflects dynamic fatty acid levels depending on short-term dietary intake and exercise similar to glycogen, which represents the second major energy storage form next to fatty acids. In agreement with our findings it has been already demonstrated in rats based on tissue extraction and biochemical analysis that the fatty acid profile of skeletal muscle is strongly influenced by short-term dietary changes (33). It has also been observed by 1H MRS that IMCL levels differ significantly between identical muscles in different subjects as well as intra-individually within 1 week intervals, with a half life time of 28 h (34). The IMCL level correlates with muscular activity since it dropped to only half of the concentration at rest after 1 day of hiking. That might explain the large standard deviation in our measurements, which is observed and reported by in vivo 13C MRS for the first time in the present study. Compared to the bone marrow, the lower percentage of the middle methylene group in skeletal muscle (Figure 3) especially for vegetarians and vegans might result from shorter chain length or a lower degree of saturation of muscle lipids. The lower degree of saturation is supported by the evidence of the existence of higher percentages of unsaturated fatty acids (Table 3 and Figure 3). The shorter chain length in these two dietary groups is also supported by the methylene to methyl ratios in Table 3. Since peak 3 resides near the end (carboxylic acid, Table 2) of the fatty acid chain, its higher percentage (Figure

Chapter 4: Fatty acids at 3T

91

3) proves again the existence of short chains in the muscle. To sum up the above findings, it seems that the muscle lipids reflect short term dietary intake and energy consumption based on exercise with shorter chain length and a lower degree of saturation compared to the lipids in the adipose tissue, which are processed for long term high energy storage with longer chains and higher saturation. Gender-related difference among different groups is observed especially in the bone marrow. While male omnivores have higher concentrations of saturated lipids and lower concentrations of unsaturated lipids compared to male vegans as shown in Figure 2 and Figure 5, opposite results between female omnivores and vegans are shown for saturated lipids in Figure 5. For vegetarians the concentrations are mixed and fall into the middle range. The higher concentrations of saturated lipids in female vegans result into a higher average concentration among vegans compared to omnivores. The gender differences might reflect gender specific nutrition behavior for instance a higher intake of vegetables and fruits and a lower intake of meat among female omnivores compared to male omnivores, but more samples should be collected in order to look into this gender dependent difference in more detail. T1 and T2 corrections during proton PRESS localization for enhancement factors as well as absolute concentrations were adopted from literature (28). Proton T1 and T2 relaxation times were only distinguished by different tissue types: bone marrow and muscle, but could not be assigned to individual fatty acid component. Small deviation might be brought into the quantification scheme. The relaxation is similar in bone marrow and subcutaneous adipose tissue, but quite different in skeletal muscle (6,31). Therefore different it is important to use tissue specific relaxation constants when absolute molar concentrations are required.

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4.5 Conclusion The J-refocused proton PRESS localized DEPT sequence for in vivo 13C MRS was applied to characterize fatty acids from the calf tibial bone marrow and skeletal muscle among three dietary groups, with the optically transmitted and inductively coupled ERETIC as reference signal for compound quantification. Indicated by both relative and absolute molar concentrations, the fatty acid profile in the bone marrow is stable and reflects the long-term diet, while the fatty acids in the muscle are related to the metabolic activities and short-term dietary intake. Quantitative SNR-enhanced and localized 13C MRS based on the suggested protocol provides a unique and reliable opportunity to noninvasively study lipid metabolism in metabolically distinct tissue types in clinical research and diagnosis.

4.6 Acknowledgement We thank the HARTMANN MÜLLER foundation for medical research (grant 1433) and the ZIHP (Zurich Center for Integrative Human Physiology) for fundings, and Johannes Slotboom and Roland Kreis for providing TDFDFit.

Chapter 5: Cholesterol at 7T

93

Chapter 5:

Cholesterol Detection in Adipose Tissue by Natural Abundance in vivo 13C MRS at 7T

Adjusted from: ISMRM proceeding 2012: 1762 and ESMRMB 2012 : 94.

Chapter 5: Cholesterol at 7T

94

5.1 Introduction As a ubiquitous component of all animal tissues, cholesterol has vital structural roles in membranes and in lipid metabolism. Abnormal cholesterol levels are strongly associated with cardiovascular disease. Adipose tissue is a major site of cholesterol storage [1]. A variety of nutritional and metabolic alteration may influence the adipose tissue cholesterol level, such as age, dietary cholesterol load, which has been studied in rats by chemical analysis. Localized natural abundance 13C magnetic resonance spectroscopy (MRS) has been shown to be a powerful noninvasive technique for the study of lipids in vivo [2]. 13C MRS has been applied to study the major classes of unsaturated and saturated fatty acids, like mono- and polyunsaturated fatty acids, ω-1, ω-2, ω-3, C2, C3, allylic and CH2 envelop [3]. In this work, detection of cholesterol is reported for the first time noninvasively in human calf adipose tissue by 13C MRS at 7T, with J-refocused 1

H PRESS localized DEPT sequence combined with broadband decoupling.

5.2 Methods Measurements were performed on a 7T Philips Achiena MRI system using a quadrature dual-tuned

13

C/1H partial volume extremity coil. A J-refocused 1H

PRESS localized DEPT sequence as shown in Fig.3 right, with a 90o RF pulse inserted in the middle of the double echo PRESS localization part, was used. 1

H-1H scalar couplings are completely refocused by the 90o pulse as long as

TE1=TE2, while the chemical shifts are refocused by each echo pulse. The offset of the 13C frequency was centered at 30ppm of the CH2 envelop region

Chapter 5: Cholesterol at 7T

95

and the offset of 1H frequency was centered at 1.3ppm for both PRESS localization and DEPT enhancement pulses. 32ms PRESS echo time was used where TE1=TE2=16ms. Polarization transfer echo time was optimized (τ 3.7 ms) corresponding to the 1H-13C coupling constants in lipids covering from 125 Hz to 145 Hz. Waltz16 decoupling with 18μT pulses was used to fulfil the SAR limit. 13C spectra were acquired by averaging 192 FIDs with TR 10s for a total scan time of 32min. The sequence was applied to detect lipids in the calf adipose tissue (Fig.1 left) of two healthy subjects in a total of 5 measurements (twice on one subject and three times on the other subject).

Figure 1 localized voxel in adipose tissue with partial volume coil at 7T (left) and Jrefocused 1H PRESS DEPT sequence (right).

5.3 Results and Discussion Natural abundance 13C MR spectra acquired with the proposed J-refocused 1H PRESS localized DEPT sequence from the left calf of two healthy subjects are

96

Chapter 5: Cholesterol at 7T

shown in Fig. 2. Eight well established peak assignments are labeled in green with their chemical shifts marked in black which are in agreement with literature values (Table 1). In addition, there are also five obvious peaks observed in both subjects consistently across all measurements, which have not been reported yet. The three peaks at 28.28ppm, 24.34ppm and 24.03ppm (Fig. 2; yellow) are tentatively assigned to cholesterol in accordance to literature values [7, 8]. The other two peaks at 33.24ppm and 30.90ppm (Fig.2; blue) are assigned to the allylic carbon atoms C13 trans and C18 respectively (Table 1) [4, 5 and 6]. The C13 trans peak at 33.24ppm has a slightly different chemical shift compared to a previous reported trans oleic acid peak (32.78ppm, [3]) and might stem from a isoprene unit or acetlylenic fatty acids with two olefinic groups adjacent to it [6]. The diallylic peak at 25.78ppm [3], however, could be barely seen probably due to the echo timing parameters which are sensitive factors in PRESS localized DEPT enhancement.

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97

Figure 2 The J-refocused 1H PRESS DEPT 13C spectra from adipose tissue of two healthy subjects at 7T, with cholesterol marked as yellow.

Figure 3 The zoomed region from 22.5 ppm to 32.5 ppm of the spectra.

Chapter 5: Cholesterol at 7T

98

Table 1 Peak assignments of J-refocused 1H PRESS DEPT 13C spectra at 7T.

a

most refer to AOCS lipid library and Ref. [3]

5.4 Conclusion 13

C MRS has the potential of detecting cholesterol and other lipid components

from adipose tissue and studying the interaction between cholesterol storage and whole body metabolism.

Chapter 6: Conclusion and Outlook

Chapter 6:

Conclusion and Outlook

99

Chapter 6: Conclusion and Outlook

100

Conclusion and Outlook The thesis aimed at reliable application of quantitative, SNR enhanced and localized natural abundance

13

C MRS in physiological studies. The goal was

achieved by developing the optically transmitted and inductively coupled ERETIC method, introducing the J-refocused proton PRESS localized DEPT sequence and applying the combined method on dietary related fatty acids detection from human calf muscle and bone marrow. Previous work showed that a calibrated synthetic MRS-like signal transmitted through an optical fiber and inductively coupled into a transmit/receive coil represents a reliable reference standard for in vivo 1H MRS quantification on a clinical platform. We introduced a related implementation that enables simultaneous proton decoupling and ERETIC based metabolite quantification and hence extends the applicability of the ERETIC method to in vivo 13C MRS and other multinuclear MRS. The ERETIC signal intensity stability was 100.65±4.18% over 3 months including measurements with and without proton decoupling. Glycogen and unsaturated fatty acid (UFA) concentrations measured with the ERETIC method were in excellent agreement with internal creatine and external phantom reference methods, showing a difference of 1.85±1.21% for glycogen and 1.84±1.00% for UFA between ERETIC and creatine-based quantification, while the deviation between external reference and creatine-based quantification are 6.95±9.52% and 3.19±2.60% respectively. Proton PRESS localization has been combined with DEPT (Distortionless Enhanced Polarization Transfer) in multinuclear MR spectroscopy to overcome the signal contamination problem in ISIS combined DEPT especially for lipid detection. However, homo-nuclear proton scalar couplings reduce the DEPT enhancement by modifying the spin coherence distribution under J modulation

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101

during proton PRESS localization. Herein a J-refocused proton PRESS localized DEPT sequence is presented for obtaining simultaneously enhanced and localized signals from a large number of metabolites by in vivo

13

C MR

spectroscopy. The suppression of J-modulation during PRESS and the substantial recovery of signal enhancement by J-refocused PRESS localized DEPT were demonstrated theoretically by product operator formalism, numerically by the spin density matrix simulations for different scalar coupling conditions, and experimentally with a glutamate phantom at various echo times. Applying the sequence for localized detection of saturated and unsaturated fatty acids in the calf bone marrow and skeletal muscle of healthy subjects yielded high signal enhancements simultaneously obtained for all components. Natural abundance 13C MRS has been previously applied to study in vivo lipid composition from the subcutaneous adipose tissue. Resulting from the intrinsic low sensitivity of

13

C MRS, surface coils or half-volume coils without

localization scheme are most commonly used and only relative concentrations of different fatty acid subgroups are assessed. In our work, the proposed Jrefocused proton PRESS localized DEPT sequence is applied to achieve reliably localized and SNR enhanced signals by in vivo

13

C MRS using a

volume coil, and combined with optically transmitted and inductively coupled ERETIC as a reference for absolute quantification. The proposed method is used to assess the fatty acids from human calf tibial bone marrow and skeletal muscle among omnivores, vegetarians and vegans. Both relative and absolute molar metabolite concentrations were evaluated and analyzed. Seen from the concentration values and statistical analysis, the fatty acid profile in the bone marrow is stable and reflects the long-term diet while the fatty acids in the muscle are related to the short-term metabolic activity. The results demonstrate

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102

the applicability of quantitative, SNR enhanced and localized

13

C MRS for

large scale noninvasive investigation of the impact of dietary intake, physical exercise or pathology on fatty acid metabolism and related diagnosis. As a ubiquitous component of all animal tissues, cholesterol has vital structural roles in membranes and in lipid metabolism.

13

C MRS has been applied to

study the major classes of unsaturated and saturated fatty acids, like mono- and polyunsaturated fatty acids, ω-1, ω-2, ω-3, C2, C3, allylic and CH2 envelop. By applying our J-refocused 1H PRESS localized DEPT sequence combined with broadband decoupling, detection of cholesterol is reported for the first time noninvasively in human calf adipose tissue by

13

C MRS at 7T. Further

investigation could verify this assignment by cross-validation against mass spectrometry or magic angle spinning NMR spectroscopy based on tissue samples obtained by biopsy. Another potential application of J-refocused PRESS DEPT sequence is in

13

C infusion studies to investigate TCA and

neurotransmitter cycling. In conclusion, natural abundance 13C MRS has the potential of detecting lipid components from muscle, bone marrow and adipose tissue to study long-term and short-period whole body metabolism.

Appendix

103

Appendix Appendix 1 The complete 32-step phase cycle is listed as following: For the six pulses on the proton resonance: 90o :

(x, x, -x, -x, y, y, -y, -y, -x, -x, x, x, –y, -y, y, y, x, x, -x, -x, y, y, -y, -y, -x, -x, x, x, -y, -y, y, y)

o

180 :

(x, x, x, x, -x, -x, -x, -x, x, x, x, x, -x, -x, -x, -x, x, x, x, x, -x, -x, -x, -x, x, x, x, x, -x, -x, -x, -x)

o

90 :

(y, y, -y, -y, -x, -x, x, x, -y, -y, y, y, x, x, -x, -x, y, y, -y, -y, -x, -x, x, x, -y, -y, y, y, x, x, -x, -x)

o

180 :

(x, x, x, x, -x, -x, -x, -x, y, y, y, y, -y, -y, -y, -y, -x, -x, -x, -x, x, x, x, x, -y, -y, -y, -y, y, y, y, y)

180o :

(y, y, -y, -y, -x, -x, x, x, -y, -y, y, y, x, x, -x, -x, y, y, -y, -y, -x, -x, x, x, -y, -y, y, y, x, x, -x, -x)

β:

(y, -y, -y, y, -x, x, x, -x, -y, y, y, -y, x, -x, -x, x, y, -y, -y, y, -x, x, x, -x, -y, y, y, -y, x, -x, -x, x)

For the two pulses on the carbon resonance: 90o :

(x, x, -x, -x, y, y, -y, -y, -x, -x, x, x, –y, -y, y, y, x, x, -x, -x, y, y, -y, -y, -x, -x, x, x, -y, -y, y, y)

o

180 :

(y, y, -y, -y, -x, -x, x, x, -y, -y, y, y, x, x, -x, -x, y, y, -y, -y, -x, -x, x, x, -y, -y, y, y, x, x, -x, -x)

Appendix

104 For acquisition : (x, -x, -x, x, y, -y, -y, y, x, -x, -x, x, y, -y, -y, y, x, -x, -x, x, y, -y, -y, y, x, -x, -x, x, y, -y, -y, y)

Appendix

105

Appendix 2 Product operator calculation of J-refocused proton PRESS DEPT: J-refocused proton PRESS: πJ II

o

TE1

×2I1z I2z

90 I x 2 I1z  → -I1y  → -I1y cos(πJ II

TE1 TE1 )+2I1x I 2z sin(πJ II ) 2 2

TE πJ IS 1 ×2I1z Sz 2

→ TE1 TE1 TE1 TE1 TE1 TE1 TE1 TE1 )cos(πJ IS )+2I1x Sz cos(πJ II )sin(πJ IS )+2I1x I 2z sin(πJ II )cos(πJ IS )+4I1y I 2z Sz sin(πJ II )sin(πJ Is ) 2 2 2 2 2 2 2 2

-I1y cos(πJ II o

180 I x  →

I1y cos(πJ II πJ II

TE1 TE1 TE1 TE1 TE1 TE1 TE1 TE1 )cos(πJ IS )+2I1x Sz cos(πJ II )sin(πJ IS )-2I1x I 2z sin(πJ II )cos(πJ IS )+4I1y I 2z Sz sin(πJ II )sin(πJ Is ) 2 2 2 2 2 2 2 2

TE1 ×2I1z I2z 2

→ TE1 TE1 TE1 TE1 TE1 )cos(πJ IS )-2I1x I 2z cos(πJ II )sin(πJ II )cos(πJ IS ) 2 2 2 2 2 TE TE TE TE TE1 1 1 1 1 +2I1x Sz cos 2 (πJ II )sin(πJ IS )+4I1y I 2z Sz cos(πJ II )sin(πJ II )sin(πJ IS ) 2 2 2 2 2 TE1 TE1 TE1 TE1 TE1 -2I1x I 2z cos(πJ II )sin(πJ II )cos(πJ IS )-I1y sin 2 (πJ II )cos(πJ IS ) 2 2 2 2 2 TE1 TE1 TE1 TE1 TE1 +4I1y I 2z Sz cos(πJ II )sin(πJ II )sin(πJ Is )-2I1x Sz sin 2 (πJ II )sin(πJ Is ) 2 2 2 2 2 TE1 TE1 TE1 TE1 =I1y cos(πJ II TE1 )cos(πJ IS )-2I1x I 2z sin(πJ II TE1 )cos(πJ IS )+2I1x Sz cos(πJ II TE1 )sin(πJ IS )+4I1y I 2z Sz sin(πJ II TE1 )sin(πJ IS ) 2 2 2 2 I1y cos 2 (πJ II

TE1

πJ IS

×2I1zSz

2  →

TE1 TE1 TE1 )-2I1x Sz cos(πJ II TE1 )cos(πJ IS )sin(πJ IS ) 2 2 2 TE TE TE1 1 1 -2I1x I 2z sin(πJ II TE1 )cos 2 (πJ IS )-4I1y I 2z Sz sin(πJ II TE1 )cos(πJ IS )sin(πJ IS ) 2 2 2 TE1 TE1 TE1 +2I1x Sz cos(πJ II TE1 )cos(πJ IS )sin(πJ IS )+I1y cos(πJ II TE1 )sin 2 (πJ IS ) 2 2 2 TE1 TE1 TE1 +4I1y I 2z Sz sin(πJ II TE1 )cos(πJ IS )sin(πJ IS )-2I1x I 2z sin(πJ II TE1 )sin 2 (πJ IS )=I1y cos(πJ II TE1 )-2I1x I 2z sin(πJ II TE1 ) 2 2 2 I1y cos(πJ II TE1 )cos 2 (πJ IS

90o I

y  → I1y cos(πJ II TE1 )+2I1z I 2x sin(πJ II TE1 )

πJ II

TE 2

×2I1z I2z

2  →

I1y cos(πJ II TE1 )cos(πJ II π J IS

TE 2 TE 2 TE 2 TE 2 ) )-2I1x I 2z cos(πJ II TE1 )sin(πJ II )+2I1z I 2x sin(πJ II TE1 )cos(πJ II )+I 2y sin(πJ II TE1 )sin(πJ II 2 2 2 2

TE2 ⋅2 I1 z S z 2

→ TE2 TE2 TE2 TE2 ) cos(π J IS ) − 2 I1x S z cos(π J II TE1 ) cos(π J II ) sin(π J IS ) 2 2 2 2 TE2 TE2 TE2 ) cos(π J IS ) − 4 I1 y I 2 z S z cos(π J II TE1 ) sin(π J II ) sin(π J IS −2 I1x I 2 z cos(π J II TE1 ) sin(π J II 2 2 2 TE2 TE2 TE2 +2 I1z I 2 x sin(π J II TE1 ) cos(π J II ) cos(π J IS ) + 4 I1z I 2 y S z sin(π J II TE1 ) cos(π J II ) sin(π J IS 2 2 2 TE2 TE2 TE2 TE2 + I 2 y sin(π J II TE1 ) sin(π J II ) cos(π J IS ) − 2 I 2 x S z sin(π J II TE1 ) sin(π J II ) sin(π J IS ) 2 2 2 2 I1 y cos(π J II TE1 ) cos(π J II

π J IS

TE2

TE2 ) 2 TE2 ) 2

⋅2 I 2 z S z

2  →

TE2 TE2 TE2 TE2 ) cos(π J IS ) − 2 I1x S z cos(π J II TE1 ) cos(π J II ) sin(π J IS ) 2 2 2 2 TE2 TE2 TE2 TE2 −2 I1x I 2 z cos(π J II TE1 ) sin(π J II ) cos(π J IS ) − 4 I1 y I 2 z S z cos(π J II TE1 ) sin(π J II ) sin(π J IS ) 2 2 2 2 TE2 TE TE TE2 TE2 2 2 ) cos 2 (π J IS ) + 4 I1z I 2 y S z sin(π J II TE1 ) cos(π J II ) cos(π J IS ) sin(π J IS ) +2 I1z I 2 x sin(π J II TE1 ) cos(π J II 2 2 2 2 2 TE2 TE2 TE2 TE2 TE2 ) ) cos(π J IS ) sin(π J IS ) − 2 I1z I 2 x sin(π J II TE1 ) cos(π J II ) sin 2 (π J IS +4 I1z I 2 y S z sin(π J II TE1 ) cos(π J II 2 2 2 2 2 TE2 TE2 TE2 TE2 TE2 + I 2 y sin(π J II TE1 ) sin(π J II ) cos 2 (π J IS ) − 2 I 2 x S z sin(π J II TE1 ) sin(π J II ) cos(π J IS ) sin(π J IS ) 2 2 2 2 2 TE2 TE2 TE2 TE2 TE2 ) −2 I 2 x S z sin(π J II TE1 ) sin(π J II ) cos(π J IS ) sin(π J IS ) − I 2 y sin(π J II TE1 ) sin(π J II ) sin 2 (π J IS 2 2 2 2 2 I1 y cos(π J II TE1 ) cos(π J II

Appendix

106 TE2 TE2 TE2 TE2 ) cos(π J IS ) − 2 I1x S z cos(π J II TE1 ) cos(π J II ) sin(π J IS ) 2 2 2 2 TE2 TE2 TE2 TE2 −2 I1x I 2 z cos(π J II TE1 ) sin(π J II ) cos(π J IS ) − 4 I1 y I 2 z S z cos(π J II TE1 ) sin(π J II ) sin(π J IS ) 2 2 2 2 TE2 TE2 ) cos(π J IS TE2 ) + 4 I1z I 2 y S z sin(π J II TE1 ) cos(π J II ) sin(π J IS TE2 ) +2 I1z I 2 x sin(π J II TE1 ) cos(π J II 2 2 TE2 TE2 + I 2 y sin(π J II TE1 ) sin(π J II ) cos(π J IS TE2 ) − 2 I 2 x S z sin(π J II TE1 ) sin(π J II ) sin(π J IS TE2 ) 2 2 I1 y cos(π J II TE1 ) cos(π J II

o

180 I x  →

TE2 TE2 TE2 TE2 ) − 2 I1x S z cos(π J II TE1 ) cos(π J II ) sin(π J IS ) ) cos(π J IS 2 2 2 2 TE2 TE2 TE2 TE2 +2 I1x I 2 z cos(π J II TE1 ) sin(π J II ) cos(π J IS ) − 4 I1 y I 2 z S z cos(π J II TE1 ) sin(π J II ) sin(π J IS ) 2 2 2 2 TE2 TE2 ) sin(π J IS TE2 ) −2 I1z I 2 x sin(π J II TE1 ) cos(π J II ) cos(π J IS TE2 ) + 4 I1z I 2 y S z sin(π J II TE1 ) cos(π J II 2 2 TE2 TE2 − I 2 y sin(π J II TE1 ) sin(π J II ) cos(π J IS TE2 ) − 2 I 2 x S z sin(π J II TE1 ) sin(π J II ) sin(π J IS TE2 ) 2 2 − I1 y cos(π J II TE1 ) cos(π J II

π J II

TE2

⋅2 I1 z I 2 z

2 →

TE2 TE2 TE2 TE2 TE2 ) sin(π J II ) cos(π J IS ) ) cos(π J IS ) + 2 I1x I 2 z cos(π J II TE1 ) cos(π J II 2 2 2 2 2 TE2 TE2 TE2 TE2 TE2 2 −2 I1x S z cos(π J II TE1 ) cos (π J II ) sin(π J IS ) − 4 I1 y I 2 z S z cos(π J II TE1 ) cos(π J II ) sin(π J II ) sin(π J IS ) 2 2 2 2 2 TE2 TE2 TE2 TE2 TE2 2 ) cos(π J IS ) + I1 y cos(π J II TE1 ) sin (π J II ) cos(π J IS ) +2 I1x I 2 z cos(π J II TE1 ) cos(π J II ) sin(π J II 2 2 2 2 2 TE2 TE2 TE2 TE2 TE2 −4 I1 y I 2 z S z cos(π J II TE1 ) cos(π J II ) sin(π J II ) sin(π J IS ) + 2 I1x S z cos(π J II TE1 ) sin 2 (π J II ) sin(π J IS ) 2 2 2 2 2 TE TE TE 2 2 2 ) sin(π J II ) cos(π J IS TE2 ) −2 I1z I 2 x sin(π J II TE1 ) cos 2 (π J II ) cos(π J IS TE2 ) − I 2 y sin(π J II TE1 ) cos(π J II 2 2 2 TE2 TE2 TE2 2 +4 I1z I 2 y S z sin(π J II TE1 ) cos (π J II ) sin(π J IS TE2 ) − 2 I 2 x S z sin(π J II TE1 ) cos(π J II ) sin(π J II ) sin(π J IS TE2 ) 2 2 2 TE2 TE2 TE2 2 ) cos(π J IS TE2 ) + 2 I1z I 2 x sin(π J II TE1 ) sin (π J II ) cos(π J IS TE2 ) − I 2 y sin(π J II TE1 ) cos(π J II ) sin(π J II 2 2 2 TE2 TE2 TE2 −2 I 2 x S z sin(π J II TE1 ) cos(π J II ) sin(π J II ) sin(π J IS TE2 ) − 4 I1z I 2 y S z sin(π J II TE1 ) sin 2 (π J II ) sin(π J IS TE2 ) 2 2 2 TE2 TE2 ) + 2 I1x I 2 z cos(π J II TE1 ) sin(π J II TE2 ) cos(π J IS ) = − I1 y cos(π J II TE1 ) cos(π J II TE2 ) cos(π J IS 2 2 TE2 TE2 −2 I1x S z cos(π J II TE1 ) cos(π J II TE2 ) sin(π J IS ) − 4 I1 y I 2 z S z cos(π J II TE1 ) sin(π J II TE2 ) sin(π J IS ) 2 2 − I 2 y sin(π J II TE1 ) sin(π J II TE2 ) cos(π J IS TE2 ) − 2 I1z I 2 x sin(π J II TE1 ) cos(π J II TE2 ) cos(π J IS TE2 ) − I1 y cos(π J II TE1 ) cos 2 (π J II

−2 I 2 x S z sin(π J II TE1 ) sin(π J II TE2 ) sin(π J IS TE2 ) + 4 I1z I 2 y S z sin(π J II TE1 ) cos(π J II TE2 ) sin(π J IS TE2 ) π J IS

TE2

⋅2 I1 z S z

2  →

TE2 TE2 TE2 ) sin(π J IS ) ) + 2 I1x S z cos(π J II TE1 ) cos(π J II TE2 ) cos(π J IS 2 2 2 TE2 TE2 TE2 2 +2 I1x I 2 z cos(π J II TE1 ) sin(π J II TE2 ) cos (π J IS ) + 4 I1 y I 2 z S z cos(π J II TE1 ) sin(π J II TE2 ) cos(π J IS ) sin(π J IS ) 2 2 2 TE2 TE2 TE2 2 ) − I1 y cos(π J II TE1 ) cos(π J II TE2 ) sin (π J IS ) −2 I1x S z cos(π J II TE1 ) cos(π J II TE2 ) cos(π J IS ) sin(π J IS 2 2 2 TE2 TE2 TE2 −4 I1 y I 2 z S z cos(π J II TE1 ) sin(π J II TE2 ) cos(π J IS ) sin(π J IS ) + 2 I1x I 2 z cos(π J II TE1 ) sin(π J II TE2 ) sin 2 (π J IS ) 2 2 2 TE2 TE2 ) + 2 I 2 x S z sin(π J II TE1 ) sin(π J II TE2 ) cos(π J IS TE2 ) sin(π J IS ) − I 2 y sin(π J II TE1 ) sin(π J II TE2 ) cos(π J IS TE2 ) cos(π J IS 2 2 TE2 TE2 −2 I1z I 2 x sin(π J II TE1 ) cos(π J II TE2 ) cos(π J IS TE2 ) cos(π J IS ) − 4 I1z I 2 y S z sin(π J II TE1 ) cos(π J II TE2 ) cos(π J IS TE2 ) sin(π J IS ) 2 2 TE2 TE2 ) sin(π J IS TE2 ) − I 2 y sin(π J II TE1 ) sin(π J II TE2 ) sin(π J IS ) sin(π J IS TE2 ) −2 I 2 x S z sin(π J II TE1 ) sin(π J II TE2 ) cos(π J IS 2 2 TE2 TE2 +4 I1z I 2 y S z sin(π J II TE1 ) cos(π J II TE2 ) cos(π J IS ) sin(π J IS TE2 ) − 2 I1z I 2 x sin(π J II TE1 ) cos(π J II TE2 ) sin(π J IS ) sin(π J IS TE2 ) 2 2 − I1 y cos(π J II TE1 ) cos(π J II TE2 ) cos 2 (π J IS

Appendix π J IS

TE2

⋅2 I 2 z S z

2 →

TE2 TE TE ) + 2 I1x S z cos(π J II TE1 )cos(π J II TE2 )cos(π J IS 2 )sin(π J IS 2 ) 2 2 2 TE TE TE +2 I1x I 2 z cos(π J II TE1 )sin(π J II TE2 )cos 2 (π J IS 2 ) + 4 I1 y I 2 z S z cos(π J II TE1 )sin(π J II TE2 ) cos(π J IS 2 )sin(π J IS 2 ) 2 2 2 TE TE TE −2 I1x S z cos(π J II TE1 )cos(π J II TE2 )cos(π J IS 2 )sin(π J IS 2 ) − I1 y cos(π J II TE1 )cos(π J II TE2 )sin 2 (π J IS 2 ) 2 2 2 TE TE TE −4 I1 y I 2 z S z cos(π J II TE1 )sin(π J II TE2 )cos(π J IS 2 )sin(π J IS 2 ) + 2 I1x I 2 z cos(π J II TE1 )sin(π J II TE2 )sin 2 (π J IS 2 ) 2 2 2 TE TE TE − I 2 y sin(π J II TE1 )sin(π J II TE2 )cos(π J IS TE2 )cos 2 (π J IS 2 ) + 2 I 2 x S z sin(π J II TE1 )sin(π J II TE2 )cos(π J IS TE2 )cos(π J IS 2 )sin(π J IS 2 ) 2 2 2 TE TE TE +2 I 2 x S z sin(π J II TE1 )sin(π J II TE2 )cos(π J IS TE2 )cos(π J IS 2 )sin(π J IS 2 ) + I 2 y sin(π J II TE1 )sin(π J II TE2 )cos(π J IS TE2 )sin 2 (π J IS 2 ) 2 2 2 TE TE TE −2 I1z I 2 x sin(π J II TE1 )cos(π J II TE2 )cos(π J IS TE2 )cos 2 (π J IS 2 ) − 4 I1z I 2 y S z sin(π J II TE1 )cos(π J II TE2 )cos(π J IS TE2 )cos(π J IS 2 )sin(π J IS 2 ) 2 2 2 TE TE TE −4 I1z I 2 y S z sin(π J II TE1 )cos(π J II TE2 )cos(π J IS TE2 )cos(π J IS 2 )sin(π J IS 2 ) + 2 I1z I 2 x sin(π J II TE1 )cos(π J II TE2 )cos(π J IS TE2 )sin 2 (π J IS 2 ) 2 2 2 TE TE TE −2 I 2 x S z sin(π J II TE1 )sin(π J II TE2 )cos 2 (π J IS 2 )sin(π J IS TE2 ) − I 2 y sin(π J II TE1 )sin(π J II TE2 )cos(π J IS 2 )sin(π J IS 2 )sin(π J IS TE2 ) 2 2 2 TE TE TE − I 2 y sin(π J II TE1 )sin(π J II TE2 )cos(π J IS 2 )sin(π J IS 2 )sin(π J IS TE2 ) + 2 I 2 x S z sin(π J II TE1 )sin(π J II TE2 )sin 2 (π J IS 2 )sin(π J IS TE2 ) 2 2 2 TE TE TE +4 I1z I 2 y S z sin(π J II TE1 )cos(π J II TE2 )cos 2 (π J IS 2 )sin(π J IS TE2 ) − 2 I1z I 2 x sin(π J II TE1 )cos(π J II TE2 )cos(π J IS 2 )sin(π J IS 2 )sin(π J IS TE2 ) 2 2 2 TE TE TE −2 I1z I 2 x sin(π J II TE1 )cos(π J II TE2 )cos(π J IS 2 )sin(π J IS 2 )sin(π J IS TE2 ) − 4 I1z I 2 y S z sin(π J II TE1 )cos(π J II TE2 )sin 2 (π J IS 2 )sin(π J IS TE2 ) 2 2 2 = − I1 y cos(π J II TE1 )cos(π J II TE2 ) + 2 I1x I 2 z cos(π J II TE1 )sin(π J II TE2 ) − I1 y cos(π J II TE1 )cos(π J II TE2 )cos 2 (π J IS

− I 2 y sin(π J II TE1 )sin(π J II TE2 )(cos 2 (π J IS TE2 ) + sin 2 (π J IS TE2 )) − 2 I1z I 2 x sin(π J II TE1 )cos(π J II TE2 )(cos 2 (π J IS TE2 ) + sin 2 (π J IS TE2 )) +2 I 2 x S z sin(π J II TE1 )sin(π J II TE2 )cos(π J IS TE2 )sin(π J IS TE2 ) − 2 I 2 x S z sin(π J II TE1 )sin(π J II TE2 )cos(π J IS TE2 )sin(π J IS TE2 ) −4 I1z I 2 y S z sin(π J II TE1 )cos(π J II TE2 )cos(π J IS TE2 )sin(π J IS TE2 ) + 4 I1z I 2 y S z sin(π J II TE1 )cos(π J II TE2 )cos(π J IS TE2 )sin(π J IS TE2 ) = − I1 y cos(π J II TE1 )cos(π J II TE2 ) + 2 I1x I 2 z cos(π J II TE1 )sin(π J II TE2 ) − I 2 y sin(π J II TE1 )sin(π J II TE2 ) − 2 I1z I 2 x sin(π J II TE1 )cos(π J II TE2 )

107

Appendix

108 DEPT: π J II τ ⋅2 I1 z I 2 z − I1 y  → − I1 y cos(π J II τ ) + 2 I1x I 2 z sin(π J II τ )

π J ISτ ⋅2 I1 z S z   → − I1 y cos(π J II τ ) cos(π J ISτ ) + 2 I1x S z cos(π J II τ )sin(π J ISτ ) + 2 I1x I 2 z sin(π J II τ ) cos(π J ISτ ) + 4 I1 y I 2 z S z sin(π J II τ )sin(π J ISτ ) 180o I

y  → − I1 y cos(π J II τ ) cos(π J ISτ ) − 2 I1x S z cos(π J II τ )sin(π J ISτ ) + 2 I1x I 2 z sin(π J II τ ) cos(π J ISτ ) − 4 I1 y I 2 z S z sin(π J II τ )sin(π J ISτ )

90 S x  → − I1 y cos(π J II τ ) cos(π J ISτ ) + 2 I1x S y cos(π J II τ )sin(π J ISτ ) + 2 I1x I 2 z sin(π J II τ ) cos(π J ISτ ) + 4 I1 y I 2 z S y sin(π J II τ )sin(π J ISτ ) o

detectable ⇒ 2 I1x S y cos(π J II τ )sin(π J ISτ ) + 4 I1 y I 2 z S y sin(π J II τ )sin(π J ISτ ) π J ISτ ⋅2 I1 z S z   → 2 I1x S y cos(π J II τ )sin(π J ISτ ) + 4 I1 y I 2 z S y sin(π J II τ )sin(π J ISτ ) π J ISτ ⋅2 I 2 z S z  → 2 I1x S y cos(π J II τ ) cos(π J ISτ )sin(π J ISτ ) − 4 I1x I 2 z S x cos(π J II τ )sin 2 (π J ISτ )

+4 I1 y I 2 z S y sin(π J II τ ) cos(π J ISτ )sin(π J ISτ ) − 2 I1 y S x sin(π J II τ )sin 2 (π J ISτ ) π J II τ ⋅2 I1 z I 2 z  → 2 I1x S y cos 2 (π J II τ ) cos(π J ISτ )sin(π J ISτ ) + 4 I1 y I 2 z S y cos(π J II τ )sin(π J II τ ) cos(π J ISτ )sin(π J ISτ )

−4 I1x I 2 z S x cos 2 (π J II τ )sin 2 (π J ISτ ) − 2 I1 y S x cos(π J II τ )sin(π J II τ )sin 2 (π J ISτ ) +4 I1 y I 2 z S y cos(π J II τ )sin(π J II τ ) cos(π J ISτ )sin(π J ISτ ) − 2 I1x S y sin 2 (π J II τ ) cos(π J ISτ ) sin(π J ISτ ) −2 I1 y S x cos(π J II τ )sin(π J II τ )sin 2 (π J ISτ ) + 4 I1x I 2 z S x sin 2 (π J II τ )sin 2 (π J ISτ ) 2 I1x S y cos(π J II 2τ ) cos(π J ISτ )sin(π J ISτ ) + 4 I1 y I 2 z S y sin(π J II 2τ ) cos(π J ISτ )sin(π J ISτ ) −4 I1x I 2 z S x cos(π J II 2τ )sin 2 (π J ISτ ) − 2 I1 y S x sin(π J II 2τ )sin 2 (π J ISτ ) 180o S

y  → 2 I1x S y cos(π J II 2τ ) cos(π J ISτ )sin(π J ISτ ) + 4 I1 y I 2 z S y sin(π J II 2τ ) cos(π J ISτ )sin(π J ISτ )

+4 I1x I 2 z S x cos(π J II 2τ )sin 2 (π J ISτ ) + 2 I1 y S x sin(π J II 2τ )sin 2 (π J ISτ ) βI

y 2( I1x cos β − I1z sin β ) S y cos(π J II 2τ ) cos(π J ISτ )sin(π J ISτ ) + 4 I1 y ( I 2 z cos β + I 2 x sin β ) S y sin(π J II 2τ ) cos(π J ISτ )sin(π J ISτ ) →

+4( I1x cos β − I1z sin β )( I 2 z cos β + I 2 x sin β ) S x cos(π J II 2τ ) sin 2 (π J ISτ ) + 2 I1 y S x sin(π J II 2τ )sin 2 (π J ISτ ) detectable ⇒ −2 I1z S y sin β cos(π J II 2τ ) cos(π J ISτ )sin(π J ISτ ) − 4 I1z I 2 z S x cos β sin β cos(π J II 2τ )sin 2 (π J ISτ ) π J ISτ ⋅2 I1 z S z   → −2 I1z S y sin β cos(π J II 2τ ) cos 2 (π J ISτ )sin(π J ISτ ) + S x sin β cos(π J II 2τ ) cos(π J ISτ )sin 2 (π J ISτ )

−4 I1z I 2 z S x cos β sin β cos(π J II 2τ ) cos(π J ISτ )sin 2 (π J ISτ ) − 2 I 2 z S y cos β sin β cos(π J II 2τ )sin 3 (π J ISτ ) π J ISτ ⋅2 I 2 z S z  → −2 I1z S y sin β cos(π J II 2τ ) cos3 (π J ISτ )sin(π J ISτ ) + 4 I1z I 2 z S x sin β cos(π J II 2τ ) cos 2 (π J ISτ )sin 2 (π J ISτ )

+ S x sin β cos(π J II 2τ ) cos 2 (π J ISτ )sin 2 (π J ISτ ) + 2 I 2 z S y sin β cos(π J II 2τ ) cos(π J ISτ )sin 3 (π J ISτ ) −4 I1z I 2 z S x cos β sin β cos(π J II 2τ ) cos 2 (π J ISτ )sin 2 (π J ISτ ) − 2 I1z S y cos β sin β cos(π J II 2τ ) cos(π J ISτ )sin 3 (π J ISτ ) −2 I 2 z S y cos β sin β cos(π J II 2τ ) cos(π J ISτ )sin 3 (π J ISτ ) + S x cos β sin β cos(π J II 2τ ) sin 4 (π J ISτ ) J II τ ⋅2 I1 z I 2 z π →

−2 I1z S y sin β cos(π J II 2τ ) cos3 (π J ISτ )sin(π J ISτ ) + 4 I1z I 2 z S x sin β cos(π J II 2τ ) cos 2 (π J ISτ )sin 2 (π J ISτ ) + S x sin β cos(π J II 2τ ) cos 2 (π J ISτ )sin 2 (π J ISτ ) + 2 I 2 z S y sin β cos(π J II 2τ ) cos(π J ISτ )sin 3 (π J ISτ ) −4 I1z I 2 z S x cos β sin β cos(π J II 2τ ) cos 2 (π J ISτ )sin 2 (π J ISτ ) − 2 I1z S y cos β sin β cos(π J II 2τ ) cos(π J ISτ )sin 3 (π J ISτ ) −2 I 2 z S y cos β sin β cos(π J II 2τ ) cos(π J ISτ )sin 3 (π J ISτ ) + S x cos β sin β cos(π J II 2τ ) sin 4 (π J ISτ ) τ=

1

2 J IS  → cos(π J ISτ ) = 0 sin(π J ISτ ) =1

= S x cos β sin β cos(π J II 2τ )

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Acknowledgements

Acknowledgements Here I would like to gratefully acknowledge all the people who have supported me, helped me and walked besides me during these five years. I am indebted to many people for making the time working on my PhD at the Institute for Biomedical Engineering in Switzerland an unforgettable experience. First of all, I wish to express my sincere gratitude to Prof. Dr. Peter Boesiger for providing us with the truly invaluable and unique research atmosphere. To work at IBT has been a real pleasure and fortune to me. You have oriented and inspired me with your incredible ability to balance each aspect in academic research and cooperation.

Apart from the effort of myself, the achievement of this project depends largely on the valuable supervision of Dr. Anke Henning, who has always been patient and encouraging in time of new ideas and difficulties. Your extensive insights and your hard work have set me an example. You made me feel a friend besides a supervisor, which I appreciate from my heart.

Sincere thanks to Prof. Markus Rudin for the co-examiner and interdisciplinary contact during course and with Hoenggerberg group. I would also like to thank Matteo Pavan and Dr. Susanne Heinzer for the big support and help of transferring the ERETIC technique from both hardware and software aspects.

124

Acknowledgements

I would also like to convey thanks to Dr. Roger Lüchinger, Urs Sturzenegger, Michael Wyss and Dr. Dieter Meier for solving computer, scanning and my special hardware problems.

A big thank you to our secretary Marianne Berg for helping me out from the plentiful insurance forms in German and for organizing our social activities. Furthermore, I am really grateful to the spectroscopy group. Most of all, I want to thank my first office mate Dr. Susanne Heinzer for helping me get into the life in Switzerland easily at the beginning and Alexander Fuchs for answering my questions patiently and discussing with me frequently. It has been a wonderful pleasure to work and have fun together with Andreas Hock, Arianne Fillmer, Thomas Kirchner, Niklaus Zölch, Milan Scheidegger and Erin MacMillan. Both scientific inspiration and personal joy have been shared among this enjoyable group. You are not only colleagues, but have also been part of my life which I will fondly remember for the rest of my life.

Many thanks go to all the other doctoral and post doctoral students at IBT for the advices, lunch chatting and leisure activities after work. I would like to thank my office mates during the last several months Jelena Curcic, Robert Vorburger and Tobias Hahn for sharing and easing the last period of this path with the hearty laughter. I would like to give a big thank you to Dr. Carolin Reischauer for spreading out your warm and positive energy. A special thank you to Dr. Bertram Wilm for making me feel more at home at the beginning with the Chinese related conversations. I have greatly enjoyed the close contact with Dr. Andrea Rutz and Dr. Marco Piccirelli. A big thank you to the whole MR group for such a pleasant working atmosphere: Benjamin Dietrich,

Acknowledgements

125

Christian Stoeck, Dr. Christoph Barmet, Jan Paska, Johanna Vannesjö, Johannes Schmidt, Prof. Dr. Klaas Prüssmann, Kilian Weiss, Lars Kasper, Dr. Hendrik Mandelkow, Dr. Martin Bührer, Max Häberlin, Dr. Robert Manka, Rodolf Fischer, Prof. Dr. Sebastian Kozerke, Verena Knobloch, Dr. Viton Vitanis and whoever I have forgotten to mention but accompanied me by one or another occasional contact.

I take immense pleasure thanking my lovely living mates Critina Zunzunegui, Katrin Orlowski, Katrin Merz, Nadja Olini and Ola Hola for the comforting and sharing of our sadness and happiness on the special journey of life.

I am also indebted to the Chinese friends who have completed my abroad life besides doctoral research. I want to thank Dr. Jianhua Feng, Dr Jianyong Wen and Dr. Fang Sun for taking care of me like a family and the valuable support and advices. I am very grateful to my wonderful friends Dr. Ningbo Yu, Jingyi Rao, Xiang Li, Kai Li, Yufei Li, Dr. Xiaojun Wang, Dr. Yaowu Xing, Dr. Weixun Yan, Yijian Gong, Jialin Zhang, Lujing Su and many companions from the Chinese associations like Dr. Ming Wu, Dr. Zhonghai Li, Dr. Wei Fan, Dr. Wei Hu, Xuebin Fu, Cen Nan and whom I really don’t have enough space to name one by one. I appreciate all the great times we experienced together no matter in badminton, skiing, hiking, travelling, activities organizing or just pure drinking. I thank Shan Yang for being part of my life during the last period of PhD. Finally, yet importantly, I wish to express my deepest thanks to my beloved parents and the whole big family for their infinite and unreserved understanding and support. I want to thank my mother for her amazingly optimistic mind to

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Acknowledgements

take me out of any pressure and turndown. I want to thank my father for always being there with insightful advices when I am confused. （最后，也是最重要的，感谢我挚爱的父母以及庞大的家族，感谢你们毫无保留 的理解和支持。妈妈那惊人的乐观态度总能陪伴我走出压力和挫折，而在我迷茫 困惑的时候，适时出现的是爸爸和他富有洞察力的建议。）

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Curriculum Vitae I was born on June 13th 1981 as daughter of Baoyan Chen and Danhong Li and grew up in Shenyang (Liaoning Province, China). In fall 2000, I started my studies in the institute of Biomedical Engineering at the Zhejiang University in Hangzhou (Zhejiang Province, China). After graduated with a Bachelor degree in fall 2005, I continued my Master study in Biomedical Engineering at the Zhejiang University with specialization in Bioinformatics. During my studies I worked as software engineer in the lab of Prof. Huilong Duan and thus got acquainted with medical devices programming. In 2007, I graduated with a Master thesis on Classification Analysis Methods for Microarray Data. Since September 2007 I joined the Biophysics group of Professor Peter Boesiger at the Institute for Biomedical Engineering of the university and ETH Zurich. My research was focused on methodology of carbon-13 magnetic resonance spectroscopy. During my leisure time, I enjoy traveling, reading, swimming and outdoor activities.

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Publications

129

List of Publications X. Chen, Peter Boesiger, Anke Henning, Quantification of fatty acids in human calf adipose tissue and muscle by

13

C MRS using J-refocused PRESS DEPT

and ERETIC, Proc Intl Soc Mag Reson Med 2013. X. Chen, Peter Boesiger, Anke Henning, J-refocused 1H PRESS DEPT for localized

13

C MR Spectroscopy, NMR Biomed., 2013 Feb 25 (Epub ahead of

proint). X. Chen, Peter Boesiger, Anke Henning, Cholesterol detection with Jrefocused 1H PRESS DEPT by in vivo

13

C MRS, oral presentation, Proc

ESMRMB 2012. X. Chen, Peter Boesiger, Anke Henning, Noninvasive characterization of fatty acids in calf adipose tissue and muscle by 13C MR spectroscopy, SSBE 2012. X. Chen, Peter Boesiger, Anke Henning, J-refocused 1H PRESS combined with DEPT for localized saturated fatty acids detection by in vivo

13

C MRS,

Proc Intl Soc Mag Reson Med 2012. X. Chen, Peter Boesiger, Anke Henning, Cholesterol detection in adipose tissue by natural abundance in vivo 13C MRS at 7T, Proc Intl Soc Mag Reson Med 2012. X. Chen, Peter Boesiger, Anke Henning et al, Optically transmitted and inductively coupled Electric Reference to Access In Vivo Concentrations for

Publications

130 quantitative

proton-decoupled

13

C

Magnetic

Resonance

Spectroscopy,

Magnetic Resonance in Medicine Journal 67: 1-7 (2012). X. Chen, Peter Boesiger, Anke Henning et al, Quantification of unsaturated fatty acids by PRESS localized-DEPT enhanced

13

C MRS and ERETIC in

human skeletal muscle, oral presentation, Proc ESMRMB 2011. X. Chen, Peter Boesiger, Anke Henning et al, Combination of DEPT and PRESS for detection of UFA in posterior and medial thigh muscle by 13C MRS at 7T, oral presentation, Proc Intl Soc Mag Reson Med 2011. X. Chen, Anke Henning, P. Boesiger et al, Muscle group specific quantification of unsaturated fatty acids by localized DEPT-enhanced 13C MRS and ERETIC, oral presentation, BMT 2010. X. Chen, Anke Henning, P. Boesiger et al, ERETIC-based glycogen quantification using SNR-enhanced and localized 13C MRS, Proc Intl Soc Mag Reson Med 2010. X. Chen, Anke Henning, P. Boesiger et al, Muscle group specific quantification of unsaturated fatty acids by localized DEPT-enhanced 13C MRS and ERETIC, Proc Intl Soc Mag Reson Med 2010.