The Journal of Nutrition

Supplement: 8th Workshop on the Assessment

of Adequate and Safe Intake of Dietary Amino Acids

Determination of the Tolerable Upper Intake

Level of Leucine in Adult Men1–3
Paul B. Pencharz,4–7* Rajavel Elango,8 and Ronald O. Ball5, 7

4
The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada; 5Department of Nutritional Sciences, and 6Paediatrics, University
of Toronto, Toronto, ON, Canada; 7Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada;
and 8Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada

Abstract
Leucine is purported to improve athletic performance. Therefore, the BCAA, especially leucine, are popular as dietary
supplements among strength-training athletes. There are, however, concerns regarding possible adverse effects of excessive

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leucine intake. The objective of the current study was to determine the metabolic and adverse effects of the acute ingestion of
very high intakes of leucine supplements. Five healthy men (20–35 y) each received graded stepwise increases in leucine
intakes of 50, 150, 250, 500, 750, 1000, and 1250 mg
kg21
d21 corresponding to the Estimated Average Requirement, and
Estimated Average Requirement 33, 35, 310, 315, 320, and 325 to a total of 29 studies. The graded stepwise approach
was used rather than a randomization of leucine intake to minimize the possibility of severe adverse effects. Participants were
given a maintenance diet for 2 d prior to each leucine level containing 1 g
kg21
d21 of protein and 1.73 measured the resting
metabolic rate. Leucine oxidation was determined using L-[1–13C]-leucine and the appearance of 13CO2 (calculated as F13CO2)
in breath. A range of markers was used to monitor for adverse effects, including glucose, insulin, alanine aminotransferase, and
ammonia. Plasma leucine concentrations significantly increased beyond an intake of 500 mg
kg21
d21. The metabolic limit
to oxidize leucine was between 550 and 700 mg
kg21
d21. An increase in blood ammonia concentrations was observed at
leucine intakes >500 mg
kg21
d21. There were no changes in liver alanine aminotransferase. Glucose concentrations fell
(P < 0.004) but remained within the normal range and without any change in insulin. This study is the first to our knowledge
to directly estimate the safe upper limit of leucine intake in humans and raises concerns that intakes >550 mg
kg21
d21
or ;39 g/d may be a risk to health. It is important to note that these are acute studies, where each participant was exposed
to graded increases in leucine intake. Longer term adaptation was not studied. J. Nutr. 142: 2220S–2224S, 2012.

Introduction
The use of dietary supplements is on the rise, especially among
athletes. Reports suggest that almost 85% of professional
1
Published in a supplement to The Journal of Nutrition. Presented at the 8th
Workshop on the Assessment of Adequate and Safe Intake of Dietary Amino Acids,
held in Washington, DC, November 10–11, 2011. The conference was sponsored by
the International Council on Amino Acid Science (ICAAS) and the International Life
Sciences Institute (ILSI) Research Foundation. The Organizing Committee for the
workshop included Sidney M. Morris, Jr, Dennis M. Bier, Luc A. Cynober, Motoni
Kadowaki, and Andrew G. Renwick. The views expressed in these papers are not
necessarily those of the Supplement Coordinator or Guest Editors. The Supplement
Coordinator for this supplement was DÕAnn Finley, University California, Davis.
Supplement Coordinator disclosures: DÕAnn Finley received travel support and
compensation from ICAAS for editorial services provided for this supplement
publication. The supplement is the responsibility of the Guest Editor to whom the
Editor of The Journal of Nutrition has delegated supervision of both technical
conformity to the published regulations of The Journal of Nutrition and general
oversight of the scientific merit of each article. The Guest Editor for this supplement
was Harry Dawson. Guest Editor disclosure: Harry Dawson had no conflicts to
disclose. Publication costs for this supplement were defrayed in part by the payment
of page charges. This publication must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 USC section 1734 solely to indicate this fact. The opinions
expressed in this publication are those of the authors and are not attributable to the
sponsors or the Publisher, Editor, or Editorial Board of The Journal of Nutrition.
* To whom correspondence should be addressed. E-mail: paul.pencharz@
sickkids.ca.
athletes consume some form of nutritional supplements, with
the most popular ones being vitamins, antioxidants, and protein,
including creatine and amino acids (1). In the United States,
~3.4% of the general population uses amino acid supplements,
62% on a daily basis (2), and thus we must be concerned that
these individuals may consume excessive amounts of amino acids.
Excessive intakes of free amino acid may have adverse effects;
however, there are very few data to either confirm or deny this
position. Some amino acids have specific metabolic functions in
addition to the requirements for protein synthesis, e.g., stimula-
tion of protein synthesis by leucine, synthesis of catecholamines
from aromatic amino acids, methyl and sulfur donation from
sulfur amino acids, NO from arginine, etc. Therefore, dietary
supplementation with specific amino acids in excess of the
requirement for protein synthesis may have adverse effects, or it
may be beneficial in some situations. For these reasons, additional
knowledge is necessary regarding the highest possible intake of
each amino acid at which no adverse effect occurs (3,4).
2
Supported by a grant from the International Council on Amino Acid Science
(ICAAS). P.B. Pencharz, R. Elango, and R.O. Ball received travel funds from the
ICAAS to attend the workshop.
3
Author disclosures: P. B. Pencharz, R. Elango, and R. O. Ball, no conflicts of interest.

2220S ã 2012 American Society for Nutrition.

First published online October 17, 2012; doi:10.3945/jn.112.160259.
 The BCAA include leucine, valine, and isoleucine and are
popular as dietary supplements among strength-training athletes
(5). Leucine in particular has been shown to stimulate protein
synthesis and promote anabolism (6) and is widely used by athletes
who think it helps improve performance. Most of the initial studies
to test for leucine supplementation and effect of performance were
negative (7,8), although recent studies seem to suggest benefits with
leucine supplementation (9,10), particularly when combined with
whey protein, which has a high leucine concentration (11,12).
Furthermore, the most recent focus has been on leucine supple-
mentation on decreasing exercise-induced muscle damage (13–16),
which shows promising results and has potential for numerous
clinical applications. Thus, there is a critical and urgent need to
identify the safe upper limit of leucine that can be consumed.
A review of the literature can be conducted based on the numerous
leucine supplementation studies conducted to identify a safe intake.
Fernstrom (17) earlier summarized studies in athletes, normal
adults, and patients with clinical disorders and reported that
intakes of 15–60 g
d21 (;200–850 mg
kg21
d21 for a 70-kg
man) of total BCAA did not result in adverse event outcomes
for the variables monitored. A considerable problem with
interpreting the reported human studies is the great variability in
approaches; these experiments included supplements of all 3
BCAA at different doses, different ratios among the BCAA,
different routes of infusion/feeding (i.v. vs. oral), and different
athletic training regimes. Thus, there is a need to identify direct
approaches to determine a safe upper limit of leucine intake.
Recently, we proposed one such approach (18) and the following
article describes our approach with the experimental evidence (19).
Concept behind UL of amino acid oxidation
Markers for identifying excess intake of an amino acid should have
very specific dose-response characteristics. In particular, variation
with intake should display an inflection point that would identify
the onset of the amino acid excess situation (18). Previously, in
neonatal piglets, we observed an upper inflection point in the dose-
response curve for phenylalanine retention and 14CO2 production
from phenylalanine oxidation with graded phenylalanine intake
(20). Piglets received graded phenylalanine intakes ranging from
0.2 to 1.2 g
kg21
d21. The apparent phenylalanine balance,
which was calculated as the difference between phenylalanine
intake and oxidation, increased linearly between 0.2 and 0.5 g
phenylalanine
kg21
d21. Apparent phenylalanine balances did
not differ for intakes between 0.5 and 0.8 g phenylalanine
kg21

d21. At the highest phenylalanine intakes, the apparent phenyl-
alanine balance was significantly higher than the plateau values.
Phenylalanine balance reflects the net rate of accretion or retention
of phenylalanine in body tissues. The increase in phenylalanine
balance beyond the intake of 0.8 g
kg21
d21 indicated that
oxidative processes were unable to keep pace with the increasing
intake of phenylalanine. The inflection point at 0.8 g
kg21
d21
was the ‘‘metabolic limit’’ to oxidize or catabolize phenylalanine in
neonatal piglets and was supported by the observation of signi-
ficantly higher plasma concentrations of phenylalanine and tyro-
sine in the piglets receiving 1.2 mg
kg21
d21 (20). Once the
maximum level of phenylalanine oxidation was reached, the
plasma phenylalanine concentrations and retention rose rapidly.
Hence, we reasoned that a suitable marker to define the tolerable
upper intake level (UL) for a dietary amino acid would be the level
at which the maximum oxidation level was reached (Fig. 1). This
approach was initially described and proposed by our group during
the 3rd Amino Acid Assessment Workshop (21) and subsequently
reinforced during the 5th (22) and 7th Amino Acid Assessment
Workshops (18).
FIGURE 1 Schematic to describe oxidative response to increasing
amino acid intake [Adapted with permission from (18)].
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Leucine UL in animal models
Sakai et al. (23) used the above-described approach to identify
leucine excess in rats. They identified the metabolic limit by
measuring 13CO2 production from U-13C-leucine with increas-
ing leucine intake in the excess range. The maximum limit to
oxidize excess leucine was reached at 10% of dietary intake or
8900 mg
kg21
d21; the oxidation achieved a plateau and this
inflection point was identified as the UL of leucine intake in rats.
The same authors, in an earlier study using a similar strain of
rats that received a diet similar in composition, observed
significant growth inhibition in rats fed 15% leucine or 12,400
mg leucine/kg (23). This suggests that the inflection point at
which the maximum limit to oxidize excess leucine is reached is
an early marker to identify the potential for an adverse event (in
this case, growth inhibition) and may more appropriately
identify the UL.
Tsubuku et al. (24) in a controlled experiment studied oral
supplements of leucine excess in 4-wk-old rats and estimated
;3500 mg
kg21
d21 as the no observed adverse effect level
based on body weight, food consumption, and hematological
measurements. Similarly, Mawatari et al. (25) in 10-wk-old female
rats estimated that oral leucine at 1000 mg
kg21
d21 did not
affect the outcome of pregnancy and did not cause fetal toxicity.
Leucine UL in adult humans
We recently reported a study of the effects of graded doses of
leucine in healthy young men aimed at defining the UL of leucine in
the acute phase (19). Participants received increased dietary leucine
in a graded stepwise intake of 50, 150, 250, 500, 750, 1000, and
1250 mg
kg21
d21 corresponding to the Estimated Average
Requirement and Estimated Average Requirement 33, 35, 310,
315, 320, and 325 on separate study days (26). All study days
were separated by a minimum of 2 wk to ensure a sufficient
washout period between the leucine excess study day diets.
Blood and urine biochemical variables. Baseline and hourly
vital signs (blood pressure, heart rate, body temperature) and
blood glucose (One Touch Ultra) were measured during the
study day to ensure patient safety and did not reveal any
significant changes. Alanine aminotransferase, as a marker of
liver function, blood urea, creatinine, electrolytes, CBC, urinary
creatinine, or urea, did not show any significant changes due
to increased leucine intakes. Blood ammonia concentrations at
Upper limit of leucine intake 2221S
the end of all study days were higher (P < 0.0001) than fasting
blood ammonia concentrations, except at a leucine intake of
50 mg
kg21
d21. With increasing intakes of leucine, blood
ammonia concentrations rose (P < 0.0001) by the end of the study
day and were above the normal range of 35 mmol/L after leucine
intakes of 500 mg
kg21
d21. When blood ammonia concen-
trations at the end of the study were beyond the upper limit of the
normal range, participants were contacted and requested to come
back to the clinical investigational unit the next day to provide a
blood sample to check ammonia concentrations. In all cases, the
blood ammonia concentrations were within the normal range
(<35 mmol/L) when tested on the following day, indicating that
the adverse effect due to increased leucine intake was not observed
when the participants returned to their normal diet. Plasma
glucose was decreased at all intakes of leucine compared with 50
mg
kg21
d21 (P = 0.0004), from 6.2 6 0.3 mmol/L (mean 6
SEM) to a low of 4.7 6 0.2 mmol/L, although all values stayed
within the normal range of 3.3–6.1 mmol/L. Leucine has been
suggested to act as an insulin secretagogue (6,27,28), but we did
not observe significant changes in plasma insulin due to increasing
leucine intakes.
Plasma BCAA concentrations were measured in all participants,
because previous studies have documented that increased intakes of
leucine decrease the plasma concentrations of valine and isoleucine
(29–32). The plasma leucine concentration significantly increased
and plasma valine and isoleucine concentrations significantly
decreased with increasing leucine intakes, as previously reported
(31). The BCAA share a common catabolic pathway with the
branched-chain ketodehydrogenase controlling the irreversible cat-
abolic step, which commits the carbon skeleton of the BCAA to the
TCA cycle. Leucine concentrations have been shown to stimulate
branched-chain ketodehydrogenase as well as to compete with the
other 2 BCAA for metabolism in vivo (33); this phenomenon,
referred to as BCAA antagonism, is well documented (31).
13
L-[1- C]Leucine oxidation and estimation of the UL of
leucine. Three baseline and 4 plateau breath samples were
collected for measurement of leucine oxidation during each
study day. The oxidation of L-[1-13C]leucine to 13CO2,
calculated as F13CO2, responded to increasing leucine intakes
(P < 0.0001). F13CO2 increased with increasing leucine intakes
up to 500 mg
kg21
d21, after which oxidation remained at
a plateau up to intakes of 1250 mg
kg21
d21. Using 2-phase
linear regression analysis (34,35), a breakpoint in F13CO2 was
identified at 550 mg
kg21
d21 (r2 = 0.85) (19). This
breakpoint identifies the maximum oxidative potential in
adult humans to dispose of excess dietary leucine intake. The
lower and upper 95%CI was calculated as 454 and 647
mg
kg21
d21, respectively.
Analysis of results from leucine UL study in adult
humans
The primary objective of our recent study (19) was to define the
metabolic capacity to dispose of excess leucine intake in vivo in
adult men. Oxidation of L-[1-13C]leucine to 13CO2 in breath
(F13CO2) was the primary endpoint measured with increasing
intakes of dietary leucine. F13CO2 increased with increasing
intakes of leucine until 500 mg
kg21
d21, after which oxida-
tion remained at a plateau. Two-phase linear regression analysis
identified a breakpoint at a leucine intake of 550 mg
kg21
d21
and this represents the maximum leucine oxidative potential in
vivo in adult men (19). There was a concomitant increase in blood
ammonia concentrations beyond the normal range (<35 mmol/L)
in all participants with intakes of leucine >500 mg
kg21
d21.
2222S Supplement
There is an increasing risk of adverse effects, as observed with the
increasing ammonia levels, with leucine intakes beyond the meta-
bolic capacity to oxidize leucine. Our study results are acute in
nature and it is unknown whether chronic consumption of leucine in
adult humans will result in a similar upper limit of leucine oxidation.
Mechanism to explain hyperammonemia observed with
increasing leucine intakes
Leucine is an activator of glutamate dehydrogenase, which results
in a-ketoglutarate and ammonia production. It has been reported
that leucine at concentrations >800 mmol/L activates glutamate
dehydrogenase to dispose of the surplus amino acids (36,37).
Intracellular glutamate concentrations are very high (5–10 mmol/
L) compared with extracellular concentrations (30–50 mmol/L).
Although amino acids can be converted to glutamate via
transamination reactions, glutamine serves as the major precursor
for glutamate via the phosphate-dependent glutaminase, which is
also regulated by the energy potential (36). Thus, flux into
glutamate comes from glutamine, especially when leucine con-
centrations increase (as it did in the current study, up to 2000
mmol/L), leading to increased glutamine synthesis.
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An additional explanation is also possible. Due to the increased
accumulation of isovaleryl CoA, which is formed from leucine
catabolism, inhibition of N-acetylglutamate synthase (NAGS) has
been shown to occur in isovaleric aciduria (38). NAGS is an
activator of carbamoyl-phosphate synthetase-1, which regulates
the urea cycle (38). With a decrease in NAGS, carbamoyl-
phosphate synthetase-1 activation would not have occurred and
this is also shown by the lack of increase in urea concentrations in
both blood and urea in our current study.
Summary
The DRI proposed by the Institute of Medicine defines the UL as
the highest average daily nutrient intake level that is likely to pose
no risk of adverse health effects to almost all individuals in the
general population. As intake increases beyond the UL, the
potential risk of adverse effects may increase. Based on the above
definition and the maximum oxidative potential observed in the
present study, the measured breakpoint (550 mg
kg21
d21) may
or may not be the UL for leucine intake in all individuals, because
blood ammonia concentrations were greater than the normal range
at 500 mg
kg21
d21 of leucine intake. Furthermore, the current
study was conducted with an acute supplement of dietary leucine in
normal adult men, so whether chronic leucine supplementation in
normal/trained athletes will provide similar results is unknown.
An analysis of the current habitual leucine intake was
conducted in strength-training athletes who are chronic amino
acid supplement users. The average protein intake in these athletes
was ;2 g
kg21
d21 (39). The mean leucine content in food is
;15% (40). Therefore, for an athlete weighing 80 kg, the dietary
leucine intake is 300 mg
kg21
d21. Available BCAA supple-
ments contain a maximum 1800 mg/serving (41), with a
recommended regimen of 3 doses/d, whic equals ;67.5 mg

kg21
d21. Thus, the habitual total exposure to leucine in adults
consuming 2 g
kg21
d21and the amino acid supplement is
;367 mg
kg21
d21. These calculations reveal that most
people, including athletes, consume less than the UL for leucine
oxidation determined in the current study (550 mg
kg21
d21).
However, there is probably a range in protein and leucine intake
among athletes, with some consuming more than the recommen-
ded dose and thus at potential risk of adverse effects. The impact of
chronic consumption by humans of excess leucine less than, at, or
greater than the maximum oxidation limit for nonadapted
individuals remains unknown. A high chronic intake may either
reduce the risk of adverse effects by increasing the basal leucine
oxidation rate or increase the risk of adverse effects by gradual
accumulation of metabolic events associated with excess intake.
In conclusion, with increasing intakes of leucine, a clear dose
response in leucine oxidative capacity, measured as F13CO2 from
the oxidation of L-[1-13C]leucine, was observed. The maximum
oxidative potential for leucine in vivo under acute feeding
conditions was identified using 2-phase linear regression analysis
with a breakpoint of 550 mg
kg21
d21 or 39 g
d21.
Significant increases in blood ammonia concentrations were
observed at leucine intakes >500 mg
kg21
d21. Unfortunately,
we have data only at leucine intakes of 250 and 500 mg
kg21

d21. At a leucine intake of 250 mg
kg21
d21, the mean fed-
state ammonia concentrations are within normal limits, but at a
leucine intake of 500 mg
kg21
d21, the mean ammonia
concentration is beyond the normal limits. We did try nonlinear
regression analysis on the fed-state ammonia results and did not
obtain a significant model. It could therefore be argued that when
chronic ingestion studies are designed, leucine intake levels should
be in the range of 250–300 mg
kg21
d21. Further, intakes
>550 mg
kg21
d21 may be a risk to health, because this is the
upper limit of oxidative capacity in normal adult humans. The
current study is the first to our knowledge to identify a metabolic
endpoint with a clear inflection in the dose-response curve as a
measure for identifying a UL for amino acids in humans at which
there may be increasing risk. It is important to appreciate that
based on the current acute leucine ingestion studies, chronic high-
dose leucine ingestion studies need to be performed. Further, such
studies need to be considered in young women as well as in elderly
women and men.
Acknowledgments
P.B.P., R.E., and R.O.B. participated in writing the manuscript.
All authors read and approved the final manuscript.
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