Determinants of Alpha-aminoadipic Acid (2-AAA) and Relationship to Diabetes: Study 2
Phase: NA
Conditions: Healthy
Sponsor: Vanderbilt University Medical Center

Protocol Version #:3 
 
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Determinants of Alpha-aminoadipic Acid (2-AAA) and Relationship 
to Diabetes: Study 2 
 
 
 
 
ClinicalTrials.gov Identifier: NCT04417218 
 
 
 
 
 
 
 
Document Date: 13 July 2020 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol Version #:3 
 
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Determinants of alpha-aminoadipic acid (2-AAA) and relationship to diabetes: 
 
Study 2 
 
 
 
Principal Investigator 
 
Jane F. Ferguson, PhD 
 
Division of Cardiovascular Medicine 
Vanderbilt University Medical Center 
 
2220 Pierce Ave, PRB 354 
Nashville TN 37232 
 
 
 
 
Co-Investigator 
 
Heidi J. Silver, PhD 
 
Division of Gastroenterology, Hepatology and Nutrition 
Medical Arts Building, Suite 514 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table of Contents 
 

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Contents 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 Statistical Considerations 
11.0 Privacy/Confidentiality Issues 
12.0 Follow-up and Record Retention 
13.0 References 
14.0 Appendices 
            

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1.0 
Background 
 
Diabetes is a major global health concern, associated with significantly increased 
mortality and high incidence of co-morbidities1. Given the high rate of cardiovascular 
and other diseases in individuals with diabetes, diabetes-related mortality may be under-
estimated and is estimated to be the third largest cause of death in the US2. Treatment 
efficacy and successful disease management varies between patients. This is in part 
attributable to the fact that diabetes is a heterogeneous disease, with multiple 
underlying causes. In a small proportion of cases of monogenic diabetes, the defects 
have been identified by genetic testing, sometimes leading to vastly improved treatment 
strategies3,4. However, the vast majority of cases of diabetes are polygenic and 
multifactorial. Despite some understanding of disease pathophysiology, and a variety of 
treatment options, the underlying causes of diabetes are unknown in most cases. A 
broader understanding of the variety of paths that lead to diabetes could identify 
alternative therapeutic targets. This may be crucial to preventing or managing disease in 
specific subsets of patients. Large-scale metabolomics studies have successfully 
identified novel candidates for diabetes, including a novel diabetes biomarker, α-
aminoadipic acid (2-AAA, Figure 1), which predicts the development of diabetes in 
humans5. In matched diabetes cases and controls from the Framingham Heart Study 
(FHS), higher plasma 2-AAA (at baseline) was 
associated with future diabetes, independent of fasting 
glucose and other known risk markers (age, sex, BMI, 
family history, diet)5.  
 
Source of 2-AAA: 2-AAA is generated from 
the catabolism of the amino acid lysine through 
the saccharopine (mitochondrial) and/or the 
pipecolic acid (peroxisomal) pathways6,7 
(Figure 2). Both pathways converge in the 
generation of 2-aminoadipate-6-semialdehyde, 
which is then further metabolized to 2-AAA in 
the cytosol8. 2-AAA is subsequently metabolized 
to 2-oxoadipate (α-ketoadipic acid), and further 
to acetyl-coA, entering the TCA cycle. Lysine is 
an essential amino acid which is acquired from 
dietary sources, with a recommended daily 
intake of 30mg/kg/day9. A portion of lysine in 
humans may be derived from the microbiome, 
with bacteria and fungi capable of de novo 
synthesis, in addition to modulation of 
bioavailability of dietary sources10.  
 
Genetics: Variation in genes encoding enzymes 
in the lysine degradation pathway (indicated in 
boxes in Figure 2) may influence 2-AAA levels 
and downstream function. The strongest 
evidence thus far exists for DHTKD1, variation in which has been associated with 2-
Ketoadipic, 2-Aminoadipic and 2-Oxoadipic Aciduria8,11, and Charcot-Marie-Tooth 
Figure 1. L-α-aminoadipic acid 
Figure 2. Multicompartmental model of L-lysine 
degradation (Adapted from Danhauser et al. 2012) 

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Disease12. DHTKD1 expression correlates with ATP production in mitochondria in vitro, 
and siRNA knockdown of DHTKD1 leads to impaired mitochondrial biogenesis and 
increased reactive oxygen species production, resulting in apoptosis and reduced cell 
growth13. Variation in mouse Dhtkd1 has been found to associate with expression of the 
gene (eQTL) and levels of protein (pQTL) in liver, as well as with serum 2-AAA levels14. 
Liver Dhtkd1 expression correlated significantly with serum 2-AAA in these mice. 
Further, liver 2-AAA was negatively associated with liver mass, fasting glucose and 
serum cholesterol while higher liver 2-AAA was associated with higher insulin sensitivity.  
 
 
 
2.0 
Rationale and Specific Aims 
 
Diabetes is a significant contributor to mortality and morbidity. The lysine-derived 
metabolite α-aminoadipic acid (2-AAA) has been identified as a novel predictor of 
diabetes development in humans, potentially identifying at-risk individuals before 
development of other known risk markers. Little is known about the function of 2-
AAA.Our preliminary investigation of human clinical, human genetic, animal and cell 
experimental data has revealed multiple lines of evidence supporting 2-AAA and its 
pathway as biologically relevant to diabetes pathophysiology. However, it is not yet clear 
whether 2-AAA is itself causal in diabetes development, or is a biomarker for altered 
metabolic processes, and many questions remain as to mechanisms of action. The 
determinants of variation in 2-AAA levels between individuals are not known. The aim of 
the first phase of this study (2-AAA Screening study, ongoing) was to measure plasma 
2-AAA levels from healthy individuals from the general population, to identify subjects 
with extremely high or low 2-AAA (defined as top and bottom 10%-25% of distribution). 
In this second phase of the study, individuals with high or low 2-AAA will be invited to 
participate in a dietary lysine modification study, to assess the effect of controlled lysine 
intake on plasma and urine 2-AAA. 
 
Specific Aim: Determine whether plasma 2-AAA levels are responsive to 
dietary intervention within individuals in the extremes of 2-AAA distribution 
in the general population. We hypothesize that plasma 2-AAA levels in a well-
nourished population are determined by genetics and gene-diet interactions and are not 
simply a reflection of dietary lysine exposure. Based on plasma 2-AAA measurements in 
our Phase 1 Screening study (ongoing, planned N=400), we will invite subjects who fall 
in the top and bottom ~10% of circulating 2-AAA (up to N=80) to return for future 
study. We will implement a dietary intervention to assess whether 2-AAA is a biomarker 
of lysine intake, or if 2-AAA levels are determined by genetic or gene-diet effects. 
Subjects will consume two different diets (“normal” and “high” lysine) in a randomized 
order (one week each), separated by a 2-week habitual diet washout period. 
 
 
3.0 
Animal Studies and Previous Human Studies 
 
2-AAA has been identified as a marker of oxidative stress in C. elegans, and in 
endothelial cells in response to hyperglycemia15, highlighting a potential mitochondrial 
phenotype, and relationship between 2-AAA and reactive oxygen species (ROS)16,17. In 

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animal models, administration of 2-AAA resulted in increased insulin levels and lower 
fasting plasma glucose, but no change in peripheral insulin sensitivity as measured by 
insulin tolerance test5. In vitro 2-AAA treatment has been shown to enhance insulin 
secretion in pancreatic β cells and islets. 2-AAA is thought to have important functions in 
the brain18, as well as in other organs, including liver and pancreas.  
 
High baseline plasma 2-AAA in healthy individuals was associated with increased future 
risk of diabetes (12-year follow-up) in FHS participants and validation samples 
(N~2,000)5. Insulin sensitizing therapy leads to reduction in 2-AAA, but not other 
putative diabetes-biomarkers, including branched-chain amino acids (BCAAs)19. 
Increased 2-AAA is found in skin in the setting of diabetes, as well as in chronic renal 
failure and sepsis, and increases with age20. Further, decreased urine 2-AAA has been 
reported in diabetes patients compared with non-diabetic14, highlighting a likely complex 
feedback relationship between 2-AAA levels and early diabetes development vs. the 
setting of established disease.  Treatment of overweight/obese adults with impaired 
fasting glucose or untreated diabetes with pioglitazone (45 mg/day) and metformin 
(1000 mg twice/day) led to a significant reduction in plasma 2-AAA and lysine, 
concurrent with an increase in insulin sensitivity19. Acute insulin infusion (7 hrs) in these 
subjects also led to a decrease in both lysine and 2-AAA19,21. The epilepsy drug 
vigabatrin, which acts through GABA transaminase inhibition, leads to increased 2-AAA 
in plasma and urine, to levels similar to those seen in alpha-aminoadipic aciduria22. Apart 
from diabetes, 2-AAA has been found to be elevated in the setting of other 
cardiometabolic or inflammatory diseases including atherosclerosis23, chronic renal 
failure and sepsis20, non-alcoholic fatty liver disease (NAFLD)24 and polycystic ovary 
syndrome (PCOS)25. There is likely some reciprocal regulation between insulin and 2-
AAA: insulin infusion in humans leads to decreased 2-AAA19,21,26, while insulin deprivation 
and hyperglycemia in the setting of type 1 diabetes leads to increased 2-AAA27. 
 
Observational data in humans does not support a relationship between dietary intake of 
protein or lysine and 2-AAA (as assessed in FHS5, and our own preliminary data). There 
are limited data on 2-AAA in response to dietary intervention. There was no change in 2-
AAA in obese subjects following 16 weeks of caloric restriction, despite weight loss28. 
However, dietary protein was not altered in this study. In a multi-diet comparison, 2-
AAA decreased on a low fat diet, and increased on a very-low carbohydrate diet29. The 
relationship between dietary lysine and 2-AAA has never been assessed through a 
randomized intervention trial. We will address this in the current study. One week of diet 
is appropriate to identify short-medium term dietary biomarkers30, but not long enough 
to affect other variables (which could alter 2-AAA through reverse causation). 
 

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We have already analyzed plasma samples for the first 122 participants of phase 1 of 
the study. As expected, there was variability in plasma 2-AAA, with some evidence for 
differences 
by sex 
(Figure 3). 
We will 
select initial 
participants 
from the 
extremes 
(by sex) of 
this group. 
Analysis of 
additional 
samples is 
pending and 
will continue 
to be 
batched 
based on 
sample accrual.  
 
 
4.0 
Inclusion/Exclusion Criteria 
 
Inclusion Criteria:  
• 
Prior participant in 2-AAA screening study. 
• 
Identified as eligible due to high or low plasma 2-AAA, in the absence of 
hyperglycemia, as defined by study team. 
 
Exclusion Criteria:  
• 
Individuals who currently use tobacco products. 
• 
Use of prescription or over-the-counter medications or dietary supplements 
which could modulate levels of 2-AAA and unwilling to discontinue use (from 24 
hours prior to first study visit until completion of study). Hormonal birth control is 
acceptable. 
• 
Follow a severely restricted diet or have food allergies, which would preclude 
adherence to study diet. 
• 
Newly diagnosed disease (since screening visit), including cardiovascular, renal, 
or liver disease, or Diabetes mellitus. 
• 
Individuals who are pregnant or lactating 
• 
Inability to provide written or electronic informed consent 
• 
Inability to fast for 8 hours 
 
 
 
 
 

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Figure 4. Overview of the proposed dietary intervention study. 
5.0 
Enrollment/Randomization 
 
We will enroll up to 80 subjects who meet the inclusion and exclusion criteria. We will 
identify potential participants based on their results from the screening study. We will 
contact eligible participants directly, by email or phone, and invite them to participate. 
 
6.0 
Study Procedures 
 
The study will be conducted as a randomized crossover dietary intervention, with 4 
separate study visits (Figure 4). Individuals will be randomized to normal or high lysine 
diet for 1 week, followed by a 2-week washout, where participants return to their 
habitual diet. They will then complete the second 1-week dietary intervention. The 
entire study period encompasses 4 weeks.  
 
 
 
 
 
 
Screening 
 
Subjects will be screened based on their 2-AAA levels and other data obtained during 
phase 1 of the study. Inclusion/exclusion criteria will be reviewed to confirm that the 
subject meets study eligibility requirements. Individuals who meet the inclusion criteria, 
and do not meet any of the exclusion criteria will be invited to participate in the study. 
Informed consent will be obtained via telephone with a phone consent witness or 
electronically. All participants will be instructed to discontinue use of over-the-counter 
medications and supplements on the day prior to the study visit. Participants will be 
asked about any major changes to their diet since their screening visit, and if any 
changes are reported, they will be asked to complete an online food frequency 
questionnaire (DHQ III) at any time before their visit (Food Frequency Questionnaire 
Instructions, Appendix A). In addition, participants will be asked to complete a 24-hour 
diet recall (ASA24) around the time of each study visit (Diet Recall Instructions, 
Appendix A). As part of the screening questionnaire, participants will be asked about 
their physical activity to ascertain their energy requirements for the dietary intervention. 
Participants will be asked to pick up a stool collection kit from the study coordinator 
(from the Preston Research Building, or another convenient location) prior to their first 
visit.  If you are a woman and are able to become pregnant, you will have a urine 
pregnancy test to make sure that you are not pregnant before you receive treatment in 
this study.    
 

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Study Visits 
There will be four separate study visits. All study visits will follow similar procedures. 
Subjects will be requested to arrive at a research room in the Preston Cancer Research 
building or the Vanderbilt Clinical Research Center on the morning of each study visit 
(~7:00 - 11:00 AM) in a fasting state (at least 8 hours, with no food or drink, excluding 
water). At the first visit, after informed consent has been reviewed and documented, 
inclusion/exclusion criteria will be reviewed to confirm that the subject meets study 
eligibility requirements. Subject’s medical history and medications will be discussed and 
documented by study personnel to assess for any changes that may potentially exclude 
the subject from participating. Study personnel will collect the subject’s blood pressure, 
heart rate, height, weight, waist and hip circumference. All subjects will be asked to 
provide a urine sample. Subjects will be asked to provide a drop of blood via fingerprick 
for measurement of fasting glucose. Venipuncture will be performed to obtain a blood 
sample (~50ml). All subjects will be asked to bring a stool sample (collected in the 24 
hours prior to each study visit) and will be given additional collection kits for subsequent 
samples at their study visits. Participants will be asked to fill out a questionnaire to 
assess general wellbeing and mental health (PHQ).  
 
Diet 
Subjects will meet with a dietician and be provided a menu, shopping list, and a gift card 
to purchase foods to prepare 3 meals and 1-2 snacks per day during the study period. 
All subjects will consume the same foods for both dietary periods (normal lysine and 
high lysine, 1 week each), with lysine supplements (5g/day) included for the high lysine 
diet period. Menus will be developed by the study dietitian (Dr. Heidi Silver, Co-I). 
Energy content of meals will be personalized for each participant based on the 
individual’s estimated daily energy requirement using the Harris Benedict Equation with 
an activity factor of 1.2. 
 
Compensation 
Subjects will receive $250 following successful completion of the study visit.  
 
Biomarkers 
Blood, urine and stool samples will be coded for subject confidentiality. Plasma, serum, 
buffy coat, stool and urine aliquots will be frozen at -80°C and stored for subsequent 
measurements. Peripheral blood mononuclear cells (PBMCs) may be isolated from blood 
for analysis. We are already storing buffy coat and extracting DNA in the first phase of 
this study. While we do not expect the DNA to change between visits, we will store 
additional buffy coat samples to allow for back-up samples for DNA extraction in the 
event of any sample failures from phase 1, or for any follow-up analyses. Samples will 
be prepared for metabolite measurement by methanol extraction and centrifugation in 
Dr. Ferguson’s laboratory using standard protocols31. 2-AAA and other metabolites and 
biomarkers (e.g. lysine, creatinine, insulin) will be measured at Vanderbilt core facilities 
(e.g. Mass Spectrometry core, Hormone Assay core). DNA from buffy coat will be 
extracted and submitted for genotyping at the Vanderbilt Technologies for Advanced 
Genomics (VANTAGE) core. Microbial DNA will be extracted from stool, prepared for 
sequencing (16S V4 or metagenomic), and sequenced at VANTAGE. For possible future 

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investigations, excess blood, urine and stool samples will be frozen and stored in Dr. 
Ferguson’s laboratory.  
 
 
Genomic Data: Genomic data will be deposited in dbGap, a controlled-access database, 
and will be made available as part of the Policy for Sharing of Data Obtained in NIH 
Supported or Conducted Genomic Studies.  All deposited data and metadata will be 
documented, per NIH guidelines. All data will be de-identified to protect study 
participants’ identities.   
 
7.0 
Risks 
 
Venous Blood draw: This is a routine procedure that is considered standard of care in 
clinical medicine. At the study visit, subjects will undergo venous blood draws. All blood 
draws will be performed by trained personnel using universal precautions to protect both 
the subject and personnel. The risks to subjects are minimal, but may include pain, 
bruising, allergic reaction, infection or bleeding at the needle stick site. A small number 
of individuals may experience dizziness, lightheadedness or fainting. These usually 
resolve without any specific medical therapy over the course of minutes to days. 
 
Fingerprick: This is a routine procedure that is considered standard of care in clinical 
medicine. Each subject will undergo a fingerprick using a sterile, single-use lancet to 
pierce the skin on the pad of fingertip and extract a single drop of blood. The risks to 
subjects are minimal, but may include pain, bruising at the puncture site, and/or 
infection or bleeding at the puncture site. These reactions usually resolve without any 
specific medical therapy over the course of minutes to days. 
 
Dietary Intervention and Lysine Supplementation: There are no known risks associated 
with dietary intervention. Lysine is expected to be well-tolerated by all participants. 
Lysine is used as a dietary supplement, available over the counter, with no known long-
term risks. The proposed dose of lysine may cause mild gastrointestinal upset in some 
subjects. This is expected to be minor and transient. 
 
Stool Sample Collection: There are no known risks associated with stool sample 
collection. Sample collection may be inconvenient for some individuals.  
 
Private Health Information: This information will be collected during the course of the 
study. However, only key study personnel will have access to this information, which will 
be stored in a HIPAA compliant, password protected REDCap database. No protected 
health information will be shared with employers, insurers, or non-research personnel.  
 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
 
Adverse events will be reported to the IRB per Vanderbilt University IRB policy. 
 
 
 

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9.0 
Study Withdrawal/Discontinuation 
 
Subjects may withdraw from the study at any time and should notify study personnel if 
they wish to withdraw from the study. Subjects may request their biological samples to 
be destroyed at any time. However, any data or biological samples that have already 
been used for research cannot be destroyed. Subjects may be discontinued from the 
study at the discretion of the investigator (possible reasons listed below). Subjects will 
receive financial compensation for completion of the visit. 
 
Possible reasons for withdrawal/discontinuation from study include, but are not limited 
to: 
• 
Noncompliance with treatment or procedures 
• 
Decision by participant/participant withdraws consent 
• 
Significant adverse event deemed by investigator to preclude continued 
participation 
 
 
10.0 Statistical Considerations 
 
The primary goal is to measure changes in 2-AAA in plasma and urine following dietary 
intervention study. At 80% power and α=0.05 we require 39 subjects to detect a diet-
induced change of 0.5 SD in 2-AAA. Although we expect that a true diet effect would be 
greater, we have chosen a low threshold to avoid Type 2 error. We expect that 
individuals in our high and low groups have a significantly different risk of future 
diabetes. To detect significant differences in disease risk by 2-AAA status at 80% power 
and α=0.05 (based on 1.6-fold increased risk of diabetes per SD of 2-AAA) we require 
76 subjects. These considerations informed our choice to include up to 80 subjects (40 
high and 40 low) for the dietary intervention study.  
 
11.0 Privacy/Confidentiality Issues 
 
Strict confidentiality will be maintained to the fullest extent by the research team, 
including keeping all data in a secure location. All specimens will be coded anonymously 
to remain confidential and identifiers will be kept in a separate, secure location.   
Samples may be shared with third parties outside of Vanderbilt for future testing but will 
remain anonymous to the recipient.  Subjects may contact the principal investigator at 
any time to request that samples be destroyed.  
 
12.0 Follow-up and Record Retention 
 
Anticipated study duration is 5 years. Research data will be maintained by the PI after 
study closure. After study closure, research data will be maintained for a minimum of 6 
years and possibly indefinitely. Data will be stored on the Vanderbilt University computer 
network in a password-protected database. Only members of the study team will have 

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access. Pertinent paper documentation will be kept in locked office and only study 
personnel will have access. Only personnel directly involved with the study will have 
access to source data and the electronic database. Genomic data will be deposited in 
dbGap, a controlled-access database, and will be made available as part of the Policy for 
Sharing of Data Obtained in NIH Supported or Conducted Genomic Studies.  All 
deposited data and metadata will be documented, per NIH guidelines. All data will be 
de-identified to protect study participants’ identities.   
 
13.0 References 
 
1.  
CDC. National Diabetes Statistics Report: Estimates of Diabetes and Its Burden in 
the United States, 2014. Atlanta, GA: US Department of Health and Human 
Services: 2014.  
2.  
Stokes A et al. Deaths Attributable to Diabetes in the United States: Comparison of 
Data Sources and Estimation Approaches. PLoS ONE. 2017;12:e0170219.  
3.  
Carmody D et al. GCK-MODY in the US National Monogenic Diabetes Registry: 
frequently misdiagnosed and unnecessarily treated. Acta Diabetol. 2016;53:703–8.  
4.  
Naylor RN et al. Cost-effectiveness of MODY genetic testing: translating genomic 
advances into practical health applications. Diabetes Care. 2014;37:202–9.  
5.  
Wang TJ et al. 2-Aminoadipic acid is a biomarker for diabetes risk. The Journal of 
Clinical Investigation. 2013;123:4309–17.  
6.  
Chang YF. Pipecolic acid pathway: the major lysine metabolic route in the rat brain. 
Biochemical and biophysical research communications. 1976;69:174–80.  
7.  
Posset R et al. Understanding cerebral L-lysine metabolism: the role of L-pipecolate 
metabolism in Gcdh-deficient mice as a model for glutaric aciduria type I. Journal 
of inherited metabolic disease. 2015;38:265–72.  
8.  
Danhauser K et al. DHTKD1 mutations cause 2-aminoadipic and 2-oxoadipic 
aciduria. American journal of human genetics. 2012;91:1082–7.  
9.  
Joint WHOFAOUNUEC. Protein and amino acid requirements in human nutrition. 
World Health Organ Tech Rep Ser. 2007;1–265, back cover.  
10.  Neis EP et al. The role of microbial amino acid metabolism in host metabolism. 
Nutrients. 2015;7:2930–46.  
11.  Stiles AR et al. New Cases of DHTKD1 Mutations in Patients with 2-Ketoadipic 
Aciduria. JIMD Rep. 2015; 
12.  Xu WY et al. A nonsense mutation in DHTKD1 causes Charcot-Marie-Tooth disease 
type 2 in a large Chinese pedigree. American journal of human genetics. 
2012;91:1088–94.  

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13.  Xu W et al. DHTKD1 is essential for mitochondrial biogenesis and function 
maintenance. FEBS letters. 2013;587:3587–92.  
14.  Wu Y et al. Multilayered genetic and omics dissection of mitochondrial activity in a 
mouse reference population. Cell. 2014;158:1415–30.  
15.  Yuan W et al. Amine metabolomics of hyperglycemic endothelial cells using 
capillary LC-MS with isobaric tagging. Journal of proteome research. 
2011;10:5242–50.  
16.  Zeitoun-Ghandour S et al. C. elegans metallothioneins: response to and defence 
against ROS toxicity. Molecular bioSystems. 2011;7:2397–406.  
17.  Lin H et al. Myeloperoxidase-mediated protein lysine oxidation generates 2-
aminoadipic acid and lysine nitrile in vivo. Free radical biology & medicine. 
2017;104:20–31.  
18.  Wu HQ et al. L-alpha-aminoadipic acid as a regulator of kynurenic acid production 
in the hippocampus: a microdialysis study in freely moving rats. Eur J Pharmacol. 
1995;281:55–61.  
19.  Irving BA et al. Effect of insulin sensitizer therapy on amino acids and their 
metabolites. Metabolism. 2015;64:720–8.  
20.  Sell DR et al. Aging, diabetes, and renal failure catalyze the oxidation of lysyl 
residues to 2-aminoadipic acid in human skin collagen: evidence for metal-
catalyzed oxidation mediated by alpha-dicarbonyls. Annals of the New York 
Academy of Sciences. 2008;1126:205–9.  
21.  Barazzoni R et al. Insulin fails to enhance mTOR phosphorylation, mitochondrial 
protein synthesis, and ATP production in human skeletal muscle without amino acid 
replacement. American journal of physiology. 2012;303:E1117-25.  
22.  Vallat C et al. Treatment with vigabatrin may mimic alpha-aminoadipic aciduria. 
Epilepsia. 1996;37:803–5.  
23.  Saremi A et al. Advanced Glycation End Products, Oxidation Products, and the 
Extent of Atherosclerosis During the VA Diabetes Trial and Follow-up Study. 
Diabetes Care. 2017;40:591–598.  
24.  Feldman A et al. Clinical and Metabolic Characterization of Lean Caucasian Subjects 
With Non-alcoholic Fatty Liver. Am J Gastroenterol. 2017;112:102–110.  
25.  Chang AY et al. Combining a nontargeted and targeted metabolomics approach to 
identify metabolic pathways significantly altered in polycystic ovary syndrome. 
Metabolism. 2017;71:52–63.  
26.  Robinson MM et al. High insulin combined with essential amino acids stimulates 
skeletal muscle mitochondrial protein synthesis while decreasing insulin sensitivity 
in healthy humans. J Clin Endocrinol Metab. 2014;99:E2574-83.  

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27.  Lanza IR et al. Quantitative metabolomics by H-NMR and LC-MS/MS confirms 
altered metabolic pathways in diabetes. PLoS ONE. 2010;5:e10538.  
28.  Johnson ML et al. Mechanism by Which Caloric Restriction Improves Insulin 
Sensitivity in Sedentary Obese Adults. Diabetes. 2016;65:74–84.  
29.  Esko T et al. Metabolomic profiles as reliable biomarkers of dietary composition. 
The American journal of clinical nutrition. 2017;105:547–554.  
30.  Hedrick VE et al. Dietary biomarkers: advances, limitations and future directions. 
Nutrition journal. 2012;11:109.  
31.  Zhu J et al. Colorectal cancer detection using targeted serum metabolic profiling. 
Journal of proteome research. 2014;13:4120–30.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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14.0 Appendices 
 
Appendix A 
 
Diet Questionnaire and Recall Instructions 
 
 
Food Frequency Questionnaire (NCI Diet History Questionnaire (DHQ) III)) 
At any time following consent, but before your study visit, please complete a food 
frequency questionnaire. You will be provided with a unique URL to access your 
questionnaire.  
Alternatively, we will provide you with a username and password, and the Study Access 
Code, which you may use to log in at: http://www.dhq3.org/respondent-login/ 
You will be given the option to download a Nutrition Report following completion of the 
questionnaire. 
 
This survey may take you 30-60 minutes to complete.  
 
 
 
24-Hour diet recall (NCI Automated Self-Administered 24-Hour (ASA24®) 
Dietary Assessment Tool) 
You will be asked to recall all of the foods and drinks consumed on the day before your 
study visit, starting at midnight on the day before your visit, and ending at midnight on 
the day of your study visit. You may complete the assessment in the morning before 
your study visit, or immediately following your study visit.  
The recall can be accessed at: https://asa24.nci.nih.gov/ 
Please log in with the username and password provided to you by the study coordinator. 
You will be given the option to download a Nutrition Report following completion of the 
questionnaire. 
 
This survey may take you 20-30 minutes to complete.