Phase 1 Study: Detection of Brown Adipose Tissue in Normal Volunteers Using a 3 Tesla (3T) Magnetic Resonance Imaging System and Hyperpolarized (HP) Xenon Gas
Phase: PHASE1
Conditions: Obesity
Sponsor: University of North Carolina, Chapel Hill

 
Phase 1 Study: Detection of Brown Adipose Tissue in Normal 
Volunteers Using a 3 Tesla (3T) Magnetic Resonance Imaging System 
and Hyperpolarized (HP) Xenon Gas 
 
 
NCT number NCT02220426 
Document Date 10/09/2014 
 

SPECIFIC AIMS: 
In spite of the many efforts to raise awareness on the health 
complications of obesity, our obesity epidemic keeps growing, adding an immense cost to our 
health care system. At a fundamental level obesity is the result of an imbalance between energy 
intake and energy expenditure1. The latter is very difficult to quantify and recent work suggests 
that in humans it is partially modulated by the activity of Brown Adipose Tissue (BAT)2,3. BAT is 
a tissue with the unique capacity to regulate energy expenditure by a process called 
thermogenesis during which this tissue burns fat to produce heat4. By preventing excessive 
accumulation of triglyceride in non-adipose tissues such as muscle and liver, this tissue also 
helps to protect against insulin resistance and type 2 diabetes5,6.  
As ways to harness this tissue to address obesity and diabetes are being investigated, its non 
invasive detection in humans remains an unmet need. The current gold standard for the 
detection of BAT activity is FDG-PET. Unfortunately this technique has obvious limitations: 1) It 
cannot be used for longitudinal studies in healthy subjects due to concerns over radiation 
exposure, 2) It is not reproducible, and 3) It fails in detecting BAT in the target population, i.e. in 
overweight and obese subjects3. Other imaging modalities like MRI and CT have been tried, but 
aside from lacking specificity and their inability to detect BAT activity, they all share the same 
insensitivity to the lipid rich BAT present in overweight and obese subjects.  
The goal of the proposed application is to provide a non-invasive imaging tool that is suitable to 
detect mass and metabolic activity of BAT in both lean and obese human subjects.  Our group 
was the first to demonstrate the use of hyperpolarized xenon gas MRI to specifically and directly 
detect BAT mass and thermogenic activity in lean and obese mouse phenotypes, with 
unprecedented sensitivity in the latter. The objective of the current proposal is to validate this 
technique in humans. To this end we propose the following aims:  
Specific Aim 1: Demonstrate BAT mass detection in human subjects. For this aim we will 
implement hyperpolarized xenon imaging on our clinical scanner and we will compare the 
images obtained before stimulation of BAT thermogenesis with those obtained during 
stimulation of BAT thermogenesis. Our working hypothesis is that, during stimulation of BAT 
thermogenesis the lipophilic gas will accumulate in the highly perfused BAT tissue enabling us 
to clearly delineate it. 
Specific Aim 2: Demonstrate detection of BAT thermogenesis in humans. For this aim we will 
measure BAT temperature by measuring the chemical shift of lipid-dissolved xenon. Our 
working hypothesis is that, during stimulation of BAT thermogenic activity, changes in BAT 
temperature would be detected as a shift in the resonance frequency of lipid-dissolved xenon.  
By the end of the proposed project we expect to have demonstrated the feasibility of HP xenon 

MRI detection of human BAT mass and thermogenic activity.  These studies will also provide 
preliminary data for powering a future clinical trial with which we aim to investigate the incidence 
and activity of this tissue in the human population. 
SIGNIFICANCE: 
Most of our knowledge on BAT comes from animal studies8. In small 
animals we know that BAT prevents obesity and improves and reverses insulin insensitivity6. In 
humans the role of this tissue is less clear9,10. This is because human BAT studies have been 
limited mainly to retrospective studies on cancer patients due to lack of safe and reliable 
detection techniques to study the small quantities of diffusely distributed BAT depots present in 
humans in vivo11. Fusion FDG-PET/CT remains the gold-standard imaging modality for non-
invasive studies of metabolically active BAT. However, this technique is intrinsically insensitive 
to BAT metabolism as only small amounts (less than 10%) of glucose are taken up by activated 
BAT11. The rest of the substrate (more than 90%) is made by fatty acids released from 
intracellular lipid storages10. As such, the activity of this tissue is clearly underestimated by 
FDG-PET scans7,12. This is especially true for overweight and obese subjects, the target 
population for BAT3,10, in whom the intracellular lipid storage is more abundant (brown fat cells 
are swollen with fat) than in lean subjects. Therefore, this tissue relies even less on circulating 
glucose for thermogenesis, further limiting its detectability by FDG-PET scans7,12,13. More 
importantly, the elevated risk of ionizing radiation and radionuclide tracer radiation exposure 
from these scans precludes repetitive BAT screening in healthy and young subjects, scans 
which are needed to correctly estimate the true prevalence of BAT, to determine the 
physiological relevance of BAT in humans14,15, and to test the efficacy of new anti-obesity 
treatments. With this proposal we aim to fulfill this need. HP xenon MRI is extremely sensitive to 
the more lipid rich and less thermogenically active BAT of obese phenotypes (see comparison 
with FDG-PET in Fig 1) and, by being non invasive, can be repeatedly used on healthy subjects 
to track BAT function over time. In addition it allows direct measurement of BAT cell 
temperature (Fig. 2) and therefore represents the only direct way we have to monitor BAT 
thermogenic activity.  
INNOVATION: 
MRI with hyperpolarized xenon-129 is a technique that is been used 
extensively in humans to detect lung ventilation function16. Here we propose for the first time to 
use hyperpolarized xenon gas MRI to detect BAT. Xenon is an ideal BAT probe for several 
reasons: A) the solubility of xenon in fatty tissue is 10 times higher than in lean tissues or 
blood17. This means that circulating xenon naturally diffuses into BAT, enabling adequate SNR 
for imaging. B) Xenon is an ideal blood perfusion tracer, and therefore ideally suited to detect 
the increase in blood flow to BAT that occurs during stimulation of BAT thermogenesis. C) 

Because xenon is not intrinsic to biological tissue, HP 129Xe can produce background free maps 
of human BAT. D) The temperature sensitivity of lipid-dissolved xenon will enable us to directly 
measure BAT temperature and monitor thermogenic activity in real time.  
This research is innovative as it uses an entirely different approach to investigate BAT. To our 
knowledge we are the very first group to use hyperpolarized xenon gas to detect BAT mass and 
to have demonstrated HP xenon MR thermometry of fatty tissues.   
APPROACH: Aim1: Demonstrate BAT mass detection in human subjects.  
Our extensive preliminary studies in rodents have shown that, upon stimulation of BAT 
thermogenesis, an enhancement of more than 10-fold of the MR signal from xenon dissolved in 
BAT is observed. This enhancement is such that a background free map of this tissue can be 
obtained in just few seconds (Fig. 1). In addition, when compared to FDG-PET images of BAT, 
hyperpolarized xenon gas images were able to clearly show BAT in both  lean and obese mice 
and, in the latter, with superior sensitivity (Fig.1b-d).   
 
 
The broad aim of this proposal is to validate this completely new approach to detect BAT in 
humans.  To this end we will perform HP 129Xe-MRI of BAT on a 3T clinical magnet using a 
dual tuned xenon/1H surface coil recently purchased for human BAT studies. The coil will be 
accurately located above the supraclavicular area to increase sensitivity to the xenon signal 
dissolved in the tissue beneath. Conventional 1H MRI, which will provide anatomical reference 
images, will be followed by HP 129Xe spectroscopy and imaging scans, similar to those used in 
our rodent studies. Before the acquisition of the xenon images, the subject will be asked to 
inhale a 1L bag of 100% hyperpolarized and enriched xenon from a Tedlar bag and to hold his 
breath for about 15-20s. During this time hyperpolarized xenon gas will diffuse from the lung 
airspaces to BAT in a perfusion-based manner. At the end of the 10 s from the beginning of gas 
inhalation we will trigger the acquisition of the MR image, which will last for about 10s. The 
Fig. 1  Detection of interscapular 
BAT by hyperpolarized xenon gas 
MRI (a-b) and FDG-PET (c-d) in a 
lean (a-c) and obese (b-d) animal. 
Hyperpolarized xenon gas is 
clearly more sensitive to the lipid-
rich BAT of obese animals (b) 
than FDG-PET. In d) glucose 
uptake by BAT is substantially 
reduced, making it difficult to 
differentiate BAT from the 
surrounding tissue.  

same protocol will be followed during stimulation of BAT thermogenesis, which will be achieved 
by an MR compatible water cooling-blanket (~15oC circulating water temperature), which will be 
positioned on the lower abdomen/upper leg part of the subjects. The ability to detect BAT by 
hyperpolarized xenon gas relies on the enhancement in blood flow to this tissue that occurs 
during stimulation of thermogenic activity. The magnitude of this enhancement seems to be the 
same, irrespective of the species and of the modality of stimulation of BAT thermogenesis (NE 
injection of mild cold exposure). Therefore we expect to detect a similar enhancement in the MR 
signal from xenon dissolved in human BAT. In addition, the use of enriched xenon versus 
naturally abundant xenon will compensate for the possible loss in xenon polarization during 
blood transit time. Pre and post stimulation scans will be compared with Matlab to detect 
differences of more than 5% in the intensity of the dissolved phase xenon images. Detectable 
BAT volume will be calculated as the sum of pixels whose intensity rose by more than 5% 
during cold stimulation multiplied by the pixel volume (1 mm3). Data will be analyzed using JMP 
Pro-9.0.0 software (SAS Institute). All P values presented will be two-tailed, and values less 
than 0.05 will be considered to indicate statistical significance. According to previous studies 
done in human brain18,19 and considering the increase in blood perfusion that occurs in BAT 
during cold stimulation20 as well as the higher solubility of xenon in fatty tissues, we expect to be 
able to obtain images with a resolution of 0.3x0.3cm, high enough to differentiate BAT from the 
surrounding tissues.  
Aim 2:  Demonstrate detection of BAT thermogenesis in humans.  
Dynamic spectroscopy data have shown the possibility to track BAT temperature over time by 
measurement of the chemical shift of lipid-dissolved xenon. We demonstrated for the first time 
MR thermometry by hyperpolarized xenon gas. This method is based on the temperature 
dependence of the chemical shift of lipid dissolved xenon, which we measured for the first time 
in vivo to be -0.2ppm/oC,(Fig 2.a), i.e. 20 times larger than that of water protons currently used 
for MR thermometry in lean tissues. This higher sensitivity of lipid-dissolved xenon to 
temperature, coupled with the lower gyromagnetic ration of xenon (which makes xenon less 
sensitive to possible magnetic field in homogeneities present in fatty tissues), allowed us to 
detect BAT thermogenic activity in mice (Fig.2.b). To this end we will acquire dynamic 
spectroscopy data from xenon dissolved in BAT during stimulation of BAT thermogenesis by 
mild cold exposure. During these scans we will track the resonance frequency of lipid-dissolved 
xenon to detect changes in BAT temperature during thermogenic activity. Although in humans 
we expect temperature changes less than 2 oC, the high sensitivity of xenon to temperature and 
the narrow line width can allow us to detect changes as small as 0.2oC.  

 
For these studies the subject will inhale 4 doses of 1L each of xenon during mild cold exposure, 
achieved with a water cooling blanket. After inhalation, the subject will be asked to hold his 
breath for 15-20 seconds, during which we will trigger the acquisition of xenon spectra. The 
spectra will then be fitted with homemade Matlab routines to extract lipid-dissolved xenon 
resonance frequency that will be used to detect any change in BAT temperature.  
The change in BAT temperature will then be analyzed as function of BMI to establish whether or 
not there is an association between BMI and BAT temperature increase (Pearson correlation 
and Spearman correlation coefficient calculation).  
This feasibility study will allow us to estimate the standard deviation for the increase in signal 
from xenon dissolved in BAT and for the increase in BAT temperature that will then be used to 
power future trials to investigate the incident of this tissue in the healthy human population and 
the association between BMI and BAT thermogenic activity. For these aims we plan on enrolling 
a total of 24 healthy subjects (12 for aim 1 and 12 for aim 2) with a BMI ranging between 18 and 
28. Twelve is the minimum sample size recommended in the absence of prior information on 
which to base our sample size calculation21. With 12 subjects per group we should be able to 
obtain positive results (with a 95% confidence interval) on at least 9 subjects if the true 
prevalence of BAT is 90%22 (expected prevalence), and on at least 3 subjects if the true 
prevalence of BAT is much smaller and close to 50% (estimated by FDG-PET scans7,23).  
 
By the end of the study we expect to have fully assessed the potential of HP xenon to detect 
BAT mass and activity and to have developed an imaging protocol that can be used in studies 
aimed to investigate the prevalence of this tissue in humans, its connection with other metabolic 
diseases such as diabetes, and to study the response to treatments aimed to counteract obesity 
by increasing BAT mass and or function. 
 
 
 
Fig.2 a) Linear temperature shift of the 
lipi-dissolved xenon frequency as 
acquired in vivo in an obese mouse. 
b. Thermogenic activity as detected by 
hyperpolarized xenon gas. The graph 
shows the resonance frequency shift of 
lipid-dissolved xenon and the 
corresponding shift in BAT temperature 
right after injection of NE.  

References 
 
1 
Himms-Hagen, J. Brown adipose tissue thermogenesis, energy balance, and obesity. 
Canadian Journal of Biochemistry and Cell Biology 62, 610-617, doi:10.1139/o84-081 
(1984). 
2 
Wang, J. et al. Ablation of LGR4 promotes energy expenditure by driving white-to-
brown fat switch. Nat Cell Biol 15, 1455-1463, doi:10.1038/ncb2867 
http://www.nature.com/ncb/journal/v15/n12/abs/ncb2867.html 
- 
supplementary-information 
(2013). 
3 
Vijgen, G. H. E. J. et al. Brown Adipose Tissue in Morbidly Obese Subjects. PLoS ONE 
6, e17247, doi:10.1371/journal.pone.0017247 (2011). 
4 
Foster, D. O. Quantitative contribution of brown adipose tissue thermogenesis to overall 
metabolism. Can J Biochem Cell Biol. 62, 618-622 (1984). 
5 
Silva, F. J. et al. Metabolically Active Human Brown Adipose Tissue Derived Stem 
Cells. STEM CELLS 32, 572-581, doi:10.1002/stem.1595 (2014). 
6 
Gunawardana, S. C. & Piston, D. W. Reversal of Type 1 Diabetes in Mice by Brown 
Adipose Tissue Transplant. Diabetes 61, 674-682, doi:10.2337/db11-0510 (2012). 
7 
Lee, P., Greenfield, J. R., Ho, K. K. Y. & Fulham, M. J. A critical appraisal of the 
prevalence and metabolic significance of brown adipose tissue in adult humans. 
American Journal of Physiology - Endocrinology And Metabolism 299, E601-E606, 
doi:10.1152/ajpendo.00298.2010 (2010). 
8 
CANNON, B. & NEDERGAARD, J. Brown Adipose Tissue: Function and Physiological 
Significance. Physiological Reviews 84, 277-359, doi:10.1152/physrev.00015.2003 
(2004). 
9 
Nedergaard, J., Bengtsson, T. & Cannon, B. Unexpected evidence for active brown 
adipose tissue in adult humans. American Journal of Physiology - Endocrinology And 
Metabolism 293, E444-E452, doi:10.1152/ajpendo.00691.2006 (2007). 
10 
Ouellet, V. r. et al. Brown adipose tissue oxidative metabolism contributes to energy 
expenditure during acute cold exposure in humans. The Journal of Clinical Investigation 
122, 545-552 (2012). 
11 
Nedergaard, J. & Cannon, B. The Changed Metabolic World with Human Brown 
Adipose Tissue: Therapeutic Visions. Cell Metabolism 11, 268-272. 
12 
Lee, P. et al. High Prevalence of Brown Adipose Tissue in Adult Humans. Journal of 
Clinical Endocrinology & Metabolism 96, 2450-2455, doi:10.1210/jc.2011-0487 (2011). 
13 
Cannon, B. & Nedergaard, J. Yes, even human brown fat is on fire! The Journal of 
Clinical Investigation 122, 486-489, doi:10.1172/jci60941 (2012). 
14 
Brix, G. et al. Radiation Exposure of Patients Undergoing Whole-Body Dual-Modality 
18F-FDG PET/CT Examinations. J Nucl Med 46, 608-613 (2005). 
15 
Huang, B., Law, M. W.-M. & Khong, P.-L. Whole-Body PET/CT Scanning: Estimation 
of Radiation Dose and Cancer Risk1. Radiology 251, 166-174 (2009). 
16 
Fain, S. B. et al. Functional lung imaging using hyperpolarized gas MRI. Journal of 
Magnetic Resonance Imaging 25, 910-923, doi:10.1002/jmri.20876 (2007). 
17 
Cherubini, A. & Bifone, A. Hyperpolarised xenon in biology. Progress in Nuclear 
Magnetic Resonance Spectroscopy 42, 1-30, doi:citeulike-article-id:4275368 
doi: 10.1016/S0079-6565(02)00052-3 (2003). 

18 
Kilian, W., Seifert, F. & Rinneberg, H. Dynamic NMR spectroscopy of hyperpolarized 
129Xe in human brain analyzed by an uptake model. Magnetic Resonance in Medicine 
51, 843-847, doi:10.1002/mrm.10726 (2004). 
19 
Kilian, W., Seifert, F. & Rinneberg, H. in International Society of Magnetic Resonance in 
Medicine    (Honolulu, Hawaii, 2002). 
20 
Orava, J. et al. Different Metabolic Responses of Human Brown Adipose Tissue to 
Activation by Cold and Insulin. Cell Metabolism 14, 272-279 (2011). 
21 
Julious, S. A. Sample size of 12 per group rule of thumb for a pilot study. 
Pharmaceutical Statistics 4, 287-291, doi:10.1002/pst.185 (2005). 
22 
van Marken Lichtenbelt, W. D. et al. Cold-Activated Brown Adipose Tissue in Healthy 
Men. 
New 
England 
Journal 
of 
Medicine 
360, 
1500-1508, 
doi:doi:10.1056/NEJMoa0808718 (2009). 
23 
Saito, M. et al. High Incidence of Metabolically Active Brown Adipose Tissue in Healthy 
Adult Humans: Effects of Cold Exposure and Adiposity. Diabetes 58, 1526-1531, 
doi:10.2337/db09-0530 (2009). 
 
 

The present project will be HIPAA compliant and will be performed under a protocol approved 
by the UNC Chapel Hill Institutional Review Board (IRB) and under an FDA IND approval.  
 
A. Proposed involvement of human subjects 
All research involving human subjects will be carried out at the University of North Carolina at 
Chapel Hill. All MRI experiments will take place in the Biomedical Research Imaging Center, 
where the PI’s lab for hyperpolarized 129Xe gas MRI  and the 3T Siemens MRI scanner is 
located. A medical technician, with knowledge and skills consistent with those provided in the 
American Heart Association’s Advanced Cardiac Life Support training will observe the subjects 
whenever they are breathing xenon gas. Supplemental oxygen (adjusted to flow at 4 
liters/minute), suction source, bag valve mask, ventilation device and an airway support device 
will be available in the MR scanner room. During scanning, a MRI compatible pulse oximeter will 
be used to monitor the subject’s heart rate and blood oxygenation. 
A total of 24 healthy subjects, without any metabolic disease, of ages 18 to 40 will be recruited 
to participate. All subjects will undergo hyperpolarized xenon-129 MR. Healthy subjects are 
considered those who have never being diagnosed with metabolic diseases, and have no 
history of any chronic lung disease such as asthma, COPD, or CF. 
Since our aim is devoted to test the efficacy of the new methodology for the detection of BAT, 
there is no justification to scan children under 18 years old. Pregnant or lactating women will be 
excluded due to potential health risks. All subjects must be able to undergo an MRI examination 
(e.g., no MRI incompatible implanted devices or other MR contra-indications).  
Dr. Branca will be present during all hyperpolarized xenon-129 MR scans.  
 
Practice xenon inhalation for subjects before placement into the MR scanner:  
Individual subject sensitivity to xenon inhalation will be practiced before MR imaging. The 
subject will be directed to breathe 1-L of undiluted xenon (natural isotopic abundance) and to 
hold his breath for 15-20s. Oxygen saturation, heart rate, and blood pressure will be 
continuously recorded. If the effects of xenon inhalation are deemed intolerable by the subject 
the amount of xenon will be lowered. If the blood oxygen saturation drops significantly (5%) 
below the subject’s baseline and/or the pulse rate rises by 40 bpm, the practice session will be 
interrupted to explore the cause. If the cause cannot be identified and rectified, the subject will 
be excluded from the study. 
 
Hyperpolarized xenon-129 MRI:  

Subjects will be positioned supine within the MR scanner. The dual tuned xenon/proton surface 
coil will be positioned above the supraclavicular area. After breathing normally for a few 
minutes, the subject will exhale completely to residual volume, and then inhale a 1-L volume of 
undiluted hyperpolarized xenon-129, followed by a breath-hold lasting from 10-15 s during and 
after which imaging will be performed. The subject will then exhale fully and breathe normal air. 
Xenon doses will be administered no less than 15 minutes apart. During the MRI exam, oxygen 
saturation, heart rate, and blood pressure will be continuously recorded. These parameters, 
along with respiratory rate and subjective symptoms, will also be assessed after each dose. 
Immediately after inhalation, the subject will also be asked to report any symptoms or effects 
from xenon.   
 
Source of material  
The research material obtained from the subjects will be the results of MR studies. This 
information will be used solely for research. 
 
Subject recruitment 
Subjects will be recruited through advertisements, posters, and/or flyers in clinics and/or 
newspapers. The research procedure will be fully described and potential volunteers will be 
offered the opportunity to ask questions and read the protocol and consent form. No individual 
in a supervisory relationship with an investigator will be a participant (employees of an 
investigator, students in classes with the investigator, or those pursuing a degree under the 
investigator’s direction). Candidates for participation will be provided with our informed consent 
form and given sufficient time to read it. A physician or a nurse coordinator will verbally review 
the major points and ask for questions. Those who wish to participate will give their consent by 
signing the form. 
 
B. Potential risks 
B1. Risks of the MR scan:  
For subjects that are properly screened for the specific contraindications to MR imaging, it is 
generally believed at this time that the health risks associated with the short-term exposure to 
radiowaves and high magnetic fields as required for an MR imaging examination are 
insignificant. 
 
B2. Risks of xenon inhalation:  

Xenon is a general anesthetic when breathed continuously for long periods of time (minutes) at 
concentrations greater than 50% (anesthetic dose ~70%). There is also the theoretical risk that 
HP 129Xe, if given in sufficiently large volumes in forms that do not include oxygen, could cause 
or exacerbate hypoxia in a subject. However, a recent clinical trial performed at Duke University, 
were the same protocol was used to image lung ventilation function, has not caused hypoxia but 
only mild, transient, xenon-related symptoms, consistent with its known anesthetic properties, 
such as dizziness, paresthesia, euphoria, and hypoesthesia that have resolved soon after 
discontinuing gas administration(1). 
 
B3. Purity of laser polarized xenon:  
The purity of the laser polarized gases is very high because: 
1. The initial source of xenon has a purity of at least 99.99%; 
2. Only nitrogen is added to the gas before laser polarization; 
3. Only room air and oxygen are added to the gas after laser polarization; 
4. All chambers into which the gases are transferred are always pumped to a vacuum before 
gas is transferred into them; and 
5. The rubidium vapor used during the polarization process is removed by allowing the polarized 
gas to pass through either a Teflon filter or a cold trap. 
Before xenon is laser polarized, the polarization chamber and connecting tubes are pumped to 
a vacuum to ensure there are no contaminants that would interfere with the polarization 
process. For noble-gas laser polarization, xenon, together with nitrogen, which acts as a buffer 
gas, are then added to the polarization cell. The xenon gas is supplied from specially obtained 
gas cylinders with a minimum purity of 99.99%. Already present in the polarization chamber is a 
thin layer of solid rubidium. During the laser polarization, the polarization chamber is heated in 
order to create an appropriate vapor pressure of rubidium. The laser then excites the rubidium 
which then exchanges its energy with the noble gas (xenon) to create the laser polarized noble 
gas. Once polarized, the noble gas is allowed to expand out of the polarization chamber into a 
glass storage cell, through a Teflon filter or cold trap that removes any remnant rubidium. Before 
the storage cell is used, however, it is pumped out to a vacuum to remove any gaseous 
contaminants that might reduce the lifetime of the polarization. Polarized xenon-129 gas is then 
transferred into a previously evacuated plastic bag. The subject then breathes the undiluted 
xenon gas through a disposable one-way valve and a connecting tube to the plastic bag. 
 
C. Adequacy of protection against risks  

 
C1. MR Safety Considerations:  
All potential subjects will be familiarized with the MR scanning procedure and will be excluded if 
they are bothered by the associated noise and confined spaces, or based on the standard 
exclusion criteria on our MR screening forms. All MR pulse sequences will conform to standard 
safety guidelines in terms of radio-frequency (RF) power deposition and rate of change of 
magnetic field (dB/dt). 
 
C2. Xenon Inhalation:  
The xenon dosing strategy is designed to minimize the previously described xenon physiological 
effects from the inhalation. In addition, a licensed medical professional will observe the subject 
whenever they are breathing xenon, and MR compatible monitoring equipment and 
supplemental oxygen and other equipment for artificial ventilation will be available. In addition, 
there will be practice sessions to assess the subject’s response to the gas mixture. If there is 
any significant discomfort by a subject, he/she will be excluded from the project. 
 
C3. Volunteer Confidentiality:  
All MRI images, as well as all records of research, will contain only a study code number and no 
subject identifying information. Research records will be kept in password protected computer 
files and will only be available to investigators. The key between subject name and study 
number will be kept on a flash drive that is stored in a locked file cabinet. 
 
D. Potential benefits of the proposed research to subjects and others and importance of 
the knowledge to be gained 
Subjects will receive no direct benefit from participating in this study. Nonetheless, the risks to 
the subjects from participating are very low as discussed above. This very low, but non-zero, 
risk to the study subjects is reasonable considering that success of proposed research will yield 
high societal benefit. Hyperpolarized-gas MRI has already shown substantial potential for 
providing medically-relevant information, unmatched by any other modality, about important 
lung diseases such as COPD and asthma. If successful, the proposed research will open a 
completely new venue for hyperpolarized xenon-129 MRI which, by virtue of producing well 
defined maps of human BAT and by measuring its temperature non invasively, can provide 
insights on the mass and function of this tissue in human population. This study will lay the 
foundation for future application of this technique for purposes such as (i) improving our 

understanding of the physiological relevance of this tissue in human populations and its 
connection with other metabolic diseases. (ii) monitoring BAT function in response to obesity 
interventions. 
 
E. Inclusion of Women and Minorities 
In keeping with the NIH policy to provide equal participation opportunities for women and for 
members of minority and ethnic groups, we will ensure that recruitment announcements are 
accessible to a diverse range of potential subjects, for instance by posting announcements at 
women’s health sites and in Spanish. 
No specific gender or racial or ethnic group will be excluded or favored. Based on the racial and 
ethnic composition estimates for the University of North Carolina at Chapel Hill area it is 
anticipated that roughly 10% of the study subjects will be from racial minorities and roughly 6% 
will be of Hispanic or Latino origin. We are aware of no evidence to suggest that the outcome of 
our evaluations of these new imaging methods would be affected by the racial or ethnic 
composition of the study population and thus, while we will take steps to ensure equal 
participation opportunities as discussed above, there is no scientific basis for targeting the 
enrollment of a specific group in numbers higher than those represented in our underlying 
population. 
 
F. Number Justification 
A total of 24 subjects will be enrolled in this study. 12 to complete specific aim 1 and 12 to 
complete specific aim 2. Twelve is the minimum sample size recommended in absence of prior 
information on which to base our sample size calculation(2). With 12 subjects per group we 
should be able to obtain positive results (with a 95% confidence interval) on at least 9 subjects if 
the true prevalence of BAT is 90%(3) (expected prevalence), and on at least 3 subjects if the 
true prevalence of BAT is much smaller and close to 50% (estimated by FDG-PET scans(4). 
 
1. 
B. Driehuys et al., Chronic Obstructive Pulmonary Disease: Safety and Tolerability of 
Hyperpolarized 129Xe MR Imaging in Healthy Volunteers and Patients. Radiology 262, 
279 (January 1, 2012, 2012). 
2. 
S. A. Julious, Sample size of 12 per group rule of thumb for a pilot study. 
Pharmaceutical Statistics 4, 287 (2005). 
3. 
W. D. van Marken Lichtenbelt et al., Cold-Activated Brown Adipose Tissue in Healthy 
Men. New England Journal of Medicine 360, 1500 (2009). 

4. 
M. Saito et al., High Incidence of Metabolically Active Brown Adipose Tissue in Healthy 
Adult Humans: Effects of Cold Exposure and Adiposity. Diabetes 58, 1526 (July 1, 2009, 
2009). 
 
 

Program Director/Principal Investigator (Last, First, Middle): 
Branca, Rosa, Tamara 
DETAILED BUDGET FOR INITIAL BUDGET PERIOD 
DIRECT COSTS ONLY 
FROM 
THROUGH 
06/01/2014 
05/31/2015 
   
  List PERSONNEL (Applicant organization only) 
  Use Cal, Acad, or Summer to Enter Months Devoted to Project 
  Enter Dollar Amounts Requested (omit cents) for Salary Requested and Fringe Benefits 
 
NAME 
ROLE ON 
PROJECT 
Cal. 
Mnths 
Acad. 
Mnths 
Summer 
Mnths 
INST.BASE 
SALARY 
SALARY 
REQUESTED 
FRINGE 
BENEFITS 
TOTAL 
 
PD/PI 
 
 
 
 
 
 
 
Research Assistant 
 
4 
4 
 
24,025 
7,600 
1,040 
8,640 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SUBTOTALS 
7,600 
1,040 
8,640 
CONSULTANT COSTS 
 
 
EQUIPMENT  (Itemize) 
 
 
SUPPLIES  (Itemize by category) 
3T scan time  
 
Enriched Xenon gas mixture 
Volunteer time  
19,968 
 
12,240 
 
1,440 
TRAVEL 
 
 
INPATIENT CARE COSTS 
 
 
 
OUTPATIENT CARE COSTS  
 
 
ALTERATIONS AND RENOVATIONS  (Itemize by category) 
 
 
OTHER EXPENSES  (Itemize by category) 
 
 
CONSORTIUM/CONTRACTUAL COSTS 
DIRECT COSTS 
 
SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) 
$ 
42,288 
CONSORTIUM/CONTRACTUAL COSTS 
FACILITIES AND ADMINISTRATIVE COSTS 
 
TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD   
$ 
42,288 
PHS 398 (Rev. 6/09) 
Page 1 
Form Page 4 

1
Budget Justification 
 
Personnel Justification: 
Zhang Le,Graduate Research Assistant (4 calendar months). The graduate Assistant will be 
involved in data collection and data analysis. 
 
Juanita Ramirez,Study Coordinator/Research (2 calendar months). She will be responsible 
for subject recruitment and screening, and will contribute to preparation and updating of human 
subject IRB protocols for this project. She will also attend the entire study session. She will be 
paid by the PI discretionary funds. 
 
Other Direct Costs  
-Scan time ($19,968) 
Imaging and spectroscopy experiments in humans will be performed in the Biomedical 
Research Imaging Center, in which the cost for 1h of scan time is set at $416. For each subject, 
the entire imaging session will last for about 2 hours. Therefore funding for a total of 48 hours of 
MRI scan time are requested.  (($416/hour scan time x 3 hours MRI session x 10subjects)  
-Subject study compensation ($1,920) 
Each subject will receive a compensation of $60 for the entire 2 hours imaging session. 
-Enriched Xenon gas mixture ($12,240)  
During the imaging study session the subject will receive two doses of 1L of HP Xenon, spaced 
40 minutes apart (40 minutes is the minimum time required to produce a 1L of hyperpolarized 
xenon gas). For the spectroscopy study the subject will receive a total of 4 0.5L of 
hyperpolarized xenon every 20 minutes. Funds are therefore requested for 4800L of 1% 
enriched 129Xe (quoted at $5,100 per 2000L cylinder by Linde specialty gas) that will lead to a 
total of 48L (2L per subject x 12 subjects) of enriched Xenon.  
The cost of the non-enriched xenon gas as well as the cost of the other gases needed for the 
xenon polarizer will be covered in its entirety by the PI funds. 
 

Program Director/Principal Investigator (Last, First, Middle): Branca, Rosa, Tamara  
PHS 398/2590 (Rev. 06/09) 
Page  1    
Biographical Sketch 
BIOGRAPHICAL SKETCH 
Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2. 
Follow this format for each person.  DO NOT EXCEED FOUR PAGES. 
 
NAME 
Branca, Rosa Tamara 
POSITION TITLE 
Assistant Professor 
eRA COMMONS USER NAME (credential, e.g., agency login) 
RTBRANCA 
EDUCATION/TRAINING  (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and 
residency training if applicable.) 
INSTITUTION AND LOCATION 
DEGREE 
(if applicable) 
MM/YY 
FIELD OF STUDY 
La Sapienza University of Rome, Italy 
BS 
05/2002 
Physics 
La Sapienza University of Rome, Italy 
PhD 
06/2006 
BioPhysics 
Duke University, US 
PostDoc 
2006-2009 
Chemistry-Biochemistry 
 
 
 
 
 
 
 
 
A. Personal Statement 
The goal of our proposed research is to validate the use of hyperpolarized xenon gas MRI for the detection of 
human BAT mass and function. My graduate research training was largely on Magnetic Resonance Spin 
Dynamics, with an emphasis on nonlinear effects, under the guidance of Dr. Warren S. Warren, first at 
Princeton University, and then at Duke University. I have been trained in hyperpolarized gas imaging during my 
postdoctoral years at Duke University, when I developed a unique imaging method based on the use of 
hyperpolarized gas and super paramagnetic iron oxide contrast agents to detect lung nodules with high 
specificity and sensitivity, which I have carried through to a funded R01 grant (R01 CA142842). The success of 
my work and my ability to conduct research independently was a major factor in my promotion to the rank of 
Assistant Research Professor in Chemistry at Duke.  Two years ago, while still at Duke, I received an R21 
grant to develop an MR-based method to detect BAT mass and function. During this time I discovered the 
possibility to detect this tissue using hyperpolarized gas. This methodology, for which an invention disclosure 
has been filed, can overcome major limitation of 1H MR and PET based methods such as partial volume 
effects. More interestingly, this methodology seems to be particularly suited to detect BAT in obese individuals, 
where brown adipocytes lose their characteristic morphology and become similar to white adipocytes. After 
moving to UNC at the beginning of the year, I continued my work on BAT imaging using hyperpolarized xenon 
gas MRI.  To this end I used most of my startup funds to purchase a gas polarizer and xenon coils, with which 
we have obtained our preliminary results in mice and rats. A surface xenon coil for the 3T, that will be used for 
both rodents and humans, has also been purchased under the R21 grant and an IND submission has been 
filed to FDA to perform these studies in humans.  
We are the first group to propose and use hyperpolarized xenon gas to image brown adipose tissue.   My 
research experience in BAT and nuclear magnetic resonance make me a unique leader of the proposed 
project. I also believe that my research will seed a much broader interest in the application of hyperpolarized 
gas imaging at UNC. 
Positions and Honors 
Positions: 
 
2002-2006 Graduate Research Assistant Dept. of BioPhysics, “La Sapienza”, Rome, Italy 
2003-2006 Visiting Graduate Student, Dept. of Chemistry, Princeton University, Princeton, NJ  
2006-2009 Post-doctoral Associate, Center for Molecular and Biomolecular Imaging, Duke University, 
Durham,NC 
2009-2011 Assistant Research Professor, Dept. of Chemistry, Duke University, Durham, NC 

Program Director/Principal Investigator (Last, First, Middle): Branca, Rosa, Tamara  
PHS 398/2590 (Rev. 06/09) 
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Biographical Sketch 
2012-       Assistant Professor, Dept. of Physics and Astronomy & Biomedical Research Imaging Center 
University of North Carolina at Chapel Hill, Chapel Hill, NC  
 
Honors: 
1996-2000 Undergraduate student grant, “La Sapienza” University, Rome, Italy 
2000 Teaching Assistant, Physic Department, “La Sapienza” University, Rome, Italy 
2002 “Summa cum laude”, BA Degree in Physics, “La Sapienza” University, Rome, Italy 
2002 Research grant, “La Sapienza” University, Rome, Italy 
2002 Doctoral research grant in Biophysics, “La Sapienza” University, Rome, Italy 
2004 Student Travel Stipend Award, Experimental Nuclear Magnetic Resonance Conference   
2004 Student Travel Stipend Award, International Society of Magnetic Resonance 
2004 Best poster presentation award for imaging in-vivo NMR, International Society of Magnetic Resonance 
2005 Student Travel Stipend Award, Experimental Nuclear Magnetic Resonance Conference   
2005 Student Travel Stipend Award, Magnetic Resonance in Medicine   
2005 Marie Curie grant, Experimental European Nuclear Magnetic Resonance Conference  
2007 Student Travel Stipend Award, Experimental Nuclear Magnetic Resonance Conference   
2007 Student Travel Stipend Award, Magnetic Resonance in Medicine   
2008 Student Travel Stipend Award, World Molecular Imaging Conference 
 
. 
C. Selected Peer-reviewed Publications 
 Most relevant to the current application 
 
1. RT Branca, T He, L Zhang, C Floyd, M Freeman, C White,  “Detection of Brown Adipose Tissue Mass and 
Thermogenic Activity by Hyperpolarized Xenon MRI”, PNAS (under review).  
2. RT Branca, WS Warren, A Khanna, S Degan, K Ugurbil, E Auerbach, R Maronpot, “In Vivo Noninvasive 
Detection of Human Brown Adipose Tissue through Non-Linear MRI”, PlosOne 8(9) (2013). 
3. A. Khanna, R.T. Branca, “Detecting brown adipose tissue activity with BOLD MRI in mice”, Magn. Reson. 
in Medicine, Journal of Magnetic Resonance in Medicine, 68(4) 2012.PMID:  22231619 
4. R.T. Branca, W.S. Warren, "In vivo brown adipose tissue detection and characterization using water-lipid 
intermolecular zero-quantum coherences", Magn Reson Med, 65 (2), 313-9 (2011). PMID: 20939093 
5. R.T. Branca, W.S. Warren, "In vivo NMR detection of diet induced changes in adipose tissue composition", 
J Lipid Res, 52 (4), 833-9 (2011). PMID: 21270099 
6. R.T. Branca, Z.I. Cleveland, B. Fubara, C. Kumar, C. Leuschner, R.R. Maronpot, W.S. Warren, B. 
Driehuys, “Molecular MRI for sensitive and specific detection of lung metastases”, PNAS 107(8):3693-7. 
PMID:  20142483 
 
Additional recent publications of importance to the field (in chronological order) 
 
7. R.T. Branca, E.R. Jenista, W.S. Warren, "Inhomogeneity-free heteronuclear iMQC", J Magn Reson, 209 
(2), 347-51 (2011). PMID: 21316278 
8. R.T. Branca, “MRI using intermolecular multiple-quantum coherences”, Methods Mol Biol. 771:241-52 
(2011). PMID: 21874482 
9. E.R. Jenista, R.T. Branca, W.S. Warren, "Absolute temperature imaging using intermolecular multiple 
quantum MRI", Int J Hyperthermia, 26 (7), 725-34 (2010) PMID: 20849265 
10. E.R. Jenista, G. Galiana, R.T. Branca, P.S. Yarmolenko, A.M. Stokes, M. W. Dewhirst, W.S. Warren, 
“Application of mixed spin iMQCs for temperature and chemical-selective imaging”, J. Magn. Reson. 204 
(2), 208-18 (2010).  PMID: 20303808 
11. W.S. Warren, E.R. Jenista, R.T. Branca, “Increasing hyperpolarized spin lifetimes through true singlet 
eigenstates”, Science 323(5922), 1711-1714 (2009). PMID: 19325112 

Program Director/Principal Investigator (Last, First, Middle): Branca, Rosa, Tamara  
PHS 398/2590 (Rev. 06/09) 
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Biographical Sketch 
12. R.T. Branca, W. S. Warren, “Solvent suppression without crosspeak attenuation in iZQC experiments”, 
Chem. Phys. Lett., 470(4-6), 325-331 (2009). 
13. R.T. Branca, Y.  M. Chen, V. Mouraviev, G. Galiana, E. Jenista, C. Kumar, C. Leuschner, W. S. Warren, 
“iDQC anisotropy map imaging for tumor tissue characterization in vivo”, Magn. Reson. in Med. 61(4), 937 - 
943 (2009). PMID: 19215050 
14. G. Galiana, R.T. Branca, E.R. Jenista, and W.S. Warren, “Accurate Temperature Imaging Based on  
Intermolecular Coherences in Magnetic Resonance,” Science 322(5900), 421-424 (2008). PMID: 
18927389 
15. R.T. Branca, G. Galiana, W. S. Warren, “Enhanced Nonlinear Magnetic Resonance signals via Square 
Wave Dipolar Fields”, J. Chem. Phys. 129, 054502 (2008). PMID: 18698909 
 
D. Research Support 
Ongoing Research Support 
 
R21 DK090758                (role: PI) 09/20/2010–08/31/2013 
Novel magnetic resonance approach to detect BAT distribution and temperature. 
The goal of this project is to develop an MR-based method to detect both BAT mass and activity in rodents  
that can be easily translated to humans.  
 
R01 CA142842-01A1                (role: PI) 
04/01/2010–01/31/2015 
Sensitive and specific molecular imaging of pulmonary nodules. 
The goal of this project is to fully develop and characterize a method that combines hyperpolarized gas MRI 
with superparamagnetic contrast agent for the detection of lung metastases 
 
Completed Research Support 
(Pilot ½ Funding) Duke Cancer Center 
Branca (Investigator)  
06/01/2008–12/31/2008 
(Pilot ½ Funding) (5U24 CA092656-08) Small Animal Imaging Resource Program  Johnson (PI)  
  
Scanned breast tumor-bearing mice after they received an injection of our contrast agent LHRH-SPION. We 
utilized 10 breast tumor mice that had already been imaged with other modalities (CT and PET) and showed 
signs of metastatic cancer cells in lungs.  
Role: Investigator