Mechanisms of the Pellagragenic Effect of Leucine: Stimulation of Hepatic Tryptophan Oxidation by Administration of Branched-Chain Amino Acids to Healthy Human Volunteers and the Role of Plasma Free Tryptophan and Total Kynurenines

Abstract

The pellagragenic effect of leucine (Leu) has been proposed to involve modulation of L-tryptophan (Trp) metabolism along the hepatic kynurenine pathway. Here, we discuss some of the mechanisms suggested and report the effects in healthy volunteers of single doses of Leu (4.05-6.75 g) administered in a 16-amino acid mixture on concentrations of plasma Trp and its kynurenine metabolites. Flux of Trp through Trp 2,3-dioxygenase (TDO) is dose-dependently enhanced most probably by Leu and can be attributed to TDO activation. Trp oxidation is better expressed using plasma total kynure-nines, rather than kynurenine, and free, rather than total, Trp. Increased hepatic Trp oxidation may be an additional mechanism of action of branched-chain amino acids in the acute Trp depletion test. Inhibition of intestinal absorption or hepatic uptake of Trp by Leu can be excluded. Potential mechanisms of the aggravation of pellagra symptoms by Leu are discussed.

Full text

23INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
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International Journal of
Tryptophan Research
Introduction
Pellagra is a disease of many causes, including nutritional
deciencies, conditions of gastrointestinal dysfunction,
alcoholism, and many drugs, all of which decrease the avail-
ability of nicotinic acid (niacin or vitamin B
3
).
1
Niacin is
present in many foods and also synthesized in the liver from
the essential amino acid L-tryptophan (Trp) in the kynure-
nine pathway of Trp degradation (Fig. 1). is pathway
accounts for .95% of total body Trp oxidation and is con-
trolled by the rst enzyme Trp 2,3-dioxygenase (TDO, for-
merly Trp pyrrolase).
2,3
Assessment of activity of TDO and
other enzymes of this pathway in humans has largely been
made by measuring urinary kynurenine metabolites, because
urinary levels far exceed those in plasma. However, with the
advent of more sensitive methods for measuring the generally
low plasma levels, more studies are now performed on plasma
samples. Previous human studies of the kynurenine pathway
in pellagra have recently been reviewed.
1
Existence on diets composed largely of maize or sorghum
led to the incidence of pellagra in India, South Africa, Southern
Europe during the 18th century, and the Southern USA during
the American civil war.
1
is is because of the low Trp content
of these staples and failure to isolate the niacin bound to poly-
saccha rides in the form of niac y tin, which cannot be hydrolyzed
by mammalian enzymes.
1,2
e symptoms of pellagra (the three
Ds: dermatitis, diarrhea, and delirium) are compounded by the
Mechanisms of the Pellagragenic Effect of Leucine: Stimulation of Hepatic
Tryptophan Oxidation by Administration of Branched-Chain Amino Acids to
Healthy Human Volunteers and the Role of Plasma Free Tryptophan and Total
Kynurenines
Abdulla A-B Badawy
1
, Sarah L. Lake
2
and Donald M. Dougherty
2
1
School of Health Sciences, Cardi Metropolitan University, Western Avenue, Cardi, Wales, UK.
2
Department of Psychiatry, University of
Texas Health Sciences Center at San Antonio, San Antonio, TX, USA.
A BST R AC T: e pellagragenic eect of leucine (Leu) has been proposed to involve modulation of L-tryptophan (Trp) metabolism along the hepatic
kynurenine pathway. Here, we discuss some of the mechanisms suggested and report the eects in healthy volunteers of single doses of Leu (4.05–6.75 g)
administered in a 16-amino acid mixture on concentrations of plasma Trp and its kynurenine metabolites. Flux of Trp through Trp 2,3-dioxygenase (TDO)
is dose-dependently enhanced most probably by Leu and can be attributed to TDO activation. Trp oxidation is better expressed using plasma total kynure-
nines, rather than kynurenine, and free, rather than total, Trp. Increased hepatic Trp oxidation may be an additional mechanism of action of branched-chain
amino acids in the acute Trp depletion test. Inhibition of intestinal absorption or hepatic uptake of Trp by Leu can be excluded. Potential mechanisms of
the aggravation of pellagra symptoms by Leu are discussed.
KEY WORDS: branched-chain amino acids, kynurenine pathway, leucine, pellagra, plasma free tryptophan, tryptophan 2,3-dioxygenase
CITATION: Badawy et al. Mechanisms of the Pellagragenic Effect of Leucine: Stimulation of Hepatic Tryptophan Oxidation by Administration of Branched-Chain Amino Acids to Healthy
Human Volunteers and the Role of Plasma Free Tryptophan and Total Kynurenines. International Journal of Tryptophan Research 2014:7 23–32 doi: 10.4137/IJTR.S18231.
RECEIVED: June 29, 2014. RESUBMITTED: September 12, 2014. ACCEPTED FOR PUBLICATION: October 3, 2014.
ACADEMIC EDITOR: Gilles Guillemin, Editor in Chief
TYPE: Original Research
FUNDING: This study was funded by grants from the USA National Institutes of Health (NIH) (R01-AA012046, RO1-AA014988, and T32-AA07565) to DMD. SLL was supported by an
NIH training grant (T32-DA031115). The Wellcome Trust funded the equipment used in this study through Project grant 069301 to AA-BB. DMD receives funding through the William and
Marguerite Wurzbach Distinguished Professorship. The authors conrm that the funder had no inuence over the study design, content of the article, or selection of this journal.
COMPETING INTERESTS: Authors disclose no potential conicts of interest.
COPYRIGHT: © the authors, publisher and licensee Libertas Academica Limited. This is an open-access article distributed under the terms of the Creative Commons CC-BY-NC 3.0
License.
CORRESPONDENCE: ABadawy@cardiffmet.ac.uk
Paper subject to independent expert blind peer review by minimum of two reviewers. All editorial decisions made by independent academic editor. Upon submission manuscript was
subject to anti-plagiarism scanning. Prior to publication all authors have given signed conrmation of agreement to article publication and compliance with all applicable ethical and
legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to
human and animal study participants, and compliance with any copyright requirements of third parties. This journal is a member of the Committee on Publication Ethics (COPE).

Badawy et al
24 INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
presence in these staples of large amounts of the branched-
chain amino acid (BCAA) leucine (Leu).
1
Various mechanisms
have been proposed to explain the pellagragenic eect of Leu
4
:
activation of liver TDO and picolinate carboxylase and inhi-
bition of kynureninase and quinolinate phosphoribosyltrans-
ferase.
4–9
ese eects may explain the decreased formation
of quinolinic acid and nicotinamide nucleotides when dietary
niacin intake is marginal
9
and the increased urinary excre-
tion of kynurenine, 3-hydroxykynurenine (3-HK), kynurenic
acid, and xanthurenic acid (XA) in pellagra patients.
10
Excess
dietary Leu may also inhibit hepatic uptake
4
and intestinal
absorption
11
of Trp.
Studies of these mechanisms have been conducted in vitro,
in isolated hepatocytes, and intact animals, or in pellagrins in
association with other nutritional deciencies that exert major
eects on this pathway. Investigation of the eects of Leu on
plasma Trp and kynurenine metabolites in normal subjects has
not been performed and may therefore throw further light on
Tryptophan
Trp dioxygenase
N´-Formylkynurenine
N´-Formylkynurenine formamidase
Kynurenine
Kynurenine aminotransferase
Kynurenic acid
Kynurenine hydroxylase
3-Hydroxykynurenine
Kynurenine aminotransferase
Xanthurenic acid
Kynureninase
3-Hydroxyanthranilic acid
3-Hydroxyanthranilic acid oxidase
Acroleyl aminofumarate
Non-enzymic cyclisation
Picolinate carboxylase
Quinolinic acid
Aminomuconic semialdehyde
Non-enzymic cyclisation Quinolinate phosphoribosyl transferase
Aminomuconic semialdehyde
dehydrogenase
Picolinic acid
Aminomuconic acid
Nicotinic acid
Nicotinate phosphoribosyltransferase
Nicotinic acid mononucleotide
Phosphorylase
Nicotinic acid-adenine
Acetyl Co-A
Nicotinamide deaminase
dinucleotide (NAAD)
NAD+ synthetase
Nicotinamide-adenine
Nicotinamide
NAD Glycohydrolase
Dinucleotide (NAD
+
)
Nicotinamide phosphoribosyl transferase
Nicotinamide methyltransferase Kynase
N
1
-Methylnicotinamide
N-Methylnicotinamide oxidases
NADP
+
NADH
N-Methylpyridone carboxamides NADPH
Figure 1. The hepatic kynurenine pathway of Trp degradation. This gure is an adaptation of Figures 1 and 2 from the review
1
titled “Pellagra and
alcoholism: a biochemical perspective” by Badawy, AA-B. Alcohol and Alcoholism 2014;49:238–50, published by Oxford University Press.

Leucine and tryptophan oxidation
25INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
these mechanisms in the absence of dietary deciencies. An
opportunity has arisen for such investigation from our previous
study of the specicity of the acute Trp depletion (ATD) test,
a powerful diagnostic tool for assessing the role of the cerebral
indolylamine serotonin in behavioral and other disorders.
12
e ATD test involves administration of an amino acid mix-
ture lacking in Trp, but rich in the three BCAAs Leu, Ile, and
Val. BCAAs and also Phe and Tyr compete with one another
and with Trp for entry into the brain and so inhibit serotonin
synthesis during the ATD test. Stimulation of protein synthe-
sis by the essential amino acids used (especially Leu; see the
Discussion) contributes to the Trp depletion. As a control, a
similar formulation supplemented with Trp is used. e ATD
formulation, however, lacks specicity because of the large
content of BCAAs
12
(see below). Specicity of the control for-
mulation has been improved by decreasing the content of the
three BCAAs by 40%, from the traditional ∼30% to 18%.
13
In the present study, we have compared aspects of Trp
metabolism and disposition and plasma kynurenine metabo-
lites in four groups of normal healthy volunteers receiving the
ATD control formulation with varying amounts of BCAAs,
and we were able to demonstrate enhanced Trp oxidation
along the kynurenine pathway, despite the added eects of the
moderate Trp load.
Subjects and Methods
Subjects. Subjects of this study were the 48 USA healthy
volunteers who took part in our previous investigation of the
specicity of the control formulation for the ATD test.
13
Sub-
jects were recruited by DMD during his professorial tenure at
Wake Forest University Health Sciences Center, NC, USA.
Subjects were divided into four groups (n = 12 each) matched
for age, gender, and ethnicity (Caucasians and African Amer-
icans). e phase of the menstrual cycle was not considered
in this study, but the gender distribution was uniform across
groups, and analysis of Trp and other parameters in our pre-
vious study
13
revealed no gender eects. Recruitment, health
screening, inclusion and exclusion criteria, and ethical approval
by the institutional review board of Wake Forest University
have all been described.
13
is research complied with the
principles of the Declaration of Helsinki, and participants gave
their written, informed consent to participate in the study.
Design. is was a double-blind study. After an overnight
fast, subjects were allocated to one of four groups to receive
a suitably avored drink containing 51.25 g of a mixture of
16 amino acids with varying BCAA content. is mixture
is based on the original control formulation of Young et al.
14
for the ATD test. Briey, in addition to the three BCAAs,
the mixture included the other three brain uptake competi-
tors Trp, Tyr, and Phe, and 10 other (non-competing) amino
acids. Table 1 gives the content of each of the six competitors
and the sum of the 10 non-competitors. e latter are summed
here for brevity, but are listed elsewhere.
13
As can be seen from
Table 1, the four groups received one of four formulations
(a between- rather than a within-group design to reduce
participant burden and attrition): the original control for-
mulation of Young et al.
14
(F0) or three other formulations
containing 20% (F1), 30% (F2), and 40% (F3) less BCAAs,
and the resulting dierences were compensated for by pro-
portionate increases in the content of each of the other 10
amino acids. Blood samples from all subjects were obtained
before and at hourly intervals for 7 hours after oral consump-
tion of the amino acid mixtures. Participants remained fast-
ing throughout the ∼7-hour study duration, following which
they received a Trp-balanced meal. Other details have been
described.
13
Laboratory procedures. Fasting plasma samples were
isolated in ethylene diamine tetra-acetic acid (EDTA) tubes
and frozen at −80 °C until transported in the frozen state to
Cardi, UK, for analysis. Plasma ultraltrates for free (non-
albumin-bound) Trp determination were prepared
15
from fresh
plasma before freezing, as frozen storage of plasma decreases
free [Trp]. Concentrations of plasma free and total Trp, kynure-
nine, and ve of its metabolites (3-HK, 3-hydroxyanthranilic
acid, kynurenic acid, XA, and anthranilic acid) were deter-
mined by high-performance liquid chromatography (HPLC)
with ultraviolet and uorimetric detection.
16
Expressions of results. Four parameters are expressed in
absolute concentrations (µM), namely, plasma free and total
Trp, kynurenine, and total kynurenines. Total kynurenines
include kynurenine, kynurenic acid, XA, anthranilic acid,
and 3-hydroxyanthranilic acid. A number of expressions are
then derived from these four parameters. ese are: (1) the
percentage free Trp (100 × [free Trp]/[total Trp]), which is an
established expression of Trp binding to albumin; (2) TDO
activity (100 × [kynurenine]/[total Trp]); (3) TDO activity
relative to free Trp (TDOF) (100 × [kynurenine]/[free Trp]);
(4) total Trp oxidation (TTOX) (100 × [total kynurenines]/
[total Trp]); (5) total Trp oxidation relative to free Trp
(TTOXF) (100 × [total kynurenines]/[free Trp]).
Table 1. Composition of the amino acid formulations.
AMINO ACID (G) FORMULA …. F0 F1 F2 F3
% DECREASE IN BCAA …. 0%
−20% −30% −40%
Trp 1.15 1.15 1.15 1.15
Phe 2.85 2.85 2.85 2.85
Tyr 3.45 3.45 3.45 3.45
Leu 6.75 5.40 4.73 4.05
Val 4.55 3.64 3.19 2.72
Ile 4.00 3.20 2.80 2.40
Other amino acids (n = 10)
28.50 31.55 33.09 34.62
Total amino acids (g) 51.25 51.25 51.25 51.25
Leu content (%) 13.17 10.54 9.23 7. 9 0
Average Leu dose (mg/kg) 96 77 68 58
Note: Details of the 10 other amino acids are in Table 2 in ref. 13.

Badawy et al
26 INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
Statistical analysis. Results were analyzed by one-way
randomized block analysis of variance (ANOVA) using SPSS
(Version 21, IBM Corporation) or Sigma Plot version 11
(Systat, UK), with which graphics were prepared. For multi-
ple group comparisons, the Holm–Sidak test is recommended
as the rst line procedure, as it is more powerful than the
Tukey or Bonferroni tests and can be used for pairwise com-
parisons and those versus a control group. When data failed
Mauchly’s test of sphericity, Greenhouse–Geisser corrections
were used to produce a conservative correction. In addition
to within-group comparisons with zero-time values, most of
the between-group comparisons presented were between data
from the F0 group (with the largest BCAA dose) and those
from the F3 group (the lowest BCAA dose) as control.
Results
In the results presented below, the sum of the total kynure-
nines does not include 3-HK, because values obtained by our
procedure
16
are generally higher than those reported in the
literature. However, the absence of 3-HK from the sum rep-
resents a conservative underestimate of the relevant eects
observed. Also in the presentation of the results, where there
are no clear BCAA dose-dependent dierences, only data
from the F0 (Leu and BCAA contents: 6.75 g and 15.35 g,
respectively) and F3 (4.05 g and 9.17 g, respectively) groups
are presented for graphic clarity and illustration of the poten-
tial level of changes caused by the highest, versus the lowest,
doses of Leu and BCAA.
Baseline parameter means. Table 2 gives the baseline
fasting mean values of the various parameters studied for all
48 subjects.
Eects of BCAAs on plasma free and total Trp con-
centrations and Trp binding. As subjects in all four groups
received a similar dose of Trp (1.15 g), both plasma free
[F(2.706, 119.084) = 99.769, P , 0.0001] and total [Trp]
[F(2.176, 95.738) = 140.087, P , 0.0001] were increased sig-
nicantly over time (P = 0.030–0.001), reaching a maximum
at 2–3 hours and returning to normal values by 6 hours. Mul-
tiple group comparisons for free Trp showed an overall sig-
nicance only at 3 hours (H = 8.458, 3; P = 0.037). e time
course of changes in free Trp in groups F0 and F3 is shown
in Figure 2A. After the initial 2 hours, the decrease in free
Trp was somewhat faster in the F0, than the F3, group, with a
signicant dierence at 3 hours (F = 8.090, 1; P = 0.009). By
contrast, total Trp did not dier signicantly between groups,
with values for the F0 and F3 groups shown in Figure 2B.
e percentage free Trp, an expression of Trp binding
to albumin, was altered as a result of the above changes in
[free Trp]. As shown in Figure 2C, the %free Trp was gener-
ally lower in the F0, compared with the F3, group, suggest-
ing that albumin-binding sites were saturated with Trp in
F3, leading to an overspill of free Trp, presumably because
Trp degradation was faster in F0. e fact that enhanced Trp
oxidation by TDO normally leads to proportionate decreases
in free and total [Trp] without inuencing Trp binding to
albumin (see the Discussion below) supports this interpreta-
tion. As was the case for free Trp, the dierence in the %free
Trp between F0 and F3 was signicant at 3 hours (H = 6.549,
1; P = 0.018). Overall, group comparisons showed a trend
toward signicance (P = 0.058) also only at 3 hours.
Eects of BCAAs on plasma kynurenine and expres-
sions of liver TDO activity. Plasma [kynurenine] rose signi-
cantly with time in all four groups [F(5.030, 221.334) =
22.790, P , 0.0001], as a result of acute Trp load leading to
increased ux of Trp through TDO. e kynurenine elevation
was signicant at all times in the F0 group, but up to 5 hours
in the F1 and up to 4 hours in the F2 and F3 groups. How-
ever, plasma [kynurenine] did not dier signicantly between
groups at any time point during the 7-hour experimental
duration. is lack of dierence is exemplied in Figure 3A
comparing the F0 and F3 groups.
Liver TDO activ it y is expressed by the plasma [kynurenine]/
[total Trp] ratio percentage. A decrease in this expression
was observed at 1 hour, but is of no signicance, as it is
because of the increase in [Trp] following Trp loading. e
increase in TDO activity in the F0 group was signicant
from 4 hours onward, whereas that in the other three groups
was transient, occurring only at 3 or 6 hours. As was the case
for [kynurenine], no signicant group dierences in TDO
were observed, as illustrated in Figure 3B for the F0 and
F3 groups.
A new expression of TDO activity was made relative to
[free Trp] (TDOF). Here, there were also no signicant dif-
ferences within or between groups, except that, at 3 hours,
the F0 and F3 TDOF means were signicantly dierent
(H = 4.675, 1; P = 0.039). ere were signicant changes in
TDOF over time [F(3.912, 172.132) = 14.449, P , 0.0001].
e time-course of changes in TDOF in these two groups is
shown in Figure 3C.
Table 2. Baseline mean values of various parameters in the
48 subjects.
PARAMETER EXPRESSION MEAN SEM
Free Trp
µM
4.78 0.22
Total Trp
µM
43.5 2.65
% Free Trp
100 × [Free Trp]/[Total Trp]
10.99 0.75
Kynurenine
µM
1.28 0.29
TDO
100 × [Kynurenine]/[total Trp]
2.94 0.26
TDOF
100 × [Kynurenine]/[Free Trp]
26.78 1.91
Total kynurenines
µM
4.22 0.69
TTOX
100 × [Total kynurenines]/
[Total Trp]
9.70 1.78
TTOXF
100 × [Total kynurenines]/
[Free Trp]
88.28 11.8 9
Abbreviations: TDO, Trp 2,3-dioxygenase relative to total Trp; TDOF, Trp
2,3-dioxygenase relative to free Trp; TTOX, total Trp oxidation relative to total
Trp; TTOXF, total Trp oxidation relative to free Trp.

Leucine and tryptophan oxidation
27INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
02468
2
4
6
8
10
12
14
16
18
20
Time (h)
[Free Trp] (µM)
F3
FO
A
*
*
02468
20
40
60
80
100
120
B
Time (h)
[Total Trp] (µM)
FO
F3
02468
8
10
12
14
16
18
20
22
C
Time (h)
Free Trp (%)
FO
F3
*
*
Figure 2. Time-course of the effects of BCAAs on plasma free and total
Trp concentrations and Trp binding in normal subjects. Fasting volunteers
received a 16-amino acid mixture at two BCAA dose levels: 15.35 g
(group F0) and 9.17 g (group F3). These correspond to Leu doses of
6.75 g and 4.05 g, respectively. Plasma samples were analyzed before
and at hourly intervals for 7 hours after oral consumption of each mixture
for free Trp (A), total Trp (B), and the %free Trp (C). Analytical and other
details are described in the Subjects and methods section. Values are
means ± SEM (bars) for 12 subjects in each group, and the signicance
of differences is described at the relevant points in the text. The asterisk
denotes signicant differences in the F0 group relative to the F3 group
(P = 0.043–0.009).
02468
1
2
3
4
5
6
7
FO
F3
Time (h)
TDO (%)
B
*
02468
10
15
20
25
30
35
40
45
FO
F3
Time (h)
TDOF (%)
*
C
Figure 3. Time-course of the effects of BCAAs on plasma kynurenine
concentration and expressions of TDO activity in normal subjects.
Details are as described in Figure 2. TDO activity is expressed in two
ways, as described in Table 2. Values for kynurenine (A), TDO (B), and
TDOF (C) are means ± SEM for 12 subjects in each group. The asterisk
denotes signicant differences in the F0 group relative to the F3 group
(P = 0.007–0.009).
02468
1.0
1.5
2.0
2.5
3.0
3.5
A
FO
F3
Time (h)
[Kynurenine] (µM)

Badawy et al
28 INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
02468
2
4
6
8
10
12
14
A
Time (h)
[Total kynurenines (µM)
FO
F3
F1
F2
*
*
*
*
*
02468
2
4
6
8
10
12
14
16
18
20
F3
FO
Time (h)
TTOX (%)
*
*
B
*
02468
0
20
40
60
80
100
120
140
C
Time (h)
TTOXF (%)
FO
F3
*
*
*
*
*
Figure 4. Time-course of the effects of BCAAs on plasma concentrations
of total kynurenines and expressions of total Trp oxidation (TTOX) in
normal subjects. Details are as described in Figure 2, except that total
kynurenines (A) were determined in groups F0 and F3 and additionally in
groups F1 and F2 receiving intermediate BCAA doses as in Table 1. TTOX
(B and C) is expressed in two ways as described in Table 2. Values are
means ± SEM for 12 subjects per group. The asterisk denotes signicant
differences in the F0 group relative to the F3 group (P = 0.007–0.009) for
TTOX and TTOXF, whereas for total kynurenines, the asterisk denotes
signicant differences relative to the F0 group (P = 0.05–0.002).
Eects of BCAAs on plasma concentrations of kynure-
nines and expressions of total Trp oxidation (TTOX). As
was the case with [kynurenine], [total kynurenines] were also
elevated signicantly [F(4.301, 189.232) = 8.698, P , 0.0001]
and maximally at 2–3 hours in all groups. However, unlike the
former, the elevation of [total kynurenines] was dose depen-
dent for all four doses of the BCAAs, with the elevation order
of groups being F0 . F1 . F2 . F3 (Fig. 4A). Multiple
group comparisons showed overall signicant dierences at
2, 3, and 4 hours (P = 0.023–0.0048). Groups F0 and F3 dif-
fered signicantly at 1, 2, 3, and 4 hours (P = 0.024–0.008). In
data not shown, individual kynurenine metabolites were also
similarly elevated in line with their sums.
Two new expressions of total Trp oxidation (TTOX) were
attempted: TTOX relative to [total Trp] and TTOXF relative
to [free Trp]. TTOX did not show overall signicant group
dierences, except at 5 hours (H = 8.383, 3: P = 0.039). How-
ever, there were signicant dierences over time [F(2.210,
97.235) = 10.722, P , 0.0001], and comparison of the F0 and
F3 groups (Fig. 4B) showed signicant dierences at 1, 3, and
4 hours (P = 0.050–0.038). More remarkable dierences were
observed with TTOXF. Here, multiple group comparisons
showed signicant group dierences at 1–5 hours (P = 0.05–
0.011), with dierences between groups F0 and F3 (Fig. 4C)
being signicant also at 1–5 hours (P = 0.033–0.005).
Comparison of areas under the curves for parameters
with signicant group dierences. To further assess the
signicance of dierences in parameters between the F0 and
F3 groups, analyses of areas under the curves (AUC) were per-
formed on all subjects in these two groups. e results averaged
in Table 3 show that for [free Trp], the 17% lower AUC for F0
was not signicant, whereas the 22% lower %free Trp in F0
was. e 20% higher TDOF (TDO activity expressed relative
to free Trp) in F0 was not signicant. Total kynurenines were
signicantly higher in F0, by 65%. e AUC for total Trp
oxidation expressed relative to total Trp (TTOX) or to free
Trp (TTOXF) showed two subjects in each group with either
very low or very high values, and these were omitted from the
analysis. AUC for the remaining 10 subjects in each group
showed signicantly and similarly higher values in F0 (139%
for TTOX and 145% for TTOXF), compared to F3. For other
parameters showing no signicant group dierences, AUC
measurements also showed no signicant dierences.
Discussion
Mechanisms of the pellagragenic eect of Leu. Based
on studies in rat liver preparations in vitro, isolated rat hepa-
tocytes, and intact rats in vivo, with very few in pellagrins and
normal humans, excessive dietary Leu has been suggested
4–11
to induce or aggravate pellagra by six mechanisms: activation
of liver (1) TDO and (2) picolinate carboxylase, inhibition of
(3) kynureninase and (4) quinolinate phosphoribosyltransferase,
and of (5) hepatic Trp uptake, and (6) intestinal Trp absorption.
Although we have addressed mainly hepatic Trp oxidation by

Leucine and tryptophan oxidation
29INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
TDO in normal humans, it is important to discuss rst the
other mechanisms.
Studies in vitro. e concentrations of Leu (1–10 mM)
required for inhibition of intestinal Trp absorption in rats
11
and that (7.6 mM) for inhibition of kynureninase activity in
rat liver preparations
9
are unlikely to be of signicance in vivo
in humans, because basal fasting plasma [Leu] (in normal
USA subjects of both genders: 109 ± 3 µM, mean ± SEM for
n = 114)
17
is increased maximally by oral Leu doses of 5.0–
13.5 g to values of 204–843 µM.
13,17–19
In subjects of the pres-
ent study, we reported
13
maximal elevations in plasma [Leu]
to 486 ± 76 µM in the F0 group (Leu dose: 6.75 g: ∼96 mg/kg
body wt) and 355 ± 42 µM in F3 (Leu dose: 4.05 g: ∼58 mg/kg
body wt). us, under our experimental conditions, elevation
of plasma [Leu] does not reach 500 µM with Leu doses of up
to 13.5 g, or ∼193 mg/kg body wt. Plasma [Leu] in normal
humans after Leu loading therefore rises to values generally
below 500 µM and certainly ,1 mM. As discussed below,
kynureninase inhibition occurs in vivo in pellagrins and Leu-
treated rats by a mechanism other than the competitive one
suggested.
9
Studies in isolated rat hepatocytes. Trp uptake by isolated rat
hepatocytes
4
is inhibited by Leu at 0.5 mM and above, with
5 mM causing only a 37% inhibition. is moderate inhibition
reects quantitatively a decreased ux of Trp through TDO.
ese high [Leu] are also unlikely to be reached in vivo,
and in fact, these authors
4
demonstrated increased ux of
Trp through TDO and subsequent steps of the kynurenine
pathway in hepatocytes from rats maintained on a high-Leu
(15 g/kg) diet.
Studies in intact rats in vivo and in pellagrins. Average
intake of the above diet (15 g of Leu/kg) corresponds to a Leu
dose (∼1.5 g/kg) that raises rat blood [Leu] to 1 mM.
20
With
this dosage,
7
the ux of Trp through kynureninase (estimated
from a tracer dose of methylene-labeled
14
C-Trp) is inhibited,
whereas that through picolinate carboxylase (estimated using
uniformly labeled benzene ring U-
14
C-Trp) is enhanced, thus
suggesting inhibition of the former, but activation of the lat-
ter, enzyme. Both eects can adequately explain the previously
reported decreased synthesis of nicotinamide dinucleotides.
Flux of U-
14
C-Trp through picolinate carboxylase has also
been demonstrated by others in intact rats in vivo
5
and hepa-
tocytes isolated from high Leu-fed rat,
4
but, as pointed out,
4
the major source of ux of Trp through picolinate carboxylase
may be the kidney, which is a richer source of this enzyme
than the liver. Inhibition of this ux by 2 mM Leu added to
hepatocytes from normal rats (not high Leu-fed)
4
may not be
physiologically signicant.
e kynureninase inhibition demonstrated in rats using
methylene
14
C-Tr p
7
may also occur in pellagrins, evidenced
indirectly from increased urinary excretion of 3-HK and
XA.
10,18
Levels of this transamination product (XA) and of
kynurenic acid (Fig. 1) rise when there is an increased ux
of Trp down the kynurenine pathway after acute Trp load-
ing or enhanced TDO activity and also after kynurenine
loading or kynureninase inhibition.
1–3
Kynureninase inhibi-
tion in pellagrins has been suggested
10
to involve decreased
activity of the pyridoxal 5′-phosphate transamination cofactor
secondarily to pyridoxine deciency. Indeed plasma pyridoxal
5′-phosphate levels are signicantly lower in pellagrins than
in controls.
18
Leu may also inhibit kynureninase by a similar
(functional vitamin B
6
deciency) mechanism. Leu is metabo-
lized initially by BCAA aminotransferase (BCAT). Excessive
dietary Leu intake could thus result in depletion of pyridoxal
phosphate. BCAT activity in rat liver is undetectable or at
best ,1% of total body enzyme activity,
21,22
but is expressed
in human liver,
23
wherein its activity is 7.7–11% of total body
activity.
24,25
e absence of BCAT from rat liver explains
the absence of inhibition of Trp ux through kynureninase
in hepatocytes from Leu-fed rats.
4
However, in the whole
animal, inhibition of kynureninase ux by high-Leu diets
7
is
most likely the result of pyridoxal phosphate depletion by the
widely distributed BCAT. Depletion of this cofactor can also
explain the low Leu tolerance in pellagrins following an acute
Leu load and its normalization by single or repeated pyridox-
ine intake.
18
Measurement of plasma and/or tissue pyridoxal
phosphate after Leu treatment should be made in future stud-
ies. Whereas kynureninase inhibition in pellagrins causes the
increased urinary excretion of 3-HK and XA, the associated
rise in urinary kynurenic acid may be caused by elevation of
kynurenine by TDO activation, the mechanism of which
was proposed
10
to involve decreased feedback inhibition by
NADPH following niacin depletion.
Relevance of the present results. It is important rst to
emphasize that BCAAs are metabolized dierently in rodents
and humans, hence the need for cautious extrapolation from
rats to humans. For example, rats metabolize BCAAs faster
than humans, with BCAT activity in human tissues being only
10% of that in the corresponding rat tissues.
24
e similarly
higher BCKA dehydrogenase in rat tissues will additionally
promote further transamination of BCAAs. However, this
does not suggest that the similar changes in Trp metabolism
Table 3. Comparison of AUC between the F0 and F3 groups for
parameters with signicant differences.
GROUP … FO F3 % DIFFERENCE
PARAMETER MEAN SEM MEAN SEM AGAINST F3 P
Free Trp (µM)
63.9 7.0 77.3 4.0
− 17%
0.117
% Free Trp 104.7 6.8 133.3 12.7
− 22%
0.038
TDOF (%) 194 22 161 20
+ 20%
0.291
Kynurenines (µM)
53.1 8.9 32.1 5.3
+ 65%
0.024
TTOX (%) 102 15 43 3
+137%
0.002
TTOXF (%) 685 34 280 34
+ 145%
0.001
Notes: AUC were determined for each subject using the Sigma Plot statistical
program, and group differences were assessed by repeated measures
ANOVA. Values are means ± SEM for 12 subjects per group for the rst four
parameters, but only 10 subjects per group for TTOX and TTOXF.

Badawy et al
30 INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
in Leu-treated rats and pellagrins are caused by dierent
mechanisms.
In the present study, the highest dose of Leu in F0
(6.75 g or on average ∼96 mg/kg body wt) is considerably
smaller than that administered to rats (∼1.5 g/kg; see above),
and it is therefore unclear if it is capable of depleting pyridoxal
5´-phosphate. Plasma 3-hydroxyanthranilic acid (the product
of the kynureninase reaction) was higher, rather than lower,
in F0, compared to F3, and other kynurenines were simi-
larly elevated with increasing doses of Leu (data not shown).
Although this suggests that the Leu doses used here do not
inhibit kynureninase activity, inhibition cannot be ruled out
if the ux of exogenously administered Trp could have had a
mitigating eect.
Eects of Leu on hepatic tryptophan oxidation. Role of
Leu. e present results (Figs. 2–4) are the rst to demon-
strate that BCAAs enhance Trp oxidation along the hepatic
kynurenine pathway in normal subjects. is enhancement is
most likely mediated by Leu, as it is the dominant BCAA in
the formulations. Neither Val nor Ile inuences rat liver TDO
activity
26
in doses (316–353 mg/kg) far greater than those
given here to F0 subjects (∼65 and 57 mg/kg respectively).
Also, a large (30 g) Val dose does not modulate plasma [Trp]
in healthy volunteers,
27
thus excluding an eect on Trp clear-
ance (degradation). Of 15 amino acids other than Trp given
acutely to rats in the above study,
26
only methionine (Met)
activated TDO at a dose (400 mg/kg) considerably higher
than those used in the present study (21 mg/kg in F0 and
26 mg/kg in F3). Met can also be excluded. Furthermore, Leu
administration decreases plasma Val and Ile, whereas neither
of these latter two amino acids lowers plasma [Leu].
20
Leu
also undergoes strong hepatic extraction.
28
Taken together, it
can be reasonably concluded that the increased production of
kynurenines is caused by Leu.
Enhanced tryptophan oxidation. e Leu enhancement of
Trp oxidation along the kynurenine pathway (Figs. 3 and 4)
could involve a simple increase in the ux of Trp down the
pathway or activation of the rst enzyme TDO. A 50 mg/kg
dose of Trp does not activate liver TDO.
29
e Trp dose used
in the present work (1.15 g or ∼16 mg/kg) is much smaller,
but undergoes ux down the pathway in all four groups, as
demonstrated in the time-course experiments (Figs. 3 and 4).
e dose-dependent increase in plasma kynurenines (Fig. 4A)
therefore suggests that the increased ux is because of Leu.
Increased ux through TDO has previously been dem-
onstrated
4
in hepatocytes isolated from high Leu-fed rats.
ese authors
4
suggested that the increased ux is because
of TDO activation, as the ratio of TDO activity to ux was
also increased in parallel. TDO activation is therefore likely
in the present study, though its mechanism(s) is dicult
to ascertain. us, the absence of increase in TDO activ-
ity by addition of Leu in vitro to cell-free preparations from
normal rat hepatocytes
4
or after administration to rats of a
350 mg/kg dose
26
excludes a direct eect of Leu. Removal of
the NADPH-mediated feedback inhibitory control of TDO,
proposed in pellagrins,
10
is a likely explanation, but cannot
be demonstrated, because of a potential reversal of NADPH
depletion by the administered Trp. e possible role of the
Leu metabolite 4-methyl-2-oxovalerate in the increased ux
of Trp through TDO in hepatocytes from high Leu-fed rats
by virtue of displacing Trp from plasma albumin-binding
sites, thereby increasing its availability to the liver, has been
ruled out
4
and is also unlikely in the present work, as plasma
free [Trp] was lower in the F0 group receiving the largest dose
of Leu (Fig. 2A). However, the observed increase in plasma
kynurenines cannot be solely because of kynureninase inhi-
bition and together with the longer duration of the kynure-
nine elevation in the F0 group suggest that TDO activity
is enhanced by Leu by an as yet unidentied mechanism(s).
One potential remaining mechanism is that of Leu enhancing
TDO synthesis. Leu plays a major role in protein synthesis,
30
and in fact, the use of Leu (BCAA) in the ATD test is in part
to stimulate protein synthesis to promote further the desired
depletion of Trp.
14
We suggest that an additional mechanism
of this depletion during the ATD test involves Leu enhancing
hepatic Trp oxidation.
Expressions of tryptophan oxidation and the role of plasma
free tryptophan and total kynurenines. TDO activation (leading
to increased Trp oxidation) normally results in proportionate
decreases in both plasma free and total [Trp], without alter-
ing Trp binding (expressed as the %free Trp).
31
However, it
is not possible to demonstrate this simple relationship in the
presence of exogenously administered Trp. Nevertheless, it is
possible to conclude that TDO is more activated in F0 than
in F3. us, whereas total [Trp] was not signicantly dierent
between groups, free [Trp] was at 3 hours (Fig. 2). Trp load-
ing saturates the plasma albumin-binding sites and the higher
%free Trp in F3 compared to F0 (Fig. 2C), despite the same
Trp dose, suggests that the larger Leu dose in the latter group
accelerated free Trp clearance.
Changes in TDO activity can also be deduced from an
increase in plasma [kynurenine] resulting in elevation of the
[kynurenine]/[Trp] ratio. Kynurenine, however, undergoes a
large renal clearance,
32,33
and only after robust TDO induction
(or Trp or kynurenine loading) could its plasma levels exceed
its rate of renal clearance. e absence of signicant group dif-
ferences in plasma kynurenine (Fig. 3A) or the [kynurenine]/
[total Trp] ratio (Fig. 3B) may be because of the inuence of
the Trp load and renal clearance, but does not exclude TDO
activation, which is suggested by the signicant elevation of
the [kynurenine]/[total Trp] ratio in F0 at 5–7 hours (Fig. 3B)
and by the signicant increase at 3 hours in the [kynurenine]/
[free Trp] ratio in F0 compared to F3 (Fig. 3C).
Using free Trp and total kynurenines may provide more
sensitive expressions of TDO activity and Trp oxidation (ux)
than total Trp and kynurenine, at least in certain circumstances,
eg, when the Trp ux through TDO is enhanced in the
absence of an apparent or robust increase in TDO activity.

Leucine and tryptophan oxidation
31INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH 2014:7
us, a clear dose-dependent elevation of [total kynurenines]
was observed (Fig. 4A), with the increase in F0 being signicant
over the rst 5 hours. As a result, the [total kynurenines]/[total
Trp] and [total kynurenines]/[free Trp] ratios were signi-
cantly higher in F0 compared with F3 over the rst 5 hours
(Fig. 4B and C). ese two expressions also showed the great-
est signicance in their AUC, followed by [total kynurenines]
(Table 3). Changes in free Trp have been shown
34
to modulate
the Trp ux through TDO in rat hepatocytes. us, the use
of plasma free Trp and total kynurenines may provide more
sensitive indices of Trp oxidation.
Role of Leu in clinical features of pellagra. Pellagra is
dened as the disease of the three Ds, namely diarrhea, derma-
titis, and dementia (or more appropriately delirium).
1
Aggra-
vation of pellagra symptoms by Leu can be attributed to the
consequences of its metabolism and its eects on Trp metabo-
lism. Leu transamination by BCAT results in conversion of
2-oxoglutarate to glutamate. Leu is therefore a nitrogen donor
for synthesis of this excitatory amino acid and the inhibitory
neurotransmitter GABA (γ-aminobutyric acid). As discussed
previously,
1
activation of the N-methyl--aspartate (NMDA)
type of glutamate receptors and enhancement of GABAergic
neurotransmission may be involved in the neurological features
of pellagra, particularly anxiety and psychosis. e decrease
in pyridoxal 5′-phosphate resulting from Leu transamination
can add further to the vitamin B
6
deciency of pellagra and
thereby aggravate the skin features. Enhanced Trp oxidation
down the kynurenine pathway by Leu can also contribute to
the aggravation of pellagra symptoms. us, increased hepatic
Trp oxidation, increased Trp utilization for protein synthesis,
and competition for cerebral Trp uptake by Leu can all induce
a decrease in Trp entry into the brain and a consequent inhibi-
tion of serotonin synthesis, which may explain the depressive
symptoms. Elevation of plasma kynurenine and the conse-
quent increase in kynurenic acid resulting from the enhanced
ux of Trp through TDO by Leu can contribute further to the
skin photosensitivity and the anxiety and psychotic features.
1
Limitations of the present study. Although this study
addresses specically hepatic Trp metabolism initiated by
TDO, we believe that any likely role of the extrahepatic Trp-
degrading enzyme indolylamine 2,3-dioxygenase (IDO),
though cannot be excluded, can only be marginal under
our experimental conditions in normal subjects, because
the combined extrahepatic tissue activity of IDO is 5–15%,
with the latter upper limit applicable to the richest source,
the placenta.
35,36
We also recognize that elevation of plasma
kynurenine metabolite levels in our study cannot be attrib-
uted solely to actions of enzymes of the hepatic kynurenine
pathway, but may involve additionally enzymes in kidney and
other tissues. Also, while renal handling of kynurenine is an
important determinant of plasma kynurenine, production
of this Trp metabolite is most likely achieved mainly in the
liver. Another limitation is lack of standardization of dosage
of the ATD test and control formulations to take account of
body weight. is has been the case since the test was used
in humans in 1985. However, more recently,
37
the use of the
test in adults according to body weight has been proposed, as
has previously been the case in children. Finally, we did not
include values for 3-HK in our calculation of total kynure-
nines, because our baseline fasting 3-HK values
16
(before Leu
administration) are far greater than those in the literature (for
a comprehensive review, see ref. 38). A discussion of this vari-
ance is outside the scope of this paper, but requires critical
appraisal of methodology and development of quantitative
techniques to measure 3-HK metabolites in addition to the
free form. However, the absence of 3-HK from our data could
only have minimized the observed group dierences in total
kynurenines.
Conclusions
In conclusion, we have demonstrated enhanced ux of Trp
through TDO by moderate doses of Leu in normal humans
that is consistent with the reported
4
enhancement in isolated
hepatocytes from rats maintained on a high-Leu diet. is
increased ux and elevation of kynurenines can be attributed
provisionally to TDO activation by the relatively moderate
doses of Leu used. Mechanisms such as impaired intestinal
absorption or hepatic uptake of Trp can be excluded. Inhibi-
tion of kynureninase by excessive dietary Leu intake in the
presence of vitamin B
6
deciency is likely to be the major
mechanism of the pellagragenic eect of Leu and its metab-
olism, and its eects of Trp degradation may aggravate the
clinical features of pellagra.
Acknowledgments
DMD gratefully acknowledges support from the William
and Marguerite Wurzbach Distinguished Professorship. We
thank Dawn M. Richard for recruitment of subjects and orga-
nization of the study part in the USA and A. Steptoe and
S. Khatun for technical assistance in the UK.
Author Contributions
DMD and AA-BB conceived and designed the experiments.
AA-BB collected and analyzed the data and wrote the rst
draft of the manuscript. DMD contributed to the writing of
the manuscript and agreed with manuscript results and conclu-
sions. SLL reviewed and revised the statistical tests. AA-BB
and DMD jointly developed the structure and arguments for
the paper and made critical revisions. All authors approved
the nal revised manuscript.
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