Vogel et al.

Orphanet Journal of Rare Diseases 2014, 9:73

http://www.ojrd.com/content/9/1/73

RESEARCH Open Access

Brain–blood amino acid correlates following

protein restriction in murine maple syrup urine
disease
Kara R Vogel1, Erland Arning2, Brandi L Wasek2, Sterling McPherson3, Teodoro Bottiglieri2 and K Michael Gibson1*

Abstract
Background: Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein
intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However,
no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in
blood is reflected in brain.
Methods: To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with
collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses.
Results: LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6%
protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not
improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU,
ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless,
numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN),
aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine
(SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice,
modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably
consistent abnormalities in GLN, ASP, and GLU.
Conclusions: Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the
outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic
neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these
disorders.
Keywords: Maple syrup urine disease (MSUD), Branched-chain keto-acid dehydrogenase (BCKDH) complex, Mouse
model, Large neutral amino acid transporter (LAT-1), Branched-chain amino acids (BCAAs), Phenylketonuria (PKU),
Large neutral aminoacidopathies, Dietary protein restriction

Background catabolism will induce release of BCAAs from muscle and

Maple syrup urine disease (MSUD; branched chain ketoa- increase the BCAA’s corresponding α-keto acids (BCKAs),
cid dehydrogenase (BCKDH) deficiency) and phenylketon- including α-ketoisocaproate (KIC, from LEU), α-keto-β-
uria (PKU) comprise the large neutral aminoacidopathies. methylvalerate (KMV, from ILE) and α-ketoisovalerate
Untreated MSUD results in accumulation of the branched- (KIV, from VAL), all of which can freely traverse the
chain amino acids (BCAAs) leucine (LEU), valine (VAL) blood–brain barrier (BBB) and transaminate to yield
and isoleucine (ILE) [1-4]. Additionally, induction of the corresponding BCAAs [5]. Treatment of MSUD
requires strict protein restriction combined with BCAA
* Correspondence: [email protected] supplementation to levels compatible with adequate
1
Experimental and Systems Pharmacology, College of Pharmacy, Washington
State University, 412 E. Spokane Falls Blvd., Pharmaceutical and Biomedical
development and growth. Although dietary intervention
Sciences Building, Room 347, P.O. Box 1495, 99210-1495 Spokane, WA, USA prevents the severe developmental delays associated
Full list of author information is available at the end of the article

© 2014 Vogel et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Vogel et al. Orphanet Journal of Rare Diseases 2014, 9:73 Page 2 of 8
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with chronic hyperleucinemia, it nonetheless presents Methods

challenges that include maintenance and adherence to Animal subjects
diet, and mounting evidence for long-term deficits in The intermediate MSUD murine model (imsud mice) has
neurocognition despite acceptable metabolic control been described, and manifests ~ 6% of residual BCKDH
determined in blood [6-16]. Additionally, over restric- enzyme function [1,2,33,34]. Animal subjects were geno-
tion of dietary amino acid intake may have untoward typed by PCR amplification. Subjects of both genders were
consequences. The preceding discussion suggests that employed, ages 10–20 days of life. Subjects were allowed
while prudent, dietary treatment alone for MSUD may ad libitum access to chow and water throughout experi-
be suboptimal. mentation. All animal experimentation was performed
The pathophysiology of MSUD has been extensively with IACUC approval (ASAF 4232–007).
reviewed [17]. Episodic increases in blood BCAAs occur
when muscle degrades protein in response to physio- Diets employed
logical stress [18-20], also increasing the level of corre- We performed pilot studies with the Teklad TD.90016
sponding BCKAs. Increased muscle BCKAs induces diet (6.9% casein = 6% protein; low protein) since it has
reversal of cytosolic transaminases and depletes tissue been employed previously for maternal protein restric-
levels of other amino acid nitrogen donors. As LEU exits tion in rodents [35]. In the latter studies, the induction
muscle and other tissues via the large neutral amino acid of serine-metabolizing enzymes was characterized in mice
transporter (LAT-2) [21], heteroexchange mechanisms receiving 2, 6 and 18% protein. Based upon empiric consid-
stoichiometrically drive import of other amino acids and erations, we felt that 2% protein would be too stringent in
increase the relative LEU blood concentration. At the imsud mice and lead to catabolic crisis. Teklad custom
BBB, elevated BCAAs may saturate the LAT-1 (see below) diets are isocaloric and balanced for nitrogen level. The
transporter and block uptake of other large neutral amino corresponding Teklad TD.91353 diet (19% protein) was
acids (LNAA; including phenylalanine (PHE), tyrosine used as control diet. Animals were on diet for 8–18 days
(TYR), methionine (MET); tryptophan (TRP); and histi- prior to sacrifice. Power analyses indicated that n = 6
dine (HIS)). Increased blood BCKAs enter the brain via subjects of each genotype (imsud+/+ and imsud−/−) would
the monocarboxylate transporter (MCT) and reverse flux be sufficient to obtain statistically significant outcomes. For
through cerebral transaminases, depleting brain glutamate brain region analyses (see below), we frequently had n = 5-
(GLU), glutamine (GLN) and gamma-aminobutyric acid 6 subjects per diet and genotype, but the smaller size of
(GABA) (among others), while enhancing production of mutant subjects often led to insufficient blood for analyt-
LEU and α-ketoglutarate [5,22-25]. These disruptions in ical studies, thereby leading to smaller n values for sera
brain amino acid homeostasis may also be accompanied (SE) and enhanced data variability. At study conclusion,
by disruptions of oxidative phosphorylation, elevated cere- subjects per genotype were n = 3-7 for SE and n = 4-8 for
bral lactate level and oxidative damage [20,26]. brain.
As noted above, LNAAs cross the BBB on the LAT-1
transporter, and accumulation of offending amino acids Tissue harvesting and analytical methodology
such as the BCAAs in MSUD can result in exclusion of At appropriate time points, animal subjects were eutha-
other LNAAs from the brain, although this has not been nized, and blood collected by cardiac puncture. The brain
rigorously evaluated in either patients or an animal model cavity was rapidly accessed on an ice-cold glass plate, with
of MSUD. Despite the specificity of the LAT-1 for LNAAs, parietal cortex (CTX), striata (STR), and cerebellum (CB)
most brain amino acid transport systems display broad rapidly excised. Harvested tissues and sera following centri-
overlap in the amino acids transported [3,4,27,28]. Here, fugation were stored at – 80°C until analyses. LNAAs and
we have employed a murine model of MSUD to address other amino acids were determined with an ultraperfor-
the question of whether control of BCAA level, as moni- mance liquid chromatography (UPLC) MassTrak amino
tored in blood, is reflected by similar correction in the acid system as previously described [36]. All values in SE
brain. Several novel therapeutic approaches to PKU have are presented in units of μmol/L, wherease all values in
emerged recently [29], yet with the exceptions of phenyl- brain regions are presented in units of nmol/gr tissue.
butyrate administration (which selectively lowers circulat-
ing BCAAs) [30] or orthotopic liver transplantation for Statistical analyses
the most severe forms of MSUD [8,15,31,32], few thera- In prior studies, we had observed depletion of GLU,
peutic advances have emerged in MSUD. The current re- GLN, ASP, serine (SER), alanine (ALA) and GABA, with
port addresses our hypothesis, and for the first time elevated glycine (GLY) in brain of imsud mice. However,
verifies in a murine model of MSUD that assessment of those studies were performed in whole brain [1,2,33,34],
metabolic control using blood studies is a poor surrogate potentially leading to artifact from mixing of regions
for disturbances of amino acid homeostasis in the brain. with high vs. low metabolite levels. In the current study
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Figure 1 Branched chain amino acids in sera (SE), cerebellum (CB), striata (STR) and cortex (CTX) as a function of protein intake (6 or 19%)
and genotype (w, wild-type; m, mutant). Two-way ANOVA (parameters: protein intake, tissue), p = ns for tissue and interaction, p < 0.05 for protein
intake. Two-tailed t-test, p < 0.05 for all mutant vs. wild-type (not shown).

we assessed discrete brain regions. Accordingly, we had ALLO in mutants with decreased protein consump-
no insight whether to expect brain regional amino acids tion were 39 (22) to 12 (3) μmol/L in SE, 66 (37) to
to be constant or different. Thus, we initially employed a 37 (24) in CB, 75 (44) to 44 (31) in STR, and 41 (29)
two-way ANOVA to assess correlations between brain to 27 (15) in CTX (brain values, nmol/gr protein).
region and protein intake, and interaction of these pa- As expected, ALLO was undetectable in wild-type
rameters, accompanied by a Holm-Sidak’s multiple com- mice with either diet. There was no significant differ-
parisons test. Our primary statistical analyses employed ence in any tissue with decreased protein intake for
a two-tailed t test within genotype as a function of pro- ALLO in mutant mice.
tein consumption. Both statistical evaluations are pre- We then turned our attention to the other LNAAs
sented in all figure legends. Comparisons of amino acid (MET, PHE, TYR and TRP) in the same regions
concentrations between blood and brain assumed a (Figure 3). For TYR, changes for mutant mice with
density of brain tissue of ~1 g/ml [37]. decreased protein intake were 94 (19) to 134 (10)
μmol/L in SE, 98 (13) to 143 (44) in CB, 57 (n = 2)-
Results 92 (26) in striata and 102 (17) to 139 (46) in CTX
For imsud mice, all BCAAs were significantly increased (brain, nmol/gr tissue). For TRP, the same values for
in comparison to littermate controls, verifying the utility mutant were 49 (5) to 57 (12) μmol/L in SE, 28 (4)
of the model (Figure 1). With decreased protein con- to 31 (8) in CB, 28 (7) to 32 (10) in STR and 25 (4)
sumption, LEU in mutant SE decreased from 1072 to 26 (9) in CTX. For MET, these values were 77
(504) (standard deviation shown in parenthesis after (16) to 82 (27) μmol/L in SE, 132 (41) to 93 (29) in
mean) to 436 (107) μmol/L, while the same decreases CB, 121 (40) to 113 (28) in STR and 146 (11) to 143
in mutant were 1026 (444) to 723 (415) in CB, 1147
(535) to 853 (648) in STR and 1017 (516) to 755
(452) in CTX (brain regions: nmol/gr tissue). ILE
changes in mutant mice with decreased protein were
488 (115) to 362 (41) μmol/L in SE, 555 (84) to 585
(226) in CB, 625 (132) to 687 (349) in SE and 583
(126) to 636 (235) in cortex (nmol/gr tissue). For
VAL, comparable changes were 1007 (441) to 434
(71) μmol/L in SE, 950 (347) to 651 (288) in CB,
1071 (421) to 758 (453) in STR and 894 (448) to 661
(351) in CTX (nmol/gr tissue).
Within tissues (SE, CB, STR and CTX), there was no
significant improvement in BCAAs with lowered protein
intake, although the trend in SE BCAAs approached sig-
nificance (t-test, 2-tailed, p = 0.074-0.125 vs. CB, STR and
CTX, p = 0.210-0.789), but variation in SDs (occasional Figure 2 Alloisoleucine levels (see Figure 1 for details). Two-way
low SE quantity for selected animals) was problematic. ANOVA, p < 0.05 for protein intake, p = ns for tissue and interaction.
Two-tailed t-test, p = ns between mutant and wild-type as a function of
Alloisoleucine (ALLO, a pathognomonic biomarker for
protein intake.
MSUD) showed similar trends (Figure 2). The changes for
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Figure 3 Other large neutral amino acids (tyrosine, tryptophan, methionine, phenylalanine) as a function of protein intake and genotype
(see Figure 1 for details). Two-tailed ANOVA, p < 0.05 for both protein intake, tissue and interaction. Two-tailed t-test (*p < 0.05), comparing M19 and
W19 in CTX and CB (TYR) and STR (TRP). Also, p < 0.05 for TYR in SE (M6 vs M19).

(29) in CTX. For PHE, the corresponding values were GLU are not depicted for SE because of the much higher
94 (15) to 86 (19) μmol/L in SE, 104 (13) to 118 (13) brain levels, which would graphically dwarf SE values, and
in CB, 99 (17) to 124 (41) in STR and 100 (12) to GABA is not quantifiable in SE. Changes in GLN in mu-
101 (14) in CTX (nmol/gr tissue). tant brain regions with decreased protein consump-
Elevated BCAA levels had no substantial effect on TRP, tion were 2590 (726) to 3021 (477) in CB, 1575 (516)
MET and PHE levels in any brain region, although SE to 2342 (503) in STR and 1862 (739) to 2064 (336)
TYR was improved (increased in mutant mice; Figure 3) in CTXD (nmol/gr tissue). For ASP, the same changes
with decreased protein consumption (without a corre- with decreased protein were 1818 (303) to 2206 (420)
sponding correction in brain regions). This finding lends in CB, 1662 (517) to 1829 (183) in STR and 1808
credence to the hypothesis that correction of blood amino (420) to 2021 (187) in CTX; for GLU, 4973 (774) to
acid content may not reflect corresponding corrections in 5045 (1263) in CB, 6238 (1264) to 6554 (407) in STR
brain. Conversely, compared to control mice receiving and 7019 (1226) to 7246 (134) in CTX; and for
identical diet, levels of TYR or TRP were depleted in CB, GABA, 1920 (207) to 2317 (378) in CB, 1958 (316) to
STR and CTX with high protein intake, but these deple- 2364 (409) in STR and 2595 (171) to 2789 (329) in
tions were not consistent across different brain regions CTX (nmol/gr tissue).
(Figure 3). With the exception of GABA (which improved with
We next surveyed all remaining physiological amino lower protein intake), GLN, ASP and GLU were not im-
acids across brain regions, and observed significant abnor- proved with lowered protein consumption (Figure 4).
malities in GLN, ASP, GLU, GABA, ASN (asparagine); Conversely, lowered protein intake normalized the level of
CIT (citrulline), SER and ALA (Figures 4, 5 and 6). For ASN in CB and the concentration of CIT in STR and
comparison purposes, concentrations for GLN, ASP and CTX, while both amino acids were significantly increased

Figure 4 Neurotransmitter amino acids (glutamine, aspartate, glutamate and GABA) as a function of protein intake and tissue (see
Figure 1 Legend). Two-way ANOVA, p < 0.05 for tissue, protein intake and interaction. Two-tailed t-test, *p < 0.05 for all mutant vs. wild-type
under both protein intake regimens.
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Figure 5 Asparagine and citrulline level as a function of protein intake and tissue. Two-way ANOVA, p < 0.05 for protein intake and interaction,
p = ns for tissue. Two-tailed t-test, *p < 0.05 between genotype with identical protein intake.

vs. control during high-protein intake (Figure 5). Similar (data not shown). SER levels in SE were not abnormal in
findings were observed for SER and ALA, in which cor- mutant mice (although they were disrupted in brain re-
rection with lowered protein consumption was observed gions; Figure 6). Conversely, disruptions of ALA levels in
in CB, but not in STR or CTX (Figure 6). Threonine SE mirrored the abnormalities observed in brain (data not
(THR) is often considered a member of the LNAA sub- shown). Of interest, abnormalities of LYS in SE mirrored
group, but we found no consistent changes in brain re- the disruptions observed in brain (Figure 7), especially in
gions, with the exception of a single instance in striata the instance of high-protein intake, perhaps suggesting
(Figure 7). Conversely, LYS is not considered a LNAA, yet that some portion of LYS is trafficked on the large-neutral
its values were significantly impacted during high-protein amino acid transporter.
consumption (Figure 7). Depletion of brain GLN, GLU and GABA have been
Since concentrations of several amino acids are much suggested to associate with transamination via accumu-
lower in SE vs. brain regions (e.g., Figures 1, 2, and 3), lated KIC in brain [5]. Accordingly, we predicted that
we did not present them graphically, as noted above. We Pahenu2 mice (representing the large neutral aminoacido-
observed no significant changes for GLU and GLN in SE pathy PKU [3,4]) would have normal GLN, GLU and
(p > 0.05), yet found brain disruptions that were uncor- GABA since KIC is not elevated in this disorder. To ad-
rected with lowered protein intake (Figure 4). Con- dress this hypothesis, we developed cohorts of Pahenu2−/−
versely, in SE we observed a significant difference for mice and parallel controls and fed them diets identical to
ASP (19% diet: imsud−/−, 59 ± 17 (n = 5) vs. imsud+/+, those given to imsud mice. We first verified the utility of
104 ± 21 (n = 7) (p < 0.05), which corrected with 6% diet our PKU model through quantitation of brain PHE
(imsud−/−, 78 ± 37 (n = 3) vs. imsud+/+, 136 ± 90 (n = 5) levels, which was significantly increased (and not cor-
(p = ns). ASN in SE was within normal limits for imsud rected even with 6% protein intake; Figure 8). Unex-
subjects, despite significant alterations in brain. For CIT, pectedly, alterations in brain regions for ASP, GLN
elevations in SE approximated those seen in brain regions and GLU in Pahenu2−/− mirrored those observed in

Figure 6 Serine and alanine levels as a function of protein intake and tissue. Two-way ANOVA, p < 0.05 for protein intake, tissue and interaction.
Two-tailed t-test, *p < 0.05 for genotype with identical protein intake.
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Figure 7 Threonine and lysine levels as a function of protein intake and tissue. Two-way ANOVA, p < 0.05 for protein intake, tissue and
interaction. Two-tailed t-test, *p < 0.05 for genotype with identical protein intake.

imsud mice (Figure 9). These changes in Pahenu2−/− Discussion

mice with decreased protein intake were: GLN: 3675 The range of means for BCAAs in SE of imsud mice re-
(381) to 4500 (258) in CB, 1964 (261) to 2308 (208) ceiving 6% dietary protein was 362-434 μM (Figure 1),
in STR and 1557 (280) to 1876 (297) in CTX (nmol/ consistent with the range of LEU for 13 intermediate
gr tissue); ASP: 2468 (443) to 2335 (98) in CB, 1758 MSUD patients under dietary intervention (379 ± 147 μM;
(293) to 1661 (109) in STR and 2216 (456) to 2044 [38]) and 282 ± 277 μM for classical MSUD patients [5]
(306) in CTX; CLU: 6781 (559) to 6833 (320) in CB, considered to be under “metabolic control”. Based on
6041 (461) to 5777 (363) in STR and 6427 (810) to these human data, 6% protein consumption normalized
6164 (637) in CTX; GABA: 2152 (279) to 2470 (283) blood BCAAs, yet we observed significant disruption of
in CB, 1735 (264) to 1470 (168) in STR and 2304 several other amino acids in the brain despite apparent
(233) to 2271 (308) in CTX (all nmol/gr tissue). The metabolic control manifested in SE. We hypothesized that
striking similarity in abnormalities between imsud−/− reduced levels of GLN, GLU, GABA and ALA in brain
and Pahenu2−/− mice in different regions of the brain, regions might be partially explained by KIC accumulation
as a function of protein intake (Figure 9), suggests ([5]; see Figures 4 and 6; not measured in the current
that other factors, in addition to brain keto-acid ac- study), yet similar anomalies in PKU mice argues against
cumulation, may be at play in the brains of animals this hypothesis (Figure 9). CIT accumulation in imsud
with large neutral aminoacidopathies, and most likely brain regions likely reflects depletion of ASP, the latter ne-
in patients as well. cessary for condensation with CIT to form argininosucci-
nate within the urea cycle.
The original studies of Pardridge and Oldendorf [39] in
the rat indicated that PHE, TRP, MET, TYR, LEU, ILE,
VAL and HIS were transported into the brain on the
LAT-1, with Km values ranging from ~ 25 to 200 μM
(highest affinity, PHE; lowest affinity HIS). Limited data
suggests that THR and LYS may also traffic on LAT-1
[3,4,40,41], yet we found no evidence for disrupted THR
levels. Conversely, we did detect decreased LYS in brain
regions of imsud subjects. LYS would be expected to be
transported predominantly via the B transporter [3], al-
though there is considerable redundancy in many of the
blood–brain transporters.
Long-term neurocognitive deficits are observed in
MSUD, even with acceptable metabolic control (as deter-
Figure 8 Phenylalanine levels as a function of protein intake mined in blood), and both animal and human data sug-
and tissue in phenylketonuric mice. Two-way ANOVA, p < 0.05 for gests that these deficits correlate with exclusion of the
protein intake, tissue and interaction. All values in mutant mice were monoamine precursors TYR and TRP from the brain due
significantly increased in comparison to control mice (p < 0.05, two
to chronic elevations of BCAAs [1,2,25,33,34]. While our
tailed t-test; not shown on graph).
studies revealed significant effects on brain TYR with diet
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Figure 9 Glutamine, aspartate, glutamate and GABA as a function of protein intake and tissue in phenylketonuric (PKU) mice. Two-way
ANOVA, p < 0.05 for tissue, protein intake and interaction. Two-tailed t-test, *p < 0.05 for mutant vs. wild-type. Note: For GABA in CTX, GABA was
also significantly decreased in mutant vs. wild-type mice. In only a single instance did lower protein result in significant correction between
mutant populations (GLN in CB).

in imsud subjects, we found no consistent effect on TRP receiving 19% protein intake; imsud−/−: Intermediate maple syrup urine
(although we did not quantify monoamine neurotransmit- disease mice; Pahenu2−/−: Phenylketonuric mice; LEU: Leucine; ILE: Isoleucine;
VAL: Valine; ALLO: Allosisoleucine; SER: Serine; CIT: Citrulline; THR: Threonine;
ters). Alternatively, our data supports the premise that ALA: Alanine; LYS: Lysine; HIS: Histidine; GLN: Glutamine; ASP: Aspartate;
long-term neurocognitive deficits in MSUD patients may GLU: Glutamate; GABA: 4-aminobutyrate; PHE: Phenylalanine; TYR: Tyrosine;
also correlate with depletion of amino acid neurotransmit- MET: Methionine; TRP: Tryptophan; ASN: Asparagine.
ters (ASP, GLU and GABA). Based upon the preliminary Competing interests
studies in the current report, our next objectives are to The authors declare that they have no competing interests.
investigate LEU-depleted diets in imsud−/− mice that are
Authors’ contributions
appropriately supplemented with ILE and VAL, a diet cor- KRV performed all animal studies, including breeding and dietary
responding to that used in MSUD patients, and to again intervention, dissection, and assisted with development of the manuscript:
investigate the amino acid outcomes both regionally in EA, BW and TB performed all analytical studies and assisted in drafting the
manuscript; SM performed all data analysis, including data evaluation,
brain and blood. Such studies will yield a better overview statistical modeling, and assisted in drafting the manuscript; KMG designed
of the expected outcomes for a diet more closely resem- the study, evaluated amino acid data, derived the hypothesis for testing and
bling that utilized in the clinic. The main conclusions developed the first draft of the manuscript. All authors read and approved
the final manuscript.
drawn from the current report include:
Acknowledgements
 The first detailed cross-correlation assessment of KRV and KMG gratefully acknowledge partial support from NIH HD58553,
NS82286, the National PKU Alliance, and a Pre-Doctoral Award from the
amino acids in brain and blood, as a function of total American Foundation for Pharmaceutical Education (AFPE).
protein intake, and the first analysis of amino acids
in discrete regions of the brain of imsud mice, Author details
1
Experimental and Systems Pharmacology, College of Pharmacy, Washington
including cerebellum, cortex and striata State University, 412 E. Spokane Falls Blvd., Pharmaceutical and Biomedical
 A clear demonstration that blood amino acid Sciences Building, Room 347, P.O. Box 1495, 99210-1495 Spokane, WA, USA.
2
analysis, and the assignment of metabolic control Institute of Metabolic Disease and Baylor Research Institute, Baylor University
Medical Center, Dallas, TX, USA. 3College of Nursing, Washington State
with respect to BCAA levels, represents only a University, Spokane, WA, USA.
partial picture of brain amino acid homeostasis
Received: 19 February 2014 Accepted: 25 April 2014
Published: 8 May 2014
These results further support the concept that dietary
intervention in MSUD is likely to be insufficient to avoid References
neurocognitive deficiencies in patients, underscoring the 1. Skvorak KJ, Dorko K, Marongiu F, Tahan V, Hansel MC, Gramignoli R, Arning
E, Bottiglieri T, Gibson KM, Strom SC: Improved amino acid, bioenergetic
urgent need for more focused therapeutics in MSUD. metabolite and neurotransmitter profiles following human amnion
epithelial cell transplant in intermediate maple syrup urine disease mice.
Abbreviations Mol Genet Metab 2013, 109:132–138.
SE: Sera; CB: Cerebellum; STR: Striata; CTX: cortex; W6: Wild-type mice 2. Skvorak KJ, Dorko K, Marongiu F, Tahan V, Hansel MC, Gramignoli R, Gibson
receiving 6% protein intake; M6: Mutant mice receiving 6% protein intake; KM, Strom SC: Placental stem cell correction of murine intermediate
W19: Wild-type mice receiving 19% protein intake; M19: Mutant mice maple syrup urine disease. Hepatology 2013, 57:1017–1023.
Vogel et al. Orphanet Journal of Rare Diseases 2014, 9:73 Page 8 of 8
http://www.ojrd.com/content/9/1/73

3. Vogel KR, Arning E, Wasek BL, Bottiglieri T, Gibson KM: Non-physiological the acute phase of maple syrup urine disease and citrullinemia
amino acid (NPAA) therapy targeting brain phenylalanine reduction: encephalopathies in newborn calves. J Neurochem 1992, 59:582–590.
pilot studies in PAHENU2 mice. J Inherit Metab Dis 2013, 36:513–523. 25. Zinnanti WJ, Lazovic J, Griffin K, Skvorak KJ, Paul HS, Homanics GE, Bewley
4. Vogel KR, Arning E, Wasek BL, Bottiglieri T, Gibson KM: Characterization of MC, Cheng KC, Lanoue KF, Flanagan JM: Dual mechanism of brain injury
2-(Methylamino)Alkanoic acids capacity to restrict blood–brain and novel treatment strategy in maple syrup urine disease. Brain 2009,
Phenylalanine transport in Pahenu2 Mice: preliminary findings. Molec 132(Pt 4):903–918.
Genet Metab 2013. Aug 15 [Epub ahead of print]. 26. Sitta A, Ribas GS, Mescka CP, Barschak AG, Wajner M, Vargas CR:
5. Strauss KA, Wardley B, Robinson D, Hendrickson C, Rider NL, Puffenberger Neurological damage in MSUD: the role of oxidative stress. Cell Mol
EG, Shellmer D, Moser AB, Morton DH: Classical maple syrup urine disease Neurobiol 2013. Nov 13. [Epub ahead of print].
and brain development: principles of management and formula design. 27. Choi TB, Pardridge WM: Phenylalanine transport at the human blood–
Mol Genet Metab 2010, 99(4):333–345. brain barrier. Studies with isolated human brain capillaries. J Biol Chem
6. le Roux C, Murphy E, Hallam P, Lilburn M, Orlowska D, Lee P: 1986, 261(14):6536–6541.
Neuropsychometric outcome predictors for adults with maple syrup 28. Smith QR, Momma S, Aoyagi M, Rapoport SI: Kinetics of neutral amino
urine disease. J Inherit Metab Dis 2006, 29:201–202. acid transport across the blood–brain barrier. J Neurochem 1987,
7. Muelly ER, Moore GJ, Bunce SC, Mack J, Bigler DC, Morton DH, Strauss KA: 49:1651–1658.
Biochemical correlates of neuropsychiatric illness in maple syrup urine 29. Ney DM, Blank RD, Hansen KE: Advances in the nutritional and
disease. J Clin Invest 2013, 123:1809–1820. pharmacological management of phenylketonuria. Curr Opin Clin Nutr
8. Mazariegos GV, Morton DH, Sindhi R, Soltys K, Nayyar N, Bond G, Shellmer Metab Care 2014, 17(1):61–68.
D, Shneider B, Vockley J, Strauss KA: Liver transplantation for classical 30. Brunetti-Pierri N, Lanpher B, Erez A, Ananieva EA, Islam M, Marini JC, Sun Q,
maple syrup urine disease: long-term follow-up in 37 patients and Yu C, Hegde M, Li J, Wynn RM, Chuang DT, Hutson S, Lee B:
comparative United network for organ sharing experience. J Pediatr 2012, Phenylbutyrate therapy for maple syrup urine disease. Hum Mol Genet
160(1):116–121.e1. 2011, 20(4):631–640.
9. Morton DH, Strauss KA, Robinson DL, Puffenberger EG, Kelley RI: Diagnosis 31. Popescu I, Dima SO: Domino liver transplantation: how far can we push
and treatment of maple syrup disease: a study of 36 patients. Pediatrics the paradigm? Liver Transpl 2012, 18:22–28.
2002, 109:999–1008. 32. Strauss KA, Mazariegos GV, Sindhi R, Squires R, Finegold DN, Vockley G,
10. Simon E, Schwarz M, Wendel U: Social outcome in adults with maple Robinson DL, Hendrickson C, Virji M, Cropcho L, Puffenberger EG, McGhee
syrup urine disease (MSUD). J Inherit Metab Dis 2007, 30:264. W, Seward LM, Morton DH: Elective liver transplantation for the treatment
11. Schönberger S, Schweiger B, Schwahn B, Schwarz M, Wendel U: of classical maple syrup urine disease. Am J Transplant 2006, 6(3):557–564.
Dysmyelination in the brain of adolescents and young adults with 33. Skvorak KJ, Hager EJ, Arning E, Bottiglieri T, Paul HS, Strom SC, Homanics GE,
maple syrup urine disease. Mol Genet Metab 2004, 82:69–75. Sun Q, Jansen EE, Jakobs C, Zinnanti WJ, Gibson KM: Hepatocyte
12. Klee D, Thimm E, Wittsack HJ, Schubert D, Primke R, Pentang G, Schaper J, transplantation (HTx) corrects selected neurometabolic abnormalities in
Mödder U, Antoch A, Wendel U, Cohnen M: Structural white matter murine intermediate maple syrup urine disease (iMSUD). Biochim Biophys
changes in adolescents and young adults with maple syrup urine Acta 2009, 1792:1004–1010.
disease. J Inherit Metab Dis 2013. Jan 25. [Epub ahead of print]. 34. Skvorak KJ, Paul HS, Dorko K, Marongiu F, Ellis E, Chace D, Ferguson C,
13. Packman W, Mehta I, Rafie S, Mehta J, Naldi M, Mooney KH: Young adults Gibson KM, Homanics GE, Strom SC: Hepatocyte transplantation improves
with MSUD and their transition to adulthood: psychosocial issues. phenotype and extends survival in a murine model of intermediate
J Genet Couns 2012, 21:692–703. maple syrup urine disease. Mol Ther 2009, 17:1266–1273.
14. Carecchio M, Schneider SA, Chan H, Lachmann R, Lee PJ, Murphy E, Bhatia 35. Antflick JE, Baker GB, Hampson DR: The effects of a low protein diet on
KP: Movement disorders in adult surviving patients with maple syrup amino acids and enzymes in the serine synthesis pathway in mice.
urine disease. Mov Disord 2011, 26(7):1324–1328. Amino Acids 2010, 39:145–153.
15. Shellmer DA, DeVito DA, Dew MA, Noll RB, Feldman H, Strauss KA, Morton 36. Narayan SB, Ditewig-Meyers G, Graham KS, Scott R, Bennett MJ: Measure-
DH, Vockley J, Mazariegos GV: Cognitive and adaptive functioning after ment of plasma amino acids by Ultraperformance® Liquid Chromatog-
liver transplantation for maple syrup urine disease: a case series. Pediatr raphy. Clin Chem Lab Med 2011, 49:1177–1185.
Transplant 2011, 15(1):58–64. 37. Barber TW, Brockway JA, Higgins LS: The density of tissues in and about
16. Walsh KS, Scott MN: Neurocognitive profile in a case of maple syrup the head. Acta Neurol Scand 1970, 46:85–92.
urine disease. Clin Neuropsychol 2010, 24(4):689–700. 38. Hoffmann B, Helbling C, Schadewaldt P, Wendel U: Impact of longitudinal
plasma leucine levels on the intellectual outcome in patients with classic
17. Strauss KA, Puffenberger EG, Morton DH: Maple Syrup Urine Disease. In
MSUD. Pediatr Res 2006, 59(1):17–20.
GeneReviews™ [Internet]. Edited by Pagon RA, Adam MP, Bird TD, Dolan CR,
39. Pardridge WM, Oldendorf WH: Kinetic analysis of blood–brain barrier
Fong CT, Stephens K. Seattle: University of Washington, Seattle; 2006. Jan 30
transport of amino acids. Biochim Biophys Acta 1975, 401(1):128–136.
[updated 2013 May 09].
40. Tews JK, Kim YW, Harper AE: Induction of threonine imbalance by
18. Bridi R, Braun CA, Zorzi GK, Wannmacher CM, Wajner M, Lissi EG, Dutra-Filho
dispensable amino acids: relation to competition for amino acid
CS: Alpha-keto acids accumulating in maple syrup urine disease
transport into brain. J Nutr 1979, 109(2):304–315.
stimulate lipid peroxidation and reduce antioxidant defences in cerebral
41. Tews JK, Kim YW, Harper AE: Induction of threonine imbalance by
cortex from young rats. Metab Brain Dis 2005, 20:155–167.
dispensable amino acids: relationships between tissue amino acids and
19. Funchal C, Gottfried C, De Almeida LM, Wajner M, Pessoa-Pureur R:
diet in rats. J Nutr 1980, 110(3):394–408.
Evidence that the branched-chain alpha-keto acids accumulating in
maple syrup urine disease induce morphological alterations and death
in cultured astrocytes from rat cerebral cortex. Glia 2004, 48:230–240. doi:10.1186/1750-1172-9-73
Cite this article as: Vogel et al.: Brain–blood amino acid correlates
20. Barschak AG, Marchesan C, Sitta A, Deon M, Giugliani R, Wajner M, Vargas
following protein restriction in murine maple syrup urine disease.
CR: Maple syrup urine disease in treated patients: biochemical and
Orphanet Journal of Rare Diseases 2014 9:73.
oxidative stress profiles. Clin Biochem 2008, 41:317–324.
21. O’Kane RL, Hawkins RA: Na + −dependent transport of large neutral
amino acids occurs at the abluminal membrane of the blood–brain
barrier. Am J Physiol Endocrinol Metab 2003, 285:E1167–E1173.
22. Yudkoff M, Daikhin Y, Nissim I, Horyn O, Luhovyy B, Lazarow A, Nissim I:
Brain amino acid requirements and toxicity: the example of leucine.
J Nutr 2005, 135:1531S–1538S.
23. Yudkoff M: Brain metabolism of branched-chain amino acids. Glia 1997,
21:92–98.
24. Dodd PR, Williams SH, Gundlach AL, Harper PA, Healy PJ, Dennis JA, Johnston
GA: Glutamate and gamma-aminobutyric acid neurotransmitter systems in