Randomized Cross-over Trial of Oral L-Glutamine vs Maltodextrin on Mitochondrial Function in Chronic Kidney Disease
Phase: PHASE2
Conditions: Cardiovascular Disease, Sarcopenia, Endothelial Dysfunction, Muscle Mitochondrial Function, Kidney Disease
Sponsor: University of Washington

 
 
 
 
 
 
Randomized controlled trial of glutamine in chronic kidney disease 
 
 
 
Sponsored by: 
 
National Institute of Diabetes and Digestive and Kidney Diseases 
R01DK045462 
K23DK099442-01 
 
 
Investigators 
Jonathan Himmelfarb, MD 
Kidney Research Institute 
University of Washington 
Seattle, WA USA 
 
Michael Goligorsky 
Medicine – Renal Research Institute 
New York Medical College 
Valhalla, NY, USA 
 
Bob Roshanravan, MD MSPH MS 
Kidney Research Institute 
University of Washington 
 
Bryan Kestenbaum, MD MS 
Kidney Research Institute 
University of Washington 
 
Collaborators: 
Thomas R. Ziegler, MD 
Division of Endocrinology, Metabolism and Lipids 
Director, Emory Center for Clinical and Molecular Nutrition 
Atlanta Clinical and Translational Sciences Institute (ACTSI) 
Atlanta, GA 
 
 
 
 
 
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TABLE OF CONTENTS 
 
 
LIST OF ABBREVIATIONS AND ACRONYMS ..............................................................iv 
SCHEMA ...............................................................................................................................v-vi 
 
1.0 
INTRODUCTION............................................................................................................... 1 
1.1 Background and Prior Research ......................................................................................... 1 
1.2 Rationale ............................................................................................................................ 2 
2.0 
STUDY OBJECTIVES AND DESIGN ............................................................................. 2 
2.1 Primary Objectives............................................................................................................. 2 
2.2 Secondary Objectives......................................................................................................... 3 
2.3 Study Design ...................................................................................................................... 3 
3.0 
STUDY POPULATION ..................................................................................................... 3 
3.1 Inclusion Criteria ............................................................................................................... 3 
3.2 Exclusion Criteria .............................................................................................................. 4 
3.3 Recruitment Process........................................................................................................... 4 
3.4 Co-Enrollment Guidelines ................................................................................................. 5 
3.5 Participant Retention .......................................................................................................... 5 
3.6 Participant Withdrawal ...................................................................................................... 5 
4.0 
STUDY TREATMENT/PRODUCT/INTERVENTION ................................................. 6 
4.1 Treatment/Product/Intervention Regimen(s) ..................................................................... 6 
4.2 Treatment/Product/Intervention Supply and Accountability ............................................. 6 
4.3 Adherence Assessment ...................................................................................................... 6 
4.4 Toxicity Management ........................................................................................................ 6 
4.5 Clinical Management of Pregnancy ................................................................................... 6 
4.6 Concomitant Medications .................................................................................................. 7 
5.0 
STUDY PROCEDURES .................................................................................................... 8 
6.0 
SAFETY MONITORING AND ADVERSE EVENT REPORTING .......................... 12 
6.1 Safety Monitoring ............................................................................................................ 12 
6.2 Adverse Event Definitions and Reporting Requirements ................................................ 12 
6.2.1 Adverse Event ........................................................................................................... 12 
6.2.2 Serious Adverse Event .............................................................................................. 13 
6.2.3 Adverse Event Reporting .......................................................................................... 14 
7.0 
STATISTICAL CONSIDERATIONS ............................................................................ 14 
7.1 Review of Study Design .................................................................................................. 14 
7.2 Endpoints ......................................................................................................................... 14 
7.2.1 Primary Endpoints .................................................................................................... 15 
7.2.2 Secondary Endpoints ................................................................................................ 15 
7.3 Accrual, Follow-up, and Sample Size.............................................................................. 16 
7.4 Random Assignment / Study Arm Assignment ............................................................... 16 
7.5 Blinding............................................................................................................................ 16 
7.6 Data Analysis ................................................................................................................... 16 
7.6.1 Primary Analyses ...................................................................................................... 16 
7.6.2 Secondary Analyses .................................................................................................. 17 

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8.0 
HUMAN SUBJECTS CONSIDERATIONS .................................................................. 17 
8.1 Ethical Review ................................................................................................................. 17 
8.2 Informed Consent............................................................................................................. 17 
8.3 Risks ................................................................................................................................. 17 
8.4 Benefits ............................................................................................................................ 18 
8.5 Incentives ......................................................................................................................... 18 
8.6 Confidentiality ................................................................................................................. 18 
8.7 Study Discontinuation ...................................................................................................... 19 
9.0 
LABORATORY SPECIMENS AND BIOHAZARD CONTAINMENT .................... 19 
9.1 Local Laboratory Specimens ........................................................................................... 19 
9.2 Reference Laboratory Specimens .................................................................................... 19 
9.3 Specimen Storage and Possible Future Research Testing ............................................... 19 
10.0 
REFERENCES ............................................................................................................... 20 

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Randomized controlled trial of glutamine in chronic kidney disease 
 
LIST OF ABBREVIATIONS AND ACRONYMS 
 
 
AE 
 
 
Adverse Event 
CKD  
 
Chronic kidney disease 
EAE 
 
 
Expedited Adverse Event 
EC 
 
 
ethics committee 
FDA  
 
(United States) Food and Drug Administration 
GCP 
 
 
Good Clinical Practices 
IRB 
 
 
Institutional Review Board 
LDMS  
 
Laboratory Data Management System 
LL 
 
 
Local Laboratory 
MRS/OS 
 
31P Magnetic resonance spectroscopy and optical spectroscopy 
NIDDK 
 
United States) National Institute of Diabetes and Digestive and Kidney 
NIH 
 
 
(United States) National Institutes of Health 
PAT 
 
 
Pulse amplitude tonometry 
ROC  
 
Regulatory Operations Center 
SAE 
 
 
Serious Adverse Event 
SDMC  
 
Statistical and Data Management Center 
SUSAR 
 
Suspected Unexpected Serious Adverse Reaction 
SOP 
 
 
Standard Operating Procedures 
SSP 
 
 
Study Specific Procedures  
TCA  
 
Tricarboxylic acid (Krebs) cycle 
 
 
 
 
 
 
 
 

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Randomized controlled trial of glutamine in chronic kidney disease 
 
 
SCHEMA 
 
Purpose:  
Study the impact of oral glutamine supplementation on mitochondrial 
function and endothelial cell function measured by 31P MRS/OS among 
persons with moderate-severe CKD.  
 
Design: 
Randomized, placebo controlled cross-over trial 
 
 
Study Population: 
Moderate-severe CKD with eGFR by CKD (EPI equation)  ≤ 60ml/min/1.73m2 
 
Study Size: 
20 
 
Treatment Regimen:  - Oral glutamine 0.4gm/kg/day 
 
 
 
- Placebo 
 
Study Duration: 
1 year 
 
Primary Objectives:  
• To test if glutamine supplementation improves mitochondrial function and vascular function 
as measured by 31P MRS/OS. 
 
Secondary Objectives: 
• To test the effects of oral glutamine supplementation on metabolic response profile. 
• To test if glutamine supplementation reduces inflammatory and oxidative stress biomarkers. 
• To test if glutamine improves objective isometric muscle fatigue. 
• To test if glutamine supplementation reduces proteinuria (albuminuria). 
• To test if glutamine supplementation reduces 24 hour urine urea nitrogen. 
 
Study Sites: 
University of Washington: 
 
University of Washington Medical Center Translational Center for Metabolic Imaging 
 
Kidney Research Institute: 
o KRI at Harborview Medical Center 
o KRI at Northwest Kidney Centers-Havilland 
o KRI at UWMC  

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SCHEMA 
 
 
 

 
 
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1.0 
INTRODUCTION 
1.1 
Background and Prior Research 
Chronic kidney disease is associated with vascular dysfunction leading to substantial 
cardiovascular and cerebrovascular disease burden. A major mechanism of vascular dysfunction 
is thought to be endothelial cell dysfunction due to the accumulation of homocysteine, 
asymmetric di- and mono-methylarginines (ADMA), reactive oxygen species, and/or guanidine-
compounds. Current standard of care for treating vascular disease in patients with CKD focuses 
on addressing of hypertension and proteinuria, both of which are detected late in the disease. The 
observation that endothelial cell dysfunction (ECD) develops at earlier, pre-uremic stages of 
CKD highlights the critical need for investigations into earlier preclinical mechanisms 
contributing to ECD.  
 
Defects in endothelial nitric oxide synthase (eNOS) and/or deficiency of bioavailable nitric oxide 
are the hallmarks of endothelial cell dysfunction leading to vascular disease and defective 
angiogenesis. Accumulation of toxins found in renal disease may impact endothelial cell nitric 
oxide bioavailability and eNOS function. Indeed, exposure of endothelial cells to asymmetric 
dimethylarginines (ADMA) has been shown to result in the redistribution of eNOS from the 
plasma membrane to the mitochondrion as well as result in uncoupling of eNOS with enhanced 
generation of reactive oxygen species.  Furthermore substantial evidence indicates nitric oxide 
bioavailability is linked to mitochondrial biogenesis.  Recent observations of a proteomic 
signature in early ECD modeled by using chronic L-NMMA treatment akin to elevation on 
ADMA suggest underlying mitochondrial dysfunction. Under these conditions early ECD is 
characterized by reductions in aconitase-2 and ECHS-1 (key enzymes in the mitochondrial TCA 
cycle and fatty acid beta oxidation pathways). Reactive oxygen species have been shown to play 
a central role in inactivation of aconitase. These findings may suggest that inhibition of the Krebs 
cycle and mitochondrial bioenergetics may augur the development of ECD. Deficiency in these 
enzymes and reduction in mitochondrial mass may result in a metabolic block of normal 
mitochondrial oxidative phosphorylation.  
 
Glutamine is a potent anaplerotic (capable 
of replenishing intermediates of the Kreb’s 
cycle) and a potential therapeutic agent 
bypassing the metabolic block associated 
with reduced TCA cycle activity and 
impaired mitochondrial function (figure 1).  
It is also the substrate for glutamate 
important for antioxidant glutathione 
synthesis. It has been demonstrated to 
confer metabolic benefits in other 
populations, but the data on its effect in 
patients with chronic kidney disease are 
unknown. Here we assemble a multi-
disciplinary team of investigators with 
expertise in physiology, biophysics, and 
nephrology to investigate the impact of glutamine on mitochondrial energetics and vascular 
function. 
Figure 1. Supplementation with glutamine: its conversion 
to alpha-ketoglutarate bypasses the enzymatic block in 
ECD and reinforces the truncated Krebs cycle. 

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We propose a phase II randomized, controlled cross-over trial of glutamine supplementation on 
mitochondrial energetic and endothelial function measured by non-invasive 31P magnetic 
resonance spectroscopy coupled with optical spectroscopy (MRS/OS) and pulse amplitude 
tonometry among persons with moderate-severe chronic kidney disease.  
1.2 
Rationale 
Chronic kidney disease is associated with endothelial cell dysfunction and muscle wasting 
contributing to the heightened risk of cardiovascular morbidity and mortality and functional 
limitation. Accumulation of toxins in renal disease may adversely impact endothelial cell nitric 
oxide bioavailability and endothelial Nitric Oxide Synthase (eNOS) function consequently 
heightening oxidative stress and suppressing mitochondrial biogenesis. To date no studies have 
investigated potential therapies for endothelial and muscle dysfunction in renal disease target 
mitochondrial metabolic and energetic processes.   
 
Animal studies of uremia underscore mitochondrial dysfunction as a potential precursor for 
endothelial dysfunction.  In particular, uremia has been linked to a proteomic signature indicative 
of metabolic blockage of TCA cycle activity and fatty acid beta-oxidation. Both of these 
processes are localized to the mitochondria and may suggest that decreased mitochondrial mass 
or function may augur endothelial dysfunction in renal disease.  
 
Glutamine, an anaplerotic agent and precursor to the antioxidant glutathione, is a potential 
therapeutic agent bypassing the metabolic block associated with reduced TCA cycle and 
improving antioxidant reserve. A recent dose-finding study and randomized controlled trial of 
glutamine in patients with critical illness demonstrated that an IV dose of 0.35g/kg/day by ideal 
body weight was associated with an increased muscle mitochondrial mass1 and well tolerated 
without any serious clinical or biochemical adverse events compared to placebo.2  
 
The primary goal of proposed investigation is to study the impact of oral glutamine 
supplementation on muscle mitochondrial and endothelial cell function measured mitochondrial 
energetics and vascular function using 31P MRS/OS among persons with moderate-severe CKD. 
The secondary objective is to describe the impact of oral glutamine supplementation on 
mitochondrial metabolic profile as well as inflammatory and oxidative stress biomarkers among 
persons with chronic kidney disease. 
2.0 
STUDY OBJECTIVES AND DESIGN 
2.1 
Primary Objectives 
The primary objectives of this study are to: 
• 
To test if glutamine supplementation improves mitochondrial function and 
vascular function as measured by 31P MRS/OS. 
 
 

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2.2 
Secondary Objectives 
The secondary objectives of this study are to: 
• 
To test the effects of oral glutamine supplementation on metabolic 
response profile. 
• 
To test if glutamine supplementation reduces inflammatory, oxidative 
stress biomarkers and redox proteome 
• 
To test if glutamine improves objective isometric muscle fatigue. 
• 
To test if glutamine supplementation reduces proteinuria (albuminuria). 
• 
To test if glutamine reduces 24 hour urine urea nitrogen. 
 
2.3 
Study Design 
Overview of study design 
This is a placebo-controlled, double-blind crossover trial.  There are 2 phases 14 days 
each separated by a 3-week washout period 
A brief screening visit will assess eligibility criteria, and will include informed consent. 
Qualified participants will be stratified by diabetes status and randomly assigned (1:1) to 
14 days of placebo followed by 14 days weeks of oral L-glutamine, or 14 days of oral L-
glutamine followed by 14 days of placebo, each separated by a 3-week wash-out period.   
Qualified participants will participate in 3 study visits, plus a screening visit.  Study 
outcomes will be assessed at each of these 3 visits.  
Subjects will receive 0.4 g/kg/day of L-glutamine in three divided daily doses. This dose 
was chosen based on a review of studies in the literature evaluating biological activity of 
glutamine on immune function and modulation of muscle turnover. Placebo will consist 
of identical appearing maltodextrin powder. 
3.0 
STUDY POPULATION 
Study participants will be selected from patients with moderate-severe CKD not treated 
with dialysis from the Muscle Mitochondrial Energetics and Dysfunction (MEND) study, 
a cross-sectional clinic-based study of CKD. MEND has recruited CKD and end stage 
renal disease (ESRD) patients from Nephrology clinics at Harborview Medical Center, 
University of Washington Medical Center, and the Northwest Kidney Centers.   MEND 
includes a diverse population, representing the greater population of those with kidney 
disease. Exclusion criteria for MEND include kidney transplantation, non-English 
speaking, unable to provide informed consent, or dementia.  
Participants will be selected for this study from MEND according to the criteria in 
Section 3.1 and 3.2.  They will be recruited, screened, and enrolled as described in 
Section 3.3 [and assigned to a study treatment/intervention group as described in Section 

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7.4]. Issues related to participant retention and withdrawal from the study are described in 
Sections 3.5 and 3.6, respectively. 
3.1 
Inclusion Criteria 
Criteria included in MEND: 
Additional Criteria: 
• Adults between 20 and 79 years of age 
• Diagnosis of moderate-severe CKD, defined in this study as an estimated 
glomerular filtration rate (eGFR) of ≤60ml/min/1.73m2 using the Chronic Kidney 
Disease Epidemiology Collaboration equation 
3.2 
Exclusion Criteria 
Criteria included in MEND: 
• Pregnant 
• Kidney transplantation 
• Weight >400 lbs 
• HIV infection 
• End stage liver disease with cirrhosis 
• Have physical immobility (defined by wheelchair use, oxygen-dependent COPD, 
shortness of breath after <100 steps) 
• Have implants incompatible with MRI 
• Institutionalization,  or inability to consent 
• Use medications interfering with muscle or mitochondrial function, including 
steroids, anti-psychotic,  antivirals, muscle relaxants, oral calcineurin inhibitors, 
and anti-epileptic drugs 
• Active malignancy 
• Pacemaker 
• Current participation in another interventional trial 
• Non-English speaking 
 
Additional Criteria: 
• On chronic dialysis 
• Expectation to start dialysis within 6 months or dialysis access in place. 
• Insulin dependent diabetes 
• Exercise limiting cardiopulmonary disease (e.g. angina, severe heart valve 
disease, severe COPD, coronary ischemia) 
• Use of anticoagulation (i.e. warfarin) 
• Baseline systolic blood pressure >170 or diastolic blood pressure >100 
• Inflammatory conditions (e.g. autoimmune disease, HIV) 
• Cirrhosis, active/chronic hepatitis 
• Weight >300 lbs 
• Personal history or family history of deep vein thrombosis, pulmonary embolism 
• Patients hospitalized within the past 60 days for any reason. 
 

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3.3 
Recruitment Process 
Participants enrolled in the MEND study have documented consent approving contact 
regarding participation in future studies for which MEND will be the baseline. We will 
recruit patients who have already undergone a CKD-MEND visit. CKD-MEND is a study 
of muscle mitochondrial energetics and dysfunction recruiting from the SKS and clinics. 
A comprehensive battery of testing including MRS/OS is performed. Data from the 
CKD-MEND visit performed within 180 days of entry into the current study will be used 
as baseline information.  
 
3.4 
Co-Enrollment Guidelines 
Study participants will not be allowed to enroll in other clinical trials, as the effect of 
glutamine on this population is not clear. 
 
3.5 
Participant Retention 
Every effort to retain enrolled participants will be made throughout the duration of the 
study in order to reduce bias related to loss to follow-up. Strategies for participant 
retention include: 
 
• Thorough explanation of the importance of all 2 study treatment groups to the 
overall success of the study 
• Detailed explanation of the study visit schedule and procedural requirements 
during the informed consent process, with re-emphasis at the end of each study 
visit 
• Use of appropriate and timely visit reminder mechanisms. This includes weekly 
phone calls to assess participant adherence to regimen and remind participants of 
subsequent follow up visits 
• Compensating participants for their time and effort in the study 
• Immediate and multifaceted follow-up on missed visits 
 
3.6 
Participant Withdrawal 
Participants will be allowed to voluntarily withdraw from the study for any reason and at 
any time. The investigator may withdraw participants from the study in order to protect 
their safety or if they are unwilling or unable to comply with required study procedures 
after consultation with the study sponsors and co-sponsors. Participants also may be 
withdrawn if the study sponsor, government or regulatory authorities, or site IRBs/ECs 
terminate the study before the planned end date. Reasons for withdrawal from the study 
will be recorded and a final evaluation will be made if feasible.  
 
Participants who discontinue treatment but remain in the study will continue to be 
followed-up whenever possible. 

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4.0 
STUDY TREATMENT/PRODUCT/INTERVENTION 
4.1 
Treatment/Product/Intervention Regimen(s) 
• Active treatment will consist of 0.4 g/kg/day of L-glutamine (Nutrestore, 
EMMAUS Life Sciences, Inc Torrance, CA) in three divided daily doses. This 
dose was chosen based on a review of studies in the literature evaluating blood 
absorption of oral glutamine,3 biological activity of glutamine on immune 
function and modulation of muscle turnover.  
• Placebo will consist of identical appearing maltodextrin powder. 
4.2 
Treatment/Product/Intervention Supply and Accountability 
The Investigational Drug Pharmacy pharmacist must maintain complete records of all 
study drugs/products received from the Clinical Research Products Management Center 
and drug manufacturer and subsequently dispensed to study participants. All unused 
supplies must be returned to the Clinical Research Products Management Center after the 
study is completed or terminated. 
4.3 
Adherence Assessment 
Adherence to the study treatment will be assessed by study coordinator phone call at day 
7 of the study to assess adherence and inventory method to assess remaining 
medication/placebo at the end of the study.  Adherence will be defined as consumption of 
at least 75% of the prescribed regimen. Up to 30 participants may be recruited to 
compensate for non-adherence or drop-out with the goal of 10 individuals completing in 
each treatment arm. 
4.4 
Toxicity Management 
Currently, the mechanism whereby glutamine may cause harm or toxicity is unknown. 
There have been studies demonstrating increased levels of blood urea in those treated 
with glutamine. In the event of moderate or serious adverse effects (as defined in section 
6.2) requiring treatment for such conditions as nausea, vomiting, confusion or bleeding 
thought to be related to toxicity from the study drug, the patient will be withdrawn from 
the study and adverse events reported. 
Mild adverse events that do not requiring treatment and are thought to be potentially 
related to toxicity from the drug will prompt a 50% dose reduction. 
4.5 
Clinical Management of Pregnancy 
All female participants of childbearing age who are sexually active will be asked to use 
contraception during the duration of the study and for 1 month afterwards as the effects 
of glutamine on fetal development are not clear. 
 
Any females who become pregnant during the study period will stop taking the study 
product, but will continue with study procedures with the exception of the MRS/OS 
procedures.  This is because the risks of ischemia in pregnancy are not known. 

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4.6 
Concomitant Medications 
Enrolled study participants may continue use of all concomitant medications — except 
those listed under criteria for exclusion or treatment discontinuation — during this study.   
All concomitant medications taken or received by participants within the 4 weeks prior to 
study enrollment will be reported on applicable study case report forms. In addition to 
prescribed and over-the-counter medications (vitamins, herbal remedies, and other 
traditional preparations) will be recorded. Alcohol and recreational or street drug use will 
be recorded in clinical progress notes if needed for interpretation/documentation of 
observed participant health status.  Medications used for the treatment of AEs that occur 
during study participation also will be recorded on applicable study case report forms. 
5.0 
STUDY PROCEDURES 
Schedule of visits and procedures 
An overview of the study visit and procedures schedule is presented in table 1. Presented 
below is additional information on visit-specific study procedures. Detailed instructions 
to guide and standardize all study procedures will be provided in the study-specific 
procedures manual. 
Screening visit 
Some components of the initial screening visit may be performed by telephone or in 
combination with other study visits in order to best accommodate participants’ schedules. 
Once informed consent is obtained, the subject will undergo a baseline screening 
evaluation including an assessment of demographics, smoking history, medical history, 
medication inventory, with physical examination by study personnel. If the subjects meet 
the eligibility criteria the PI or Co-I will contact the subjects’ primary physician 
regarding the subjects’ involvement in the trial. The first study visit will be scheduled 
within 28 days of the screening visit.  We will link to laboratory data from the CKD-
MEND visit. The only lab tests that are not collected in CKD-MEND needed for this trial 
are metabolomics and redox proteome testing (see below). 
Study drug dispensed at Visits 1 and 2 
The participant will receive either placebo (maltodextrin powder) or glutamine from the  
Investigational Drug Service (IDS) and instructed on how to appropriately prepare it. He 
or she will be instructed to take the medication daily starting on the day after the study 
visit. They will be instructed to consume no protein for 30 minutes after glutamine or 
placebo consumption to prevent competitive binding for absorption with other amino 
acids.  
Visit 1 – Visit 3 
The participant will have been required to fast for 8 hrs prior to each visit and abstain 
from caffeine or smoking which can effect endothelial function for 8 hours prior to the 
visit. The following data will be obtained:   

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 vital signs,  
 fasting blood collection,  
 anthropometrics (height and weight),  
 a urine pregnancy test will be given to women of child-bearing potential 
 The MRS/OS and Muscle Fatigue tests will occur at the diagnostic imaging center 
at UWMC at visits 2 and 3.  We will use the results of the MEND MRS/OS as the 
baseline for this study. Only 2 MRS/OS and Muscle Fatigue tests will be obtained 
per patient during this study because it will be assumed that there will be no 
carry-over effect of glutamine after the 3-week washout period.  
Note: We may ask the subject to repeat the MRS/OS and Muscle Fatigue tests if 
more than 180 days has passed, or if they have had a change in health status, since 
their MEND visit.  
Procedures:  
An overview of the study visit and procedures schedule is presented in Appendix I. 
Presented below is additional information on visit-specific study procedures. Detailed 
instructions to guide and standardize all study procedures across sites will be provided in 
the study-specific procedures manual. 
Blood testing for metabolic response profile (oxidative stress, inflammation, and plasma 
amino acids, metabolites, and electrolytes). Oxidative Stress and Inflammatory 
Biomarkers include: C-reactive protein and IL-6 levels will be determined using the 
automated Abbott FLX C-Reactive Protein Assay and by ELISA, respectively. Plasma 
F2-isoprostanes and isofurans will be measured by GC-MS as described by Roberts and 
Morrow at Vanderbilt University; Plasma Amino Acids, Metabolites and Electrolytes 
include: Plasma amino acid concentrations will be determined by reversed-phase HPLC 
after derivatization with phenylisothiocyanate.  
- Plasma samples will be sent to Emory University for liquid chromatography 
high-resolution mass spectrometry (LC-MS) for untargeted metabolomics analysis. 
- Clinical laboratory assays for determination of comprehensive metabolic panel 
(electrolytes, blood urea nitrogen, creatinine, glucose, albumin, total protein, calcium, 
alkaline phosphatase, ALT, AST, total bilirubin), CPK, lactate, triglyceride and ammonia 
levels will be performed at the KRI Core Research laboratory. 
 
-Testing the redox metabolome. Glutathione (GSH/GSSG) redox and cysteine 
(Cys/CySS) redox systems are oxidized in association with oxidative stress and in 
association with cardiovascular disease. Extracellar Cys/CySS redox state has been 
associated with events of early atherosclerosis, specifically monocyte adhesion to the 
endothelium and activation of the inflammatory cascade. Furthermore the redox state of 
the mitochondria represents a unique pool providing potential insight into the oxidative 
damage eventually contributing to necrosis and apoptosis. Plasma samples for all these 

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assays will be processed and sent to Emory University in Atlanta for analysis of 
mitochondrial redox control via systems of glutathione and thioredoxin models.  
24 hour urine collection for urea nitrogen: Assessment of protein turnover will be 
performed using a timed 24hr urine collection for assessment of urine urea nitrogen. In 
order to confirm adequate collection and estimate muscle mass, we will also collect urine 
creatinine. Specimens less than 1liter in volume will be repeated. 
Magnetic Resonance Spectroscopy and Optical Spectroscopy: As mentioned above, only 
2 MRS/OS will be obtained as part of this study (Visit 2 and 3). The baseline MRS/OS 
will be obtained from the MEND study.   
MRS/OS will be used to measure mitochondrial energy coupling (P/O), phosphorlylation 
capacity (ATPmax) and oxidative capacity (O2max) of the first dorsal interosseus (FDI) 
skeletal muscle.4 The selection of the FDI skeletal muscle is based on the observation of 
higher oxidative type II muscle fiber content and degree of coupling similar in extent to 
the vastus lateralis commonly used to study of mitochondrial dysfunction. 5,6 As part of 
this procedure, two functional tests will be performed: an exercise protocol to help 
determine ATPmax and a resting (ischemia) protocol to determine mitochondrial energy 
coupling and the maximal rate of mitochondrial O2 uptake (O2max). Optical spectroscopy 
measuring the delay in Hemoglobin saturation relative to Myoglobin saturation after the 
onset of reperfusion will provide an index of vascular function. These functional tests 
assist to perturb the cell physiological state in order to measure the contents and 
dynamics of key metabolic compounds important in mitochondrial energy flux. 31P MRS 
is reliable with a documented coefficient of variation for oxidative phosphorylation of 
5.1% and has a strong correlation with VO2max.7  
Muscle Fatigue: We will test muscle fatigability at each follow-up visit. We will test the 
FDI muscle and determine the isometric force generated at 70% of maximal voluntary 
contraction (MVC) against a force transducer. Using labview software to measure and 
record the force-time waveform of each contraction we will initiate the protocol at 50 
contractions per minute at 70% MVC for one minute. At each 1 minute increment will 
increase the contraction rate by 10 contractions per minute and reassess the subjects 
relative perceived exertion on a scale of 1-10 until the patient can no longer maintain the 
contraction waveform.  
Vitals and measurements 
Day 7 and 45 phone call:  
All subjects will receive a phone call by study coordinators to confirm patients are taking 
their treatment/placebo medication in order to ensure adherence. They will also receive 
reminder of their subsequent follow up visits. 
Visit 2 (day 15): 
At visit 2 the participant will be questioned regarding any concerning symptoms noted 
during the treatment period. Adherence with the treatment or placebo will be confirmed 
by evaluating the quantity of remaining placebo or glutamine in the dispensed containers. 
Repeat fasting blood collection. They will also receive a repeat MRS/OS. 
Washout (Day 16-37): 

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After the second visit, the participant will undergo a 3 week washout period prior to 
initiating the second treatment or placebo for another 2 week period. 
Day 38:  
The participant will cross-over to the opposite placebo or glutamine therapy for a two 
week course. We will phone the subject to remind them to begin taking the 
treatment/placebo. 
Visit 3 (Day 53- Final visit): 
During visit 3 the participant will be questioned regarding any concerning symptoms 
noted during the treatment period during visit 3. Adherence with the treatment or placebo 
will be confirmed by evaluating the quantity of remaining placebo or glutamine in the 
dispensed containers. Repeat testing will be performed to assess change during period 2 
of the study. Fasting blood will be collected. They will also receive a repeat MRS/OS.  
Table 1. Table of study treatments and procedures 
*Pregnancy test performed on females of childbearing age who are sexually active. 
Treatments/Procedures  
Day -90 
to 0 
(Screening) 
Day -1 
(Visit 1) 
Day   
0 - 14 
Day 15 
(Visit 2) 
Day 16-37 
Washout 
Day 38  
Day 
39-52 
Day 53 
(Visit 3) 
Visit may take this much time: 
1 hour 
3 hour 
  
3 hour 
  
(phone call) 
  
3 hour 
Informed Consent 
X 
  
  
  
  
Begin Study Product for Period 2. 
  
  
Inclusion/Exclusion 
X 
  
  
  
  
  
  
Medical History 
X 
  
  
  
  
  
  
Medication List 
X 
  
  
  
  
  
  
Demographics 
X 
  
  
  
  
  
  
Brief Physical Exam 
X 
  
  
  
  
  
  
Pregnancy test* 
X  
  
  
  
  
  
Vitals 
  
X 
  
X 
  
  
X 
Fasting Blood Draw 
  
X 
  
X 
  
  
X 
24hr urine collection 
 
 
 
X 
 
 
X 
MRS/OS** 
  
  
X 
  
  
X 
Muscle fatigue 
 
 
 
X 
 
 
X 
Study Product Dispensed 
  
X 
  
X  
  
  
  
Study Product collected 
  
  
  
X 
  
  
X 
Study Product Adherence 
  
  
  
X 
  
  
X 
Phone Call to Participant 
  
  
Day 7 
  
  
Day 45 
  

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** MRS/OS and Muscle Fatigue for baseline is already done as part of CKD-MEND visit (within 180 days).  If more 
than 180 days has passed, or if subjects have had a change in health status, these tests will be repeated at Visit 1. 
6.0 
SAFETY MONITORING AND ADVERSE EVENT REPORTING 
6.1 
Safety Monitoring  
All adverse events will be followed by the PI and study personnel until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained.  At 
the last scheduled visit, the investigator will instruct each subject to report any 
subsequent event(s) that the subject, or the subject’s personal physician, believes might 
reasonably be related to participation in this study.  The investigator will follow any 
death or adverse event occurring at any time after a subject has discontinued or 
terminated study participation that may reasonably be related to this study.  An internal 
audit will take place each month of the trial to insure that everything is well documented. 
 
As each of the first five subjects completes the cross-over (visit 3), we will submit to the 
IRB a report on how the drug was tolerated.  We will report all side effects, adverse 
events and subject concerns.  In the event that one of the first 5 subjects withdraws from 
the study for reasons related to the drug, we will report immediately to the IRB via 
modification. 
6.2 
Adverse Event Definitions and Reporting Requirements 
6.2.1 Adverse Event 
Safety information will be assessed initially by subject interview. The clinical research 
coordinator is responsible for collecting and recording all clinical data.  As these results 
are collected, all adverse events will be identified and reported to the principle 
Investigators within seven (7) days. Adverse events will be reported as described below 
(section 6.2.3). The principal investigator (PI) is responsible for evaluating each AE and 
for determining whether the AE affects the risk/benefit ratio of the study and whether 
modifications to the protocol and consent form are required.  All IRB-approved protocol 
or consent form revisions that indicate a change in risk for participants will be reported to 
the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) in a 
timely manner. 
 
Study participants will be provided a telephone number and instructed to contact the 
study clinician to report any AEs they may experience, except for life-threatening events, 
for which they will be instructed to seek immediate emergency care. Where feasible and 
medically appropriate, participants will be encouraged to seek evaluation where the study 
clinician is based, and to request that the clinician be paged or otherwise contacted upon 
their arrival. With appropriate permission of the participant, whenever possible records 
from all non-study medical providers related to AEs will be obtained and required data 
elements will be recorded on study case report forms.  All participants reporting an AE 
will be followed clinically, until the AE resolves (returns to baseline) or stabilizes. 

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Study site staff will document on study case report forms all AEs reported by or observed 
in enrolled study participants regardless of severity and presumed relationship to study 
product.  
All adverse events will be graded as follows: 
 
      Severity 
0 = No adverse event or within normal limits 
1 = Mild—did not require treatment 
2 = Moderate—resolved with treatment 
3 = Severe—required professional medical attention 
4 = Life-threatening or disabling 
5 = Death 
 
Related to study procedures (there is a reasonable possibility that the 
experience may have been caused by the study procedures) 
0 = Unrelated 
1 = Unknown 
2 = Related 
 
Unexpected event (an AE with specificity or severity not consistent with the 
risk information in the protocol/application or an AE that has not been 
previously observed) 
0 = No 
1 = Yes 
 
Serious (any AE occurring at any dose that results in death; a life-threatening 
adverse drug experience; inpatient hospitalization or prolongation of existing 
hospitalization; a persistent or significant disability/incapacity; a congenital 
anomaly/birth defect; or any important medical event that, based on medical 
judgment, jeopardizes the subject and may require medical or surgical 
intervention to prevent one of the above outcomes) 
0 = No 
1 = Yes 
 
6.2.2 Serious Adverse Event  
Serious adverse event (SAE) will be defined per U.S. Code of Federal Regulations (CFR) 
312.32 and International Conference on Harmonization (ICH), “Good Clinical Practice: 
Consolidated Guidance” (E6) and “Clinical Safety Data Management: Definitions and 
Standards for Expedited Reporting” (E2A), as AE occurring at any dose that: 
• Results in death  
• Is life-threatening 

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• Results in persistent or significant disability/incapacity 
• Is a congenital anomaly/birth defect   
• Requires inpatient hospitalization or prolongation of existing hospitalization  
This includes important medical events that may not be immediately life-threatening or 
result in death, or hospitalization but may jeopardize the patient or may require 
intervention to prevent one of the outcomes listed above. 
Per ICH SAE definition, hospitalization itself is not an adverse event, but is an outcome 
of the event. The following types of hospitalization do not require expedited reporting: 
• Any admission unrelated to an AE (e.g. for labor/delivery, cosmetic surgery, 
administrative, or social admission for temporary placement for lack of place to 
sleep) 
• Protocol-specified admission (e.g. for procedure required by protocol) 
• Admission for diagnosis or therapy of a condition that existed before receipt of 
study agent(s) and has not increased in severity or frequency as judged by the 
clinical investigator 
6.2.3 Adverse Event Reporting 
Any AE that is unexpected, related to study participation, and serious (defined in 
6.2.1) will be reported in writing to the IRB within 7 days of the PI’s first 
knowledge of the event. Any AE that is unexpected, related to study participation, 
and suggests greater risk of harm than previously known or recognized will be 
reported in writing to the IRB within 7 days of the PI’s first knowledge of the 
event.  
 
The PI will review all expedited adverse event reports. In addition, any deviations 
from protocol will be reported to the IRB and copied to the Prevention Center.  
 
A summary of all adverse events and any audit reports will be sent to the IRB.  
Any action resulting in a temporary or permanent suspension of this study (e.g., 
IRB actions, or actions by the investigators or co-investigators) will be reported to 
the appropriate NIDDK program official.  In addition, the NIDDK will be given 
notice of any actions taken by the IRB or regulatory bodies regarding the research 
and any responses to those actions. 
7.0 
STATISTICAL CONSIDERATIONS 
7.1 
Review of Study Design 
We propose a single-center double blind randomized cross-over trial of glutamine 
supplementation versus placebo. As part of the study there will be a 4-week washout 
between treatment and placebo arms.  

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7.2 
Endpoints 
Primary endpoints are change in mitochondrial energetics and endothelial function. 
Secondary endpoints are change in metabolic response profile and markers of oxidative 
stress and inflammation.  
7.2.1 Primary Endpoints 
Consistent with the primary study objective to test for glutamine’s impact on endothelial 
function and mitochondrial function, the following endpoints will be assessed: 
• 
Improvement in mitochondrial function as measured by 31P MRS/OS 
parameters of ATPmax and Oxygen uptake (O2max) and the ATPflux/O2uptake 
ratio representing the efficiency of mitochondria energy production. 
• 
Improvement in vascular function as measured by the difference in the 
spectroscopic delay in recovery of oxygenated myoglobin and oxygenated 
hemoglobin after the onset of reperfusion during optical spectroscopy. 
 
 
7.2.2 Secondary Endpoints 
Consistent with the secondary study objective to assess changes in metabolic response 
profile and markers of oxidative stress and inflammation the following endpoints will 
assessed: 
 
• Improvement in metabolic response profile suggested by:  
 
• Change in metabolomics profile using liquid chromatography high-
resolution mass spectroscopy (LC-MS). (Reference: Frediani JK. 
(2014) Plasma metabolomics in human pulmonary tuberculosis 
disease: a pilot study. PLoS ONE 9(10): e108854).  
 
• Lower levels of lactate, triglycerides. 
 
• Assessment of the integrated redox function (Reference: Jones DP. 
Measuring the poise of thiol/disulfide couples in vivo. Free Radical 
Biology and Medicine. 47(2009):1329-38.) 
 
• Plasma F2-isoprostanes will be measured by GC-MS as described by 
Roberts and Morrow; Plasma amino acids, metabolites and 
electrolytes.  Plasma amino acid concentrations will be determined by 
reversed-phase HPLC after derivatization with phenylisothiocyanate 
 
• Improvement in proteinuria (albuminuria). 
 
• Assessment of nitrogen balance and anabolism with 24hr urine for urine 
creatinine, urine urea nitrogen.  
 

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7.3 
Accrual, Follow-up, and Sample Size 
The overall sample size for this project is 20. We anticipate recruiting 1-2 participants 
weekly for total study duration of 2 years. 
Sample size was calculated based on values obtained from an estimated within individual 
coefficient of variation in values of ATP/O2 ratio from MRS/OS of approximately 15% 
and values of test-retest reliability of pulse amplitude tonometry measures of vascular 
endothelial function from a recent published study. A total of 20 patients will enter this 
two-treatment crossover study. With this sample size, the probability is over 90% that we 
will detect a meaningful treatment difference of 20% for ATP/O2 ratio at a one-sided 
0.05 significance level  (Table 2). This is based on the assumption that the within-patient 
standard deviation of the response variable is 0.375 with a mean predicted ATP/O2 ratio 
of 2.5.  Sample size was calculated using the following sample size calculator: 
http://hedwig.mgh.harvard.edu/sample_size/js/js_crossover_quant.html 
  
ATP/O2 ratio 
 
Power 
Minimal detectable difference 
80% 
90% 
20% 
6 
7 
25% 
4 
5 
33% 
4 
4 
40% 
3 
4 
 
 
 
 
7.4 
Random Assignment / Study Arm Assignment 
Investigational drug services will be responsible for randomization of participants into 
the two treatment groups.  
7.5 
Blinding 
Study participants, investigators, and research coordinators will be blinded to the study. 
The investigational drug pharmacy will receive a receive treatment/placebo designation 
in a sealed envelope on the day of study drug/placebo initiation. Subjects will not be told 
of their assignment. Early unblinding will occur to protect patient safety in the event of 
an adverse event that the investigator believes may be relate to treatment.  
7.6 
Data Analysis  
7.6.1 Primary Analyses 
The major assumption in the cross-over trial is that there is no carryover effect of first 
period into the second period.  The assumption is that a 3-week washout will be sufficient 
Table 2. Samples size required to detect treatment effects for ATP/O2 ratio from MRS/OS 
and reactive hyperemia index (RHI) from PAT in crossover design. 

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to eliminate any potential carryover effect due to glutamine. The statistical model we 
assumed for continuous data from the 2 × 2 crossover trial: 
Design 
Period 1 
Period 2 
Sequence Placebo-Gln 
A 
B 
Sequence Gln-Placebo 
B 
A 
 
From this design we construct these differences for every patient and compare the two 
sequences with respect to these differences using a two-sample paired t test. Thus, we are 
testing: 
H0 : μAB - μBA = 0 
Where:  
μAB - μBA = 2( μA - μB ) 
 
So testing  
H0 : μAB - μBA = 0, is equivalent to testing: 
H0 : μA - μB = 0 
7.6.2 Secondary Analyses 
We will test for difference in each marker of metabolic response profile, oxidative stress, 
and albuminuria in a similar fashion as above. Similar to above, we will use a two-sample 
paired t-test testing the difference in means comparing treatment to placebo. 
8.0 
HUMAN SUBJECTS CONSIDERATIONS 
The procedures in the study and model informed consent forms for subjects are to be 
reviewed by the UW Human Subjects Division (HSD). 
Subsequent to initial review and approval, the HSD will review the protocol at least 
annually.  The Investigator will make safety and progress reports to the IRB at least 
annually, and within three months of study termination or completion.  These reports will 
include the total number of participants enrolled in the study, the number of participants 
who completed the study, all changes in the research activity, and all unanticipated 
problems involving risks to human subjects or others.  
8.1 
Informed Consent 
Informed consent will be obtained from each study participant.  Consent forms will 
include the purpose of the study, description of procedures, list of risks and benefits, 
assurance of confidentiality, assurance of withdrawal without prejudice, description of 
reimbursement for trial participation ($125 plus parking and bus fare reimbursement), 
and a telephone number for answering questions about the research.  Patients are also 
given information regarding their right to privacy for personal health information based 
on HIPAA regulations, and the procedures for data sharing using de-identified data will 
be explained.  Participants will be provided with a copy of their informed consent forms 
if they are willing to receive them. 

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8.2 
Risks 
The following stated risks will be reflected in the informed consent form and 
communicated orally to prospective participants at the study site at the time of obtaining 
consent. 
 
Potential loss of confidentiality is a risk for all study participants, which is address by 
procedures at the coordinating center described below. 
All patient will have a blood draw regularly during the study for a total of four blood 
draws. The risks of phlebotomy include bleeding/bruising, infection, and discomfort. 
For the MRS study, participants may be at risk if they have implanted or non-removable 
magnetic objects within or on the body, or if loose magnetic items are attracted to the 
magnet while the subject is near or in the magnet bore.  We carefully screen subjects to 
exclude anyone who has implanted or attached magnetic items.  In addition, access to the 
magnet is strictly controlled and all magnetic objects are kept away from the magnetic 
field.  There is no intravenous contrast used in the MR study.  
During the MR study, subjects will have blood pressure cuff inflated to 50 mm Hg over 
systolic blood pressure.  This will remain in place for 12 minutes, increasing risk of 
thrombosis.  To decrease the risk of this, subjects will be screened for past history of deep 
venous thrombosis, pulmonary embolism, or history of hypercoagulability.  Family 
history for deep venous thrombosis, pulmonary embolism, or history of 
hypercoagulability will also be obtained. 
8.3 
Benefits 
There may be no direct benefits to participants in this study, however, participants and 
others may benefit in the future from information learned from this study. Specifically, 
information learned in this study may lead to the development of a safe and effective 
intervention that reduce risk of cardiovascular events and risk of disability in persons 
with CKD.   
 
8.4 
Compensation 
Participants will be compensated for their time and effort in this study, and/or be 
reimbursed for travel to study visits and time away from work. Reimbursement amounts 
will be specified in the study informed consent forms.  
8.5 
Confidentiality 
1.  All study-related information will be stored securely at the Kidney Research Institute 
(KRI) at the University of Washington in Seattle.  Access to the KRI is protected by 
individual unit keys to the elevator, the main institute, and individual offices to prevent 
tampering or unauthorized access.  The use of a Local Area Network (LAN) provides two 
additional levels of security in the unlikely event of an unauthorized user finding a 

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machine unattended or unlocked.  The LAN restricts access to the Database files via 
passwords, and the Database itself requires a password before files may be opened.   
All study participants are assigned a unique identifier that does not contain identifiable 
information.  
2. All study-related information will be stored securely at the study site. All participant 
information will be stored in locked file cabinets in areas with access limited to study 
staff.  All laboratory specimens, reports, study data collection, process, and 
administrative forms will be identified by a coded number to maintain participant 
confidentiality.  All local databases will be secured with password-protected access 
systems. Forms, lists, logbooks, appointment books, and any other listings that link 
participant ID numbers to other identifying information will be stored in a separate, 
locked file in an area with limited access.  Participant’s study information will not be 
released without the written permission of the participant.   
8.6 
Study Discontinuation 
The study also may be discontinued at any time by the IRB. 
9.0 
 LABORATORY SPECIMENS AND BIOHAZARD CONTAINMENT 
9.1 
Local Laboratory Specimens 
As described in section 5, the following types of specimens will be collected for testing at 
the Kidney Research Institute local laboratory. If the participant provides informed 
consent, his/her specimen will be stored in the KRI repository for additional studies (see 
section 9.2): 
 
• Fasting blood samples – approximately 23ml of blood per visit at baseline 
visit and post-intervention visits during each period of the study.  During the 
study a total of approximately 69 mL will be collected per subject. 
 
 
Local laboratories will perform Chemistries and pregnancy testing.  
9.2 
Reference Laboratory Specimens 
• Blood for metabolomics testing. Aliquots of EDTA preserved blood will be 
stored at -80°C and sent to Emory University (c/o Dr. Ziegler). All samples 
will be de-identified.  
• Blood for integrated redox state testing will be collected at baseline and post-
intervention visits. A 18-, 21-, or 23-guage butterfly needle will be used to 
draw up 3mL of blood into a 5ml syringe.  Enough blood will be immediately 
placed in a microcentrifuge tube containing 150µL of preservative solution to 
attain a total volume of 1.5mL. These samples will be frozen at -80°C and 
sent in bulk to Emory University (c/o Dr. Jones. See reference for more 

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details:  Jones DP. Measuring the poise of thiol/disulfide couples in vivo. Free 
Radical Biology and Medicine. 47(2009):1329-38.) 
• Blood for F2-isofuran and F2-isoprostane assays will be frozen at -80°C and 
sent in bulk to Vanderbilt University (c/o Dr. Jorge Gamboa). 
 
 
 
9.3 
Specimen Storage and Possible Future Research Testing 
Study site staff will store all blood and urine collected in this study at least through the 
end of the study. In addition, study participants will be asked to provide written informed 
consent for their blood and urine specimens to be stored in the KRI repository after the 
end of the study for possible future testing. The specimens of participants who do not 
consent to long-term storage and additional testing will be destroyed at the end of the 
study. 
 
Purpose 
Sample 
Type 
Maximum Amount 
per Sample  
Number Samples 
Total Amount 
Baseline/Post-intervention 
Blood 
- 
- 
23/46 mL 
Venous lactate (3 mL grey top) 
Blood 
3 mL 
3 
9 mL 
Renal Panel, triglycerides (green top) 
Blood 
4 mL 
3 
12 mL 
F2 isoprostanes/isofurans, 
metabolomics (EDTA) 
Blood 
10 mL 
3 
30 mL 
Redox testing (N & S tubes) 
Blood 
3 mL 
3 
9 mL 
CBC (3ml lavender top) 
Blood 
3 mL 
3 
9 mL 
Total blood collected during study 
 
 
 
69 mL 
 
 
 
 
 
Baseline/Post-intervention 
Urine 
- 
- 
n/a 
     Urine Creatinine (24 hour) 
Urine 
n/a 
2 
n/a 
     Urine albumin (24 hour) 
Urine 
n/a 
2 
n/a 
     Urine Urea Nitrogen (24 hour) 
Urine 
n/a 
2 
n/a 
     Urine pregnancy 
Urine 
10 mL 
1 
10 mL 
 
 
 
 
 

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10.0 REFERENCES 
1. 
Heyland DK, Dhaliwalm R, Day A, Drover J, Cote H, Wischmeyer P. 
Optimizing the dose of glutamine dipeptides and antioxidants in critically ill 
patients: a phase I dose-finding study. JPEN. Journal of parenteral and 
enteral nutrition. 2007;31(2):109-118. 
2. 
Heyland D, Muscedere J, Wischmeyer PE, et al. A randomized trial of 
glutamine and antioxidants in critically ill patients. N Engl J Med. 
2013;368(16):1489-1497. 
3. 
Ziegler TR, Benfell K, Smith RJ, et al. Safety and metabolic effects of L-
glutamine administration in humans. JPEN. Journal of parenteral and 
enteral nutrition. 1990;14(4 Suppl):137S-146S. 
4. 
Amara CE, Marcinek DJ, Shankland EG, Schenkman KA, Arakaki LS, 
Conley KE. Mitochondrial function in vivo: spectroscopy provides window 
on cellular energetics. Methods. 2008;46:312-318. 
5. 
Conley K, Amara C, Jubrias S, Marcinek D. Mitochondrial function, fibre 
types and ageing: new insights from human muscle in vivo. Exp Physiol. 
2007;92(2):333-339. 
6. 
Conley K, Jubrias S, Esselman P. Oxidative capacity and aging in human 
muscle. J Physiol. 2000;526:203-210. 
7. 
Larson-Meyer DE, Newcomer BR, Hunter GR, Hetherington HP, Weinsier 
RL. 31P MRS measurement of mitochondrial function in skeletal muscle: 
reliability, force-level sensitivity and relation to whole body maximal 
oxygen uptake. NMR Biomed. 2000;13(1):14-27.