Conditions: Healthy
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 2
Healthy volunteers: yes

Brief summary:
This sub-study aims to assess the effect and breakdown of lysine administration, specifically examining whether it leads to increased plasma 2-AAA in healthy humans.

Detailed description:
The significance of diabetes and related co-morbidities as considerable health concerns in the US and worldwide is clearly supported by the high incidence (estimated 9.3% of the US population), mortality burden (7th leading cause of death in the US), and rising costs ($245 billion/year). Strategies to identify individuals at high diabetic risk, and to modulate disease processes in these individuals before the onset of overt disease, would have a significant impact in reducing mortality, morbidity and healthcare costs. For this approach to be successful, early markers of disease that predict at-risk individuals before onset of dysregulated glycemic control are required, as well as discovering novel pathways for therapeutic targeting.

The purpose of the study is to investigate a novel biomarker, α-aminoadipic acid (2-AAA), which may influence the risk of diabetes. 2-AAA has been identified as a novel predictor of diabetes development in humans, identifying at-risk individuals before any detectable glucose abnormalities. 2-AAA is a naturally occurring metabolite in the body, and it has no known adverse effects at normal physiological levels. 2-AAA is generated in the body from the breakdown of lysine. Lysine is one of the twenty essential amino acids, meaning that it is essential for human function, but that our body cannot manufacture it. Thus, it is acquired from dietary sources (such as meat, eggs, soybeans and legumes), with a recommended daily intake of 30 mg/kg/day. Amino acids are the building blocks of proteins, which are what allow our cells, organs and body to maintain structure and function. The investigators are interested in whether 2-AAA is increased in the body after consumption of lysine.

The investigators' specific aim is to determine whether acute lysine administration leads to increased plasma 2-AAA in humans. Catabolism of lysine leads to generation of 2-AAA. In this study, the investigators will determine whether a single dose of 13C isotope labeled lysine leads to increased plasma 2-AAA present in the blood and urine of humans. In this sub-study, the investigators will ask 2 lean, healthy subjects (preferably individuals who participated in a previous study visit) to drink a beverage containing C-13 labeled lysine and the investigators will measure the level of 2-AAA in their blood plasma and urine at baseline (before ingestion) and serially post-ingestion. The amount of lysine subjects will be given is equivalent to that which is found in a 5 oz. serving of beef. This sub-study will allow us to further establish and understand the relationship between lysine and 2-AAA in healthy subjects, and inform future studies on how to study the effects of 2-AAA on diabetes risk.

Arms:
arm 1: Two healthy subjects will be enrolled and each will undergo study procedures at one visit. After screening and consent have been conducted over the phone, subjects will participate in the study procedures. Subjects will arrive in a fasting state (no eat or drink for 8 hours, excluding water). Following collection of blood pressure, height, weight, and a urine and blood sample, subjects will be given an oral bolus of C-13 labeled lysine (5 g) in 50 ml water. This amount of lysine is equivalent to that which is found in a 5oz. serving of beef. Subjects will provide additional urine and blood samples serially post-ingestion. Because blood draws will be collected through an IV, Normal (0.9%) Saline (NS) will be infused at a rate of approximately 10 ml/hr to flush the canula prior to each blood draw.

All subjects will undergo the same procedures and interventions.

Interventions: intervention 1: C-13 labeled Lysine intervention 2: Normal saline
Intervention descriptions: intervention 1: Carbon-13 is a stable naturally occurring heavy isotope of carbon. Inclusion of a 13C label on lysine allows for subsequent differentiation between endogenous and exogenous lysine and 2-AAA for calculation of clearance and excretion of the lysine bolus. intervention 2: Because patients will have multiple blood draws during the course of the study, subjects will have an IV placed to reduce the number of needle sticks. To keep the vein open for blood collections, we will infuse Normal (0.9%) Saline (NS) at a rate of approximately 10ml/hr in order to keep the line open.

Protocol document:
Prot_SAP_000.pdf: 
Acute lysine administration to raise 2-AAA 
 
Version 1.2 
Protocol # 
 
02-14-2017  
1 
 
 
 
The effect of acute lysine administration on α-aminoadipic acid 
 
 
Principal Investigators 
 
Jane F Ferguson, PhD 
Jonathan D Brown, MD 
Thomas J Wang, MD 
 
 
Division of Cardiovascular Medicine 
Vanderbilt University Medical Center 
 
2525 West End Ste 300-A 
Nashville TN 37203 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Acute lysine administration to raise 2-AAA 
 
Version 1.2 
Protocol # 
 
02-14-2017  
2 
 
Table of Contents 
 
Contents 
1.0  
Background ..................................................................................................................... 3 
2.0  
Rationale and Specific Aims ............................................................................................ 4 
3.0  
Animal Studies and Previous Human Studies .................................................................. 4 
4.0  
Inclusion/Exclusion Criteria.............................................................................................. 5 
5.0 
Enrollment ....................................................................................................................... 5 
6.0 
Study Procedures ............................................................................................................ 6 
7.0 
Risks of Investigational Agents (side effects) ................................................................... 7 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or 
Others ........................................................................................................................................ 9 
9.0  
Study Withdrawal/Discontinuation ..................................................................................10 
10.0 
Statistical Considerations ............................................................................................10 
11.0 Privacy/Confidentiality Issues .........................................................................................11 
12.0 Follow-up and Record Retention .......................................................................................11 
13.0 References ........................................................................................................................12 
Appendices ...............................................................................................................................15 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Acute lysine administration to raise 2-AAA 
 
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Protocol # 
 
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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 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 levels5. 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 major global health concern, associated with significantly increased mortality and 
high incidence of co-morbidities. The lysine-derived metabolite α-aminoadipic acid (2-AAA) has 
been identified as a novel predictor of diabetes development in humans, identifying at-risk 
individuals before any detectable glucose abnormalities. There is exciting preliminary evidence 
supporting the role of 2-AAA in development of diabetes, 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 aims of the study are designed to 
test feasibility of 2-AAA manipulation in humans through acute lysine administration.  
 
Specific Aim: To determine whether acute lysine administration leads to increased 
plasma 2-AAA in humans. Catabolism of lysine leads to generation of 2-AAA. To address the 
hypothesis that acute lysine ingestion leads to increased circulating and excreted 2-AAA in 
humans in vivo, we will administer lysine (10g orally) to healthy volunteers (N=10), and measure 
the level of 2-AAA in plasma and urine at baseline and serially post-ingestion.  
 
Sub-Study Aim: To measure clearance and excretion of lysine and 2-AAA using stable 
isotope tracer 13C lysine (5g orally). Carbon-13 is a stable naturally occurring heavy isotope 
of carbon. Inclusion of a 13C label on lysine allows for subsequent differentiation between 
endogenous and exogenous lysine and 2-AAA for calculation of clearance and excretion of the 
lysine bolus. 
 
 
3.0  
Animal Studies and Previous Human Studies 
The primary goal of this study is to raise circulating 2-AAA in humans. The effects of 2-AAA 
administration have not been studied in humans, with no known studies of 2-AAA 
supplementation in humans. However 2-AAA occurs naturally in the body, and has no known 
adverse effects at physiological levels. Increased dietary lysine in chickens resulted in increased 
2-AAA in muscle14, however the effect of lysine on 2-AAA in humans remains to be tested in 
healthy subjects. Older human studies have demonstrated an increase in 2-AAA following lysine 
administration in children with alpha-aminoadipic aciduria15, 16, a rare condition (< 1/1,000,000), 


Acute lysine administration to raise 2-AAA 
 
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Protocol # 
 
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with variable clinical phenotypes including intellectual disability, developmental delay, epilepsy, 
muscular hypotonia and ataxia8.Several studies have examined the effects of lysine 
supplementation, albeit without concurrent measurement of 2-AAA. Acute lysine administration 
in healthy subjects led to increased plasma lysine, lower plasma glucose, a small increase in 
insulin area, and higher glucagon. When lysine was administered in conjunction with glucose, 
relative to glucose alone there was a 44% reduction in the 2.5-h glucose area response, a 
diminished reduction in glucagon, but no change in insulin17. In T2DM subjects, 
supplementation with essential amino acids or lysine for 2 months showed some evidence of a 
decrease in postprandial plasma glucose18.Other studies have shown no effect on glucose 
tolerance19, fasting glucose20, or insulin sensitivity21. Other lysine supplementation studies have 
found variable effects on other biomarkers, including plasma lipids20, diarrhea morbidity, 
respiratory illness and CRP22, anxiety, cortisol and chromogranin-A23, and incidence of herpes 
simplex virus24-26. The stable isotope tracer 13C-lysine replaces 12C with 13C on the lysine 
molecule. Previous studies have used isotope labeled lysine to calculate amino acid flux27, 28. 
Consumption of baked beef as part of a test meal led to increased plasma 2-AAA when 
compared with a test meal including either baked or pickled herring29. 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 sensitivity30. Acute insulin infusion (7 hrs) in 
these subjects also led to a decrease in both lysine and 2-AAA30, 31. 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 aciduria.32  
 
4.0  
Inclusion/Exclusion Criteria 
 
Inclusion Criteria:  
• 
Men and women ages 18-45 years  
• 
BMI 18 to <25 kg/m2 
 
Exclusion Criteria:  
• 
Current use of prescription medications (apart from hormonal birth control) 
• 
Current use of amino acid supplements (including branched-chain amino acids) or 
supplemental protein (habitual consumption of protein powder, bars, shakes), and 
unwilling to temporarily discontinue use (1 week prior to study visit) 
• 
Individuals who currently use tobacco products or have done so in the previous 30 days 
• 
Prior or current cardiovascular disease, renal disease, or liver disease 
• 
Diabetes mellitus (taking insulin, other anti-diabetic agents, or diet-controlled) 
• 
Atrial fibrillation 
• 
Bleeding disorder or anemia 
• 
Positive pregnancy test 
• 
Women who are breastfeeding 
• 
Participation in another clinical trial within the previous 6 weeks prior to the study visit 


Acute lysine administration to raise 2-AAA 
 
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Protocol # 
 
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• 
Inability to provide written informed consent 
• 
Inability to fast for 8 hours 
5.0 
Enrollment 
 
We will enroll 10 patients (N=5 male, N=5 female) who meet the inclusion and exclusion criteria. 
In the sub-study, we will enroll 2 patients who meet the inclusion and exclusion criteria.  
 
The following recruitment approaches will be used: Vanderbilt CTSA (VICTR) Research 
Notification Distribution List, the linked email system which reaches Vanderbilt faculty and staff, 
as well as members of the Middle Tennessee community, and ResearchMatch, a national online 
registry maintained at Vanderbilt which allows people to self-register and express interest as 
research participants. In addition, we will contact lean subjects who had participated in our 
group’s prior research protocols and who had expressed interest in participating in future 
research studies. Individuals who participate in the first study (using 10g lysine) who expressed 
interest in participating in future studies may be approached about participating in the sub-study 
for comparison purposes. Subjects may also be identified by means of prescreening the medical 
record, by the research coordinator.  
 
 
6.0 
Study Procedures 
 
Screening 
Subjects will be screened via email and/or telephone. Informed consent will be obtained via 
telephone with a phone consent witness. After consent has been obtained, inclusion/exclusion 
criteria will be reviewed to confirm that the subject meets study eligibility requirements. Subjects 
who meet the inclusion criteria, and do not meet any of the exclusion criteria will be invited to 
participate in the study. Subjects who use protein or amino acid supplements but are willing to 
discontinue use for at least 1 week prior to the study may be included. Additionally, all subjects 
will be instructed to discontinue use of over-the-counter medications, alcohol, and strenuous 
exercise on the day prior to the study visit. 
 
Subjects will be requested to record their dietary intake of all foods and beverages on the day 
prior to the study visit (see Diet Record Instructions, Appendix A).  
 
 
Study Visit 
Subjects will be requested to arrive at the Vanderbilt Clinical Research Center on the morning of 
the study visit (~8:00 AM) in a fasting state (at least 8 hours, with no food or drink, excluding 
water). 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 


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personnel will collect the subject’s vital signs, height and weight prior to study interventions. All 
subjects will be asked to provide a urine sample for baseline 2-AAA measurement and will 
undergo a urine pregnancy test (if the woman is of child-bearing potential).  
 
Following the baseline urine sample, an intravenous line will be inserted into a superficial 
peripheral vein to perform blood draws.  Subjects will be asked to remain in a seated position for 
the duration of the study, but will be permitted to walk for brief periods throughout the study (e.g. 
to use the restroom). A baseline blood draw will be taken (time = 0 hr) for measurement of 
baseline plasma 2-AAA levels and related amino acids and metabolites (e.g. lysine, kynurenic 
acid).Immediately following the baseline blood draw, subjects will be given an oral bolus of L-
lysine (10g) (Ajinomoto USA Inc, Raleigh, NC) in 100ml water. This is an amount of lysine 
equivalent to that which is found in a 10oz serving of beef. A similar dose of lysine has 
previously been shown to alter the response to glucose17. Blood samples will be taken at time = 
30 minutes 1, 2, 3, and 4 hrs post-lysine administration. Normal (0.9%) Saline (NS) will be 
infused at a rate of 10 ml/hr to flush the canula prior to each blood draw, and a 5ml blood 
discard will be performed prior to each collection of samples.  Each blood draw (including 
baseline) will collect about 20cc of blood, for a total collection of 120cc, or approximately 8 
tablespoons of blood. Blood will be collected into one tube for serum (5ml) and 2 tubes for 
plasma (4ml each). Urine samples will be collected throughout the visit, in 2-hour increments (0-
2 and 2-4 hrs post-lysine). Although the timing of plasma 2-AAA increases post-lysine have 
never been measured, peak post-prandial plasma 2-AAA levels are expected to be observed 
~3hours post-lysine29. Plasma will be prepared from blood samples, and both plasma and urine 
aliquots will be frozen at -80°C and stored prior to analysis. Plasma and urine samples will be 
prepared for metabolite measurement by methanol extraction and centrifugation in Dr. 
Ferguson’s laboratory using standard protocols33. 2-AAA and other metabolites and biomarkers 
(e.g. lysine, creatinine) will be measured at Vanderbilt core facilities (e.g. Mass Spectrometry 
core).  
 
Compensation 
Subjects will receive $150 following successful completion of the study visit.  
 
Biomarkers 
Blood and urine samples will be coded for subject confidentiality. Measurements of interest will 
be performed at Vanderbilt University. For possible future investigations, excess blood and urine 
samples will be frozen and stored in Dr. Ferguson’s laboratory.  
 
6.1 Sub-Study Procedures – Lysine Tracer 
Screening 
Of subjects who successfully completed the primary study, we will invite two individuals to return 
for a second visit. If we are unable to recruit from our original enrollees, we will recruit from 
within the original pool of interested volunteers or from participants who expressed interest in 
our future studies, using the same screening procedures described above. Subjects will be 


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asked about any changes in their health and medications that have occurred since their 
participation in the lysine study. 
 
Study Visit 
Subjects will be requested to arrive at the Vanderbilt Clinical Research Center on the morning of 
the sub-study visit (~8:00 AM) in a fasting state (at least 8 hours, with no food or drink, 
excluding water). 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 vital signs, height and weight prior to 
study interventions. All subjects will be asked to provide a urine sample for baseline 2-AAA 
measurement and will undergo a urine pregnancy test (if the woman is of child-bearing 
potential).  
 
Following the baseline urine sample, an intravenous line will be inserted into a superficial 
peripheral vein to perform blood draws.  Subjects will be asked to remain in a seated position for 
the duration of the study, but will be permitted to walk for brief periods throughout the study (e.g. 
to use the restroom). A baseline blood draw will be taken (time = 0 hr) for measurement of 
baseline plasma 2-AAA levels and related amino acids and metabolites (e.g. lysine, kynurenic 
acid).Immediately following the baseline blood draw, subjects will be given an oral bolus of 13C 
L-lysine (5g) (Cambridge Isotope Laboratories, Tewksbury, MA) in 50ml water. This is an 
amount of lysine equivalent to that which is found in a 5oz serving of beef. Blood samples will 
be taken at time = 30 minutes 1, 2, 3, 4, 5 and 6 hrs post-lysine administration. Normal (0.9%) 
Saline (NS) will be infused at a rate of approximately 10 ml/hr to flush the canula prior to each 
blood draw, and a 5ml blood discard will be performed prior to each collection of samples.  Each 
blood draw (including baseline) will collect about 20cc of blood, for a total collection of about 
160cc, or approximately 11 tablespoons of blood. After a 3-5ml discard, blood will be collected 
into one tube for serum (5ml) and 2 tubes for plasma (4ml each). Urine samples will be collected 
throughout the visit, in 2-hour increments (0-2, 2-4, and 4-6 hrs post-lysine). Plasma will be 
prepared from blood samples, and both plasma and urine aliquots will be frozen at -80°C and 
stored prior to analysis. Plasma and urine samples will be prepared for metabolite measurement 
by methanol extraction and centrifugation in Dr. Ferguson’s laboratory using standard 
protocols33. 2-AAA and other metabolites and biomarkers (e.g. lysine, creatinine) will be 
measured at Vanderbilt core facilities (e.g. Mass Spectrometry core).  
 
Compensation 
Subjects will receive $150 following successful completion of the study visit.  
 
Biomarkers 
Blood and urine samples will be coded for subject confidentiality. Measurements of interest will 
be performed at Vanderbilt University. For possible future investigations, excess blood and urine 
samples will be frozen and stored in Dr. Ferguson’s laboratory.  


Acute lysine administration to raise 2-AAA 
 
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7.0 
Risks of Investigational Agents  
Lysine Administration: 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. There are 
no additional risks associated with the use of stable isotope tracer 13C lysine. The proposed 
dose of lysine may cause mild gastrointestinal upset in some subjects. This is expected to be 
minor and transient.  
 
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, allergic reaction, infection or 
bleeding at the needle stick site. These usually resolve without any specific medical therapy 
over the course of minutes to days. 
 
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. 
 
 


Acute lysine administration to raise 2-AAA 
 
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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 
Given the pilot nature of this proposal, the primary goal is to detect whether there is a 
measureable increase in 2-AAA in plasma or urine following lysine administration.  
 
Based on preliminary analyses in a separate study (N=120 healthy individuals), we expect a 
mean baseline 2-AAA of 0.00299 with a standard deviation of 0.0025 (arbitrary units based on 
mass spec measurements). Accounting for this between-subject variability, in order to detect a 
significant change in 2-AAA post-lysine, we will require a mean post-lysine 2-AAA concentration 
of 0.0055 (at 80% power, α=0.05). We expect the actual increase to be larger than this, and 
thus expect to be well-powered to detect a difference in this pilot sample. Although no data exist 
for the expected change in 2-AAA, a dietary study measuring 2-AAA following protein-rich meals 
reported a considerable post-prandial increase in 2-AAA (e.g. > 10 fold increase)29. 
 
The change in 2-AAA will be analyzed both as the overall area under the curve, and as the 
change from baseline to each subsequent timepoint (analyzed by paired T tests).  


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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 after they are obtained and 
the code key kept in a secure location.  Blood samples 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 1-2 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 access. Pertinent 
paper documentation will be kept in a 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. 
 
 
 


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13.0 References 
1. 
CDC. National Diabetes Statistics Report: Estimates of Diabetes and Its Burden in the United 
States, 2014. 2014. 
2. 
Wang TJ, Ngo D, Psychogios N, Dejam A, Larson MG, Vasan RS, Ghorbani A, O'Sullivan J, Cheng S, 
Rhee EP, Sinha S, McCabe E, Fox CS, O'Donnell CJ, Ho JE, Florez JC, Magnusson M, Pierce KA, Souza AL, 
Yu Y, Carter C, Light PE, Melander O, Clish CB and Gerszten RE. 2-Aminoadipic acid is a biomarker for 
diabetes risk. J Clin Invest. 2013;123:4309-4317. 
3. 
Wu HQ, Ungerstedt U and Schwarcz R. 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. 
4. 
Sell DR, Strauch CM, Shen W and Monnier VM. 2-aminoadipic acid is a marker of protein 
carbonyl oxidation in the aging human skin: effects of diabetes, renal failure and sepsis. The Biochemical 
journal. 2007;404:269-277. 
5. 
Wu Y, Williams EG, Dubuis S, Mottis A, Jovaisaite V, Houten SM, Argmann CA, Faridi P, Wolski W, 
Kutalik Z, Zamboni N, Auwerx J and Aebersold R. Multilayered genetic and omics dissection of 
mitochondrial activity in a mouse reference population. Cell. 2014;158:1415-1430. 
6. 
Chang YF. Pipecolic acid pathway: the major lysine metabolic route in the rat brain. Biochemical 
and biophysical research communications. 1976;69:174-180. 
7. 
Posset R, Opp S, Struys EA, Volkl A, Mohr H, Hoffmann GF, Kolker S, Sauer SW and Okun JG. 
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;38:265-272. 
8. 
Danhauser K, Sauer SW, Haack TB, Wieland T, Staufner C, Graf E, Zschocke J, Strom TM, Traub T, 
Okun JG, Meitinger T, Hoffmann GF, Prokisch H and Kolker S. DHTKD1 mutations cause 2-aminoadipic 
and 2-oxoadipic aciduria. American journal of human genetics. 2012;91:1082-1087. 
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, Dejong CH and Rensen SS. The role of microbial amino acid metabolism in host 
metabolism. Nutrients. 2015;7:2930-2946. 
11. 
Stiles AR, Venturoni L, Mucci G, Elbalalesy N, Woontner M, Goodman S and Abdenur JE. New 
Cases of DHTKD1 Mutations in Patients with 2-Ketoadipic Aciduria. JIMD Rep. 2015. 
12. 
Xu WY, Gu MM, Sun LH, Guo WT, Zhu HB, Ma JF, Yuan WT, Kuang Y, Ji BJ, Wu XL, Chen Y, Zhang 
HX, Sun FT, Huang W, Huang L, Chen SD and Wang ZG. A nonsense mutation in DHTKD1 causes Charcot-
Marie-Tooth disease type 2 in a large Chinese pedigree. American journal of human genetics. 
2012;91:1088-1094. 
13. 
Xu W, Zhu H, Gu M, Luo Q, Ding J, Yao Y, Chen F and Wang Z. DHTKD1 is essential for 
mitochondrial biogenesis and function maintenance. FEBS letters. 2013;587:3587-3592. 
14. 
Watanabe G, Kobayashi H, Shibata M, Kubota M, Kadowaki M and Fujimura S. Regulation of free 
glutamate content in meat by dietary lysine in broilers. Anim Sci J. 2015;86:435-442. 
15. 
Casey RE, Zaleski WA, Philp M, Mendelson IS and MacKenzie SL. Biochemical and clinical studies 
of a new case of alpha-aminoadipic aciduria. Journal of inherited metabolic disease. 1978;1:129-135. 
16. 
Fischer MH and Brown RR. Tryptophan and lysine metabolism in alpha-aminoadipic aciduria. Am 
J Med Genet. 1980;5:35-41. 
17. 
Kalogeropoulou D, LaFave L, Schweim K, Gannon MC and Nuttall FQ. Lysine ingestion markedly 
attenuates the glucose response to ingested glucose without a change in insulin response. The American 
journal of clinical nutrition. 2009;90:314-320. 
18. 
Natarajan Sulochana K, Lakshmi S, Punitham R, Arokiasamy T, Sukumar B and Ramakrishnan S. 
Effect of oral supplementation of free amino acids in type 2 diabetic patients-- a pilot clinical trial. Med 
Sci Monit. 2002;8:CR131-137. 


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19. 
Gater DR, Gater DA, Uribe JM and Bunt JC. Effects of arginine/lysine supplementation and 
resistance training on glucose tolerance. Journal of applied physiology. 1992;72:1279-1284. 
20. 
Hlais S, Reslan DR, Sarieddine HK, Nasreddine L, Taan G, Azar S and Obeid OA. Effect of lysine, 
vitamin B(6), and carnitine supplementation on the lipid profile of male patients with 
hypertriglyceridemia: a 12-week, open-label, randomized, placebo-controlled trial. Clin Ther. 
2012;34:1674-1682. 
21. 
Unni US, Raj T, Sambashivaiah S, Kuriyan R, Uthappa S, Vaz M, Regan MM and Kurpad AV. The 
effect of a controlled 8-week metabolic ward based lysine supplementation on muscle function, insulin 
sensitivity and leucine kinetics in young men. Clinical nutrition (Edinburgh, Scotland). 2012;31:903-910. 
22. 
Ghosh S, Smriga M, Vuvor F, Suri D, Mohammed H, Armah SM and Scrimshaw NS. Effect of 
lysine supplementation on health and morbidity in subjects belonging to poor peri-urban households in 
Accra, Ghana. The American journal of clinical nutrition. 2010;92:928-939. 
23. 
Smriga M, Ando T, Akutsu M, Furukawa Y, Miwa K and Morinaga Y. Oral treatment with L-lysine 
and L-arginine reduces anxiety and basal cortisol levels in healthy humans. Biomed Res. 2007;28:85-90. 
24. 
Thein DJ and Hurt WC. Lysine as a prophylactic agent in the treatment of recurrent herpes 
simplex labialis. Oral Surg Oral Med Oral Pathol. 1984;58:659-666. 
25. 
DiGiovanna JJ and Blank H. Failure of lysine in frequently recurrent herpes simplex infection. 
Treatment and prophylaxis. Arch Dermatol. 1984;120:48-51. 
26. 
Chi CC, Wang SH, Delamere FM, Wojnarowska F, Peters MC and Kanjirath PP. Interventions for 
prevention of herpes simplex labialis (cold sores on the lips). Cochrane database of systematic reviews 
(Online). 2015;8:CD010095. 
27. 
El-Khoury AE, Basile A, Beaumier L, Wang SY, Al-Amiri HA, Selvaraj A, Wong S, Atkinson A, Ajami 
AM and Young VR. Twenty-four-hour intravenous and oral tracer studies with L-[1-13C]-2-aminoadipic 
acid and L-[1-13C]lysine as tracers at generous nitrogen and lysine intakes in healthy adults. The 
American journal of clinical nutrition. 1998;68:827-839. 
28. 
Metges CC, El-Khoury AE, Henneman L, Petzke KJ, Grant I, Bedri S, Pereira PP, Ajami AM, Fuller 
MF and Young VR. Availability of intestinal microbial lysine for whole body lysine homeostasis in human 
subjects. Am J Physiol. 1999;277:E597-607. 
29. 
Ross AB, Svelander C, Undeland I, Pinto R and Sandberg AS. Herring and Beef Meals Lead to 
Differences in Plasma 2-Aminoadipic Acid, beta-Alanine, 4-Hydroxyproline, Cetoleic Acid, and 
Docosahexaenoic Acid Concentrations in Overweight Men. The Journal of nutrition. 2015. 
30. 
Irving BA, Carter RE, Soop M, Weymiller A, Syed H, Karakelides H, Bhagra S, Short KR, Tatpati L, 
Barazzoni R and Nair KS. Effect of insulin sensitizer therapy on amino acids and their metabolites. 
Metabolism. 2015;64:720-728. 
31. 
Barazzoni R, Short KR, Asmann Y, Coenen-Schimke JM, Robinson MM and Nair KS. 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:E1117-1125. 
32. 
Vallat C, Rivier F, Bellet H, Magnan de Bornier B, Mion H and Echenne B. Treatment with 
vigabatrin may mimic alpha-aminoadipic aciduria. Epilepsia. 1996;37:803-805. 
33. 
Zhu J, Djukovic D, Deng L, Gu H, Himmati F, Chiorean EG and Raftery D. Colorectal cancer 
detection using targeted serum metabolic profiling. Journal of proteome research. 2014;13:4120-4130. 


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Appendix A 
 
Diet Record Instructions 
 
You will need to write down ALL food and beverages consumed on the assigned day.  Please 
take the diet record with you all day during the day you are recording intake and record items at 
the same time you consume any food or beverage.  There are four columns that need to include 
information about a food or beverage you consume:  Time, Food/beverage, Amount, and 
Description/Preparation.   
 
Time:  List the time you consumed each food or beverage 
 
Food/Beverage:  Write down the name of the food or beverage.  Use brand names (e.g. Burger 
King, Big Mac, Lean Cuisine) whenever possible.  Remember to list all additions to foods and 
beverages such as cream, sugar, butter, jelly, lemon, ketchup, etc.  For mixed dishes (e.g. 
pizza, casseroles, etc) list each food item and try to include the amount for each item.  If you 
use a recipe, divide the total amount used by the number of servings you had.  For example, if 
you used .5 lb of 85% lean ground beef to make a meat loaf that made 4 servings and you had 
1 serving, the total amount of group beef you should include is 1/8 of a pound.  Or if possible, 
you may provide the recipe and list what proportion of the entire recipe you ate.   
 
Amount:  List the amount eaten or drank in ounces (oz), teaspoons (tsp), tablespoons (tbsp), 
cups (c) or in units such as 1 slice of whole wheat bread, or 1 orange.  When listing meats, list 
for cooked amounts. 
 
Description/Preparation:  Describe methods used to prepare food or beverages (ex:  baked, 
grilled).  Include any oil or other products used in the preparing of food (e.g. 1 tsp olive oil to 
sauté vegetables).  Include brand names or fast-food and restaurant names when possible 
 
Reminders List:  Please use this list to remind you to include all food and drink that you 
consume. 
 
1. Record all snacks you ate during each day. 
 
2. Record all beverages such as coffee, tea, water, milk, soda pop, juice, alcohol, etc. 
 
3. Include any items you added to your food or drink such as cream, milk, sugar, lettuce, 
tomato, ketchup, mustard, pickles, butter, margarine, etc. 
 
4. Record the time of the day when you eat any food or beverage 
 
5. Make sure to describe how each food was prepared (baked, fried, broiled, etc) 
 
BE AS DESCRIPTIVE AS POSSIBLE 


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The following is an example of a diet record.  Please note how detailed it is. 
Time 
 
Food/Beverage  
 
Amount 
 
Description 
8:00 AM 
cereal 
 
 
 
1 cup 
 
 
Cheerios 
                        
milk 
 
 
 
½ cup 
 
 
2%  
 
bread 
 
 
 
2 slices  
 
whole wheat 
 
jelly 
 
 
 
1 tbsp 
 
 
Smuckers, strawberry 
                       
 juice 
 
 
 
6 oz 
 
 
Tropica, orange, no pulp 
12:00 PM 
pizza 
 
 
 
2 slices   
16” pizza from Pizza Hut, 
thin crust with mozzarella 
cheese, tomato sauce, and 
pepperoni  
 
Coke 
 
 
 
12 oz 
 
Cookies  
 
 
3                                  chocolate chip, medium size 
7:00 PM  
Chicken  
 
 
3 oz 
 
 
baked, skinless, boneless 
 
olive oil  
 
 
1 tbsp 
 
 
used to cook chicken 
 
lemon juice 
 
 
1 tbsp 
 
 
drizzled over chicken 
 
salad 
 
 
 
1 cup 
 
 
ingredients listed below 
   lettuce  
 
 
4 leaves  
 
iceberg 
 
   red tomato 
 
 
¼ of tomato 
 
raw 
 
   cucumber 
 
 
4 slices  
 
peeled 
croutons 
6 each                              onion flavored, Pepperidge 
Farms 
 
   Dressing 
 
 
2 tbsp 
 
 
Low-fat ranch, Ken’s  
                       French fries 
 
 
¾ cup 
 
 
crispy, baked,  
10:00 PM 
   apple  
 
 
1 each  
 
red and delicious, medium 
                          coffee 
 
 
 
6 oz 
 
 
milk added (see below) 
                            milk 
 
 
 
1 tbsp 
 
 
2%  
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Name:  ______________________________________ 
 
 
Day:  ____________  
 
 
 
 
 
 
 
 
 
 
 
Date:  ____________ 
DIET RECORD 
 
Time 
Food/Beverage 
Amount 
Description