Complementary dietary treatment using lysine-free, arginine-fortified amino acid supplements in glutaric aciduria type I - A decade of experience.

Abstract

The cerebral formation and entrapment of neurotoxic dicarboxylic metabolites (glutaryl-CoA, glutaric and 3-hydroxyglutaric acid) are considered to be important pathomechanisms of striatal injury in glutaric aciduria type I (GA-I). The quantitatively most important precursor of these metabolites is lysine. Recommended therapeutic interventions aim to reduce lysine oxidation (low lysine diet, emergency treatment to minimize catabolism) and to enhance physiologic detoxification of glutaryl-CoA via formation of glutarylcarnitine (carnitine supplementation). It has been recently shown in Gcdh(-/-) mice that cerebral lysine influx and oxidation can be modulated by arginine which competes with lysine for transport at the blood-brain barrier and the inner mitochondrial membrane [Sauer et al., Brain 134 (2011) 157-170]. Furthermore, short-term outcome of 12 children receiving arginine-fortified diet showed very promising results [Strauss et al., Mol. Genet. Metab. 104 (2011) 93-106]. Since lysine-free, arginine-fortified amino acid supplements (AAS) are commercially available and used in Germany for more than a decade, we evaluated the effect of arginine supplementation in a cohort of 34 neonatally diagnosed GA-I patients (median age, 7.43years; cumulative follow-up period, 221.6patientyears) who received metabolic treatment according to a published guideline [Kölker et al., J. Inherit. Metab. Dis. 30 (2007) 5-22]. Patients used one of two AAS product lines during the first year of life, resulting in differences in arginine consumption [group 1 (Milupa Metabolics): mean=111mg arginine/kg; group 2 (Nutricia): mean=145mg arginine/kg; p<0.001]. However, in both groups the daily arginine intake was increased (mean, 137mg/kg body weight) and the dietary lysine-to-arginine ratio was decreased (mean, 0.7) compared to infants receiving human milk and other natural foods only. All other dietary parameters were in the same range. Despite significantly different arginine intake, the plasma lysine-to-arginine ratio did not differ in both groups. Frequency of dystonia was low (group 1: 12.5%; group 2: 8%) compared with patients not being treated according to the guideline, and gross motor development was similar in both groups. In conclusion, the development of complementary dietary strategies exploiting transport competition between lysine and arginine for treatment of GA-I seems promising. More work is required to understand neuroprotective mechanisms of arginine, to develop dietary recommendations for arginine and to evaluate the usefulness of plasma monitoring for lysine and arginine levels as predictors of cerebral lysine influx.

Full text

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Complementary dietary treatment using lysine-free, arginine-fortified amino acid
supplements in glutaric aciduria type I — A decade of experience
Stefan Kölkera,⁎, S.P. Nikolas Boya, Jana Heringera, Edith Müllera, Esther M. Maierb, Regina Ensenauerb,
Chris Mühlhausenc, Andrea Schluned, Cheryl R. Greenberge, David M. Koellerf,g, Georg F. Hoffmanna,
Gisela Haegea,1, Peter Burgarda,1
aDepartment of General Pediatrics, Division of Inherited Metabolic Diseases, University Hospital Heidelberg, Heidelberg, Germany
bResearch Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, Munich, Germany
cDepartment of Pediatrics, University Medical Centre, Hamburg, Germany
dDepartment of General Pediatrics and Neonatology, University Children's Hospital, Heinrich-Heine University, Düsseldorf, Germany
eDepartment of Biochemical and Medical Genetics, Winnipeg Children's Hospital, University of Manitoba, Winnipeg, MB, Canada R3A 1R9
fDepartment of Pediatrics, Doernbecher Children's Hospital, Oregon Health and Science University, Portland, OR 97239, USA
gDepartment of Molecular and Medical Genetics, Doernbecher Children's Hospital, Oregon Health and Science University, Portland, OR 97239, USA
a b s t r a c ta r t i c l e i n f o
Article history:
Received 28 March 2012
Accepted 28 March 2012
Available online xxxx
Keywords:
Glutaric aciduria type I
Arginine
Lysine
Diet
Guideline
Dystonia
The cerebral formation and entrapment of neurotoxic dicarboxylic metabolites (glutaryl-CoA, glutaric and 3-
hydroxyglutaric acid) are considered to be important pathomechanisms of striatal injury in glutaric aciduria
type I (GA-I). The quantitatively most important precursor of these metabolites is lysine. Recommended
therapeutic interventions aim to reduce lysine oxidation (low lysine diet, emergency treatment to minimize
catabolism) and to enhance physiologic detoxification of glutaryl-CoA via formation of glutarylcarnitine
(carnitine supplementation). It has been recently shown in Gcdh−/−mice that cerebral lysine influx and
oxidation can be modulated by arginine which competes with lysine for transport at the blood–brain barrier
and the inner mitochondrial membrane [Sauer et al., Brain 134 (2011) 157–170]. Furthermore, short-term
outcome of 12 children receiving arginine-fortified diet showed very promising results [Strauss et al., Mol.
Genet. Metab. 104 (2011) 93–106]. Since lysine-free, arginine-fortified amino acid supplements (AAS) are
commercially available and used in Germany for more than a decade, we evaluated the effect of arginine
supplementation in a cohort of 34 neonatally diagnosed GA-I patients (median age, 7.43 years; cumulative
follow-up period, 221.6 patient years) who received metabolic treatment according to a published guideline
[Kölker et al., J. Inherit. Metab. Dis. 30 (2007) 5–22]. Patients used one of two AAS product lines during the
first year of life, resulting in differences in arginine consumption [group 1 (Milupa Metabolics):
mean=111 mg arginine/kg; group 2 (Nutricia): mean=145 mg arginine/kg; pb0.001]. However, in both
groups the daily arginine intake was increased (mean, 137 mg/kg body weight) and the dietary lysine-to-
arginine ratio was decreased (mean, 0.7) compared to infants receiving human milk and other natural foods
only. All other dietary parameters were in the same range. Despite significantly different arginine intake, the
plasma lysine-to-arginine ratio did not differ in both groups. Frequency of dystonia was low (group 1: 12.5%;
group 2: 8%) compared with patients not being treated according to the guideline, and gross motor
development was similar in both groups. In conclusion, the development of complementary dietary
strategies exploiting transport competition between lysine and arginine for treatment of GA-I seems
promising. More work is required to understand neuroprotective mechanisms of arginine, to develop dietary
recommendations for arginine and to evaluate the usefulness of plasma monitoring for lysine and arginine
levels as predictors of cerebral lysine influx.
© 2012 Elsevier Inc. All rights reserved.
Molecular Genetics and Metabolism xxx (2012) xxx–xxx
Abbreviations: 3-OH-GA, 3-hydroxyglutaric acid; AAS, amino acid supplement; ANOVA, analysis of variance; BBB, blood–brain barrier; CAT1, cationic amino acid transporter 1;
GA, glutaric acid; GA-I, glutaric aciduria type I; GCDH, glutaryl-CoA dehydrogenase (EC 1.3.99.7); NO, nitric oxide; ORNT1 and 2, mitochondrial ornithine transporters 1 and 2; SDS,
standard deviation score.
⁎ Corresponding author at: University Children's Hospital Heidelberg, Department of General Pediatrics, Division of Inherited Metabolic Diseases, Im Neuenheimer Feld 430, D-69120
Heidelberg, Germany. Fax: +49 6221 565565.
E-mail address: Stefan.Koelker@med.uni-heidelberg.de (S. Kölker).
1Both authors contributed equally to this study.
YMGME-05266; No. of pages: 9; 4C:
1096-7192/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2012.03.021
Contents lists available at SciVerse ScienceDirect
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journal homepage: www.elsevier.com/locate/ymgme
Please cite this article as: S. Kölker, et al., Complementary dietary treatment using lysine-free, arginine-fortified amino acid supplements in
glutaric aciduria type I — A decade of experience, Mol. Genet. Metab. (2012), doi:10.1016/j.ymgme.2012.03.021

Page 2

1. Introduction
Glutaric aciduriatype I(GA-I)isa rarecerebralorganicacid disorder
first described in 1975 [1]. The disease is caused by inherited deficiency
of the homotetrameric mitochondrial flavoprotein glutaryl-CoA dehy-
drogenase (GCDH; EC 1.3.99.7), which is encoded by the GCDH gene
mapping to human chromosome locus 19p13.2 [2]. More than 200
disease-causing mutations have been identified so far [3,4]. GCDH
catalyzesthe oxidativedecarboxylationofglutaryl-CoA to crotonyl-CoA
in the final degradative pathways of L-lysine, L-hydroxylysine, and L-
tryptophan [5]. Deficiency of this enzyme results in accumulation of
glutaryl-CoA,glutaricacid(GA),3-hydroxyglutaricacid(3-OH-GA),and
glutarylcarnitine. Natural protein consists of 2–9% lysine and 0.6–2%
tryptophan and thus L-lysine is considered the quantitatively most
important precursor for GA, 3-OH-GA, and glutarylcarnitine [6]. The
concentrations of these compounds determined in body fluids of
patients and body fluids and tissues of Gcdh-deficient mice, and fruit-
eating bats showed a positive correlation to protein and lysine intake
[1,7–11]. The initial clinical presentation of affected neonates is non-
specific. The majority of untreated patients develop striatal injury
acutely during catabolic conditions or insidiously between 3 and
36 months of age [7,12–14]. Striatal injury results in a complex
movement disorder with predominant dystonia [15,16]. Antidystonic
treatment is often unsatisfactory, and life expectancy is limited in
severely disabled children [17,18]. The major aim of all therapeutic
strategies is to prevent brain injury.
Several studies highlight the role of GA, 3-OH-GA, and glutaryl-CoA
in the pathogenesis of this disease. Precipitation of excitotoxicity and
oxidative stress, and impairment of cerebral energy metabolism via
inhibition of the tricarboxylic acid cycle and the dicarboxylic acid
shuttle between astrocytes and neurons are considered mechanisms
[19–24]. In addition, disturbance of cerebral hemodynamics is thought
to synergize with these mechanisms [25]. We have recently identified
the blood–brain barrier (BBB) to play a central role by trapping
intracerebrally produced neurotoxic dicarboxylates due to low BBB
permeability [21,26–28]. Cerebral GA and 3-OH-GA concentrations of
untreated patients are 100–1000fold higher than those in plasma.
Strikingly, patients with complete loss of GCDH activity (i.e. high
excreters) and those with residual enzyme activity (i.e. low excreters)
reveal similar brain concentrations of GA and 3-OH-GA and share the
same risk for brain injury [13,29–32]. Gcdh−/−mice, an animal model
with complete loss of Gcdh activity [33], demonstrate the same steep
plasma-to-brain gradient of neurotoxic dicarboxylates [21]. Based on
these findings it has been suggested that lowering the cerebral lysine
influx and lysine oxidation is a potential neuroprotective strategy.
In neonatally diagnosed patients, the frequency of brain injury
was reduced to 0–36% compared to 90–95% in historical cohorts
[7,12,18,34–37]. Evidence-based treatment recommendations in-
clude (1) a low L-lysine diet (including the use of lysine-free,
tryptophan-reduced amino acid supplements [AAS]), (2) L-carnitine
supplementation, and (3) emergency treatment during intercurrent
illness [38,39]. Recently, we demonstrated that treatment according
to the published guideline was associated with a favorable outcome
(5% dystonia, n=37 patients) in a newborn screening cohort [40]. In
contrast, deviations from emergency treatment (100% dystonia, n=6
patients) and metabolic maintenance treatment (i.e. low lysine diet
and carnitine supplementation; 44% dystonia, n=9 patients) were
associated with a poor outcome.
In Gcdh−/−mice, the application of arginine, ornithine, and
homoarginine all lowered the cerebral concentrations of GA and 3-OH-
GA [41–43]. In analogy, the effect of low lysine diet was amplified by
add-on therapy with arginine. This can be explained by competition of
lysine and arginine at the BBB (cationic amino acid transporter 1, CAT1,
which is one of three CATs that are called system y+) and the inner
mitochondrial membrane (mitochondrial ornithine carriers, ORNT1 and
ORNT2) [43]. We have hypothesized that arginine supplementation
might also be useful for optimizing dietary treatment in GA-I patients
[44]. In fact, short-term outcome in 12 children receiving low lysine diet
fortified with arginine supports this notion [45]. The major aim of this
study is to specifically evaluate the effect of variation of dietary arginine
intake on neurological outcome and concentrations of plasma amino
acids in a prospectively followed newborn screening cohort.
2. Patients and methods
2.1. Study population
Thirty-eight children with confirmed diagnosis of GA-I who were
identified by newborn screening between 2000 and 2011 and received
metabolic treatment according to a published guideline [38,39] were
evaluated. Their neurological outcome has been published previously
[40]. Four childrenof this cohortwhohad not received either theamino
acid mixture manufactured by Milupa Metabolics or Nutricia AAS were
excludedfromtheanalysis.Dietary,biochemical,anthropometrical,and
neurological parameters were evaluated in the remaining 34 study
patients. The study was approved by the local ethics committees of all
study centers. Written informed consent was given by all parents of
study patients.
2.2. Calculation of dietary arginine intake and lysine-to-arginine ratio
The arginine contentof commerciallyavailablelysine-free,
tryptophan-reduced, arginine-fortified AAS given to study patients
was determined according to the manufacturer information (Table 1).
SHS LT-AM infant had the same high arginine content (i.e. 90 mg
arginine/g protein) as GlutarAde Junior GA-1 drink mix, an AAS
previously investigated [45]. All other AAS applied to study patients
hadalowerargininecontent(i.e.48–59 mgarginine/gprotein)whichis
similartothatofhumanmilk(i.e.51 mgarginine/gprotein)[6].AASdid
not contain ornithine. The amount of AAS consumed was prospectively
documented for all study patients from which the daily arginineAAS
supplementation and the nutritional lysine-to-arginineAASratio were
calculated. The effect of dietary treatment on biochemical and anthro-
pometricalparameterswasanalyzedbetweenages0and36 months,i.e.
duringthemostvulnerableperiodforthedevelopmentofstriatalinjury.
For breastfed infants (i.e. as long as human milk or adapted infant
formula was the only natural protein source), we also calculated the
total daily arginine intake along with other dietary and anthropo-
metric parameters in analogy to a previous study [45]. When
supplementary food is added, the amount of total arginine intake is
dependent on the varying arginine content of natural foods (Suppl.
Table 1). To estimate the total arginine intake and the nutritional
lysine-to-argininetotalratio in infants and children receiving more
than one natural protein source, we used standard dietary protocols
at ages 8 months and 3 years for ourcalculations (Suppl.Tables 2 and
3). In addition, dietary intake was regularly adjusted every 3–6 months
by specialized metabolic dietitians. Average lysine and arginine contents
of natural foods were taken from “Bundeslebensmittelschlüssel” [6], a
nutritional database provided by the German Ministry of Nutrition,
Agriculture and Consumer Protection containing average contents of
approximately 15,000 food items.
2.3. Calculation of standard deviation scores for anthropometrical
parameters
Standard deviation scores (SDS) of anthropometrical data (height,
weight, head circumference) were calculated using the LMS method
[46]. This method provides a way of obtaining normalized growth
centile standards values. Skewed distributions of the measurements
are approximated to normal distributions by power transformation.
The distribution of each covariate is summarized by three parameters,
the Box–Cox power λ, the mean μ, and the coefficient of variation σ.
2
S. Kölker et al. / Molecular Genetics and Metabolism xxx (2012) xxx–xxx
Please cite this article as: S. Kölker, et al., Complementary dietary treatment using lysine-free, arginine-fortified amino acid supplements in
glutaric aciduria type I — A decade of experience, Mol. Genet. Metab. (2012), doi:10.1016/j.ymgme.2012.03.021

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The calculation of SDS for anthropometrical parameters was based on
previous studies from Germany and Switzerland [47,48].
2.4. Assessment of neurological outcome
Neurological outcome was assessed by the frequency, age at onset
and severity of dystonia. Severity of dystonia was rated according to
the Barry-Albright dystonia scale, a 5-point ordinal scale for 8 body
regions (minimum: 0 point, maximum: 32 points) [49]. Furthermore,
we determined the age at achievement of five gross motor milestones
including head control, sitting without support, hands-and-knees
crawling, standing alone and walking alone.
2.5. Statistical analysis
SAS 9.2 (www.sas.com) for MS Windows and SPSS 18.0.1 were
used for the analyses. P valuesb0.05 were considered statistically
significant. For multiple testing, Sidak adjusted α levels were used.
For all parameters, descriptive statistical analysis was performed
including mean and standard deviation or median and range. For the
comparison of biochemical, anthropometrical and neurological
outcome parameters in patients receiving AAS from Milupa Meta-
bolics GmbH (group 1) or Nutricia GmbH (group 2), we used SAS
linear mixed models for repeated measures with the denominator
degrees of freedom approximated by the Kenward–Roger procedure,
a one-factorial repeated measures analysis of variance (ANOVA), and
Fisher's exact test. Pearson correlation was used to measure the
association between the lysine and arginineAAScontents of the diet
with corresponding plasma lysine and arginine concentrations.
Therapeutic and anthropometrical parameters obtained in breastfed
infants of the study cohort were compared to a previous study [45]
using ANOVA.
Therapy-related effects on the prevention of brain injury in two
recently published studies [40,45] were compared by a Chi-square
procedure. Expected frequencies for the data of study 1 [45] were
calculated by applying the row percentage distributions of study 2
[40] to the observed data representing the treatment periods
reported in study 1. As the distributions of the sample sizes of the
three treatment types were different between the two publications,
expected frequencies were calculated row-wise, thereby increasing
the degrees of freedom by one. In order to avoid division by zero the
“non adherence to emergency treatment” distribution in the data by
study 2 [40] was shifted from 6 vs. 0 to 5 vs. 1.
3. Results
3.1. Study population
We studied 34 children (21 girls, 13 boys) with confirmed diagnosis
of GA-I who were identified by newborn screening in Germany
between 2000 and 2011. The median age was 7.43 years (range,
0.7–10.9 years). At time point of analysis, 29 children (85%) were older
than 3 years of age, whereas two patients were younger than two years
(8and9 months),andthreepatientswerebetweentwoandthreeyears
of age (2.48, 2.82 and 2.95 years). Thecumulativefollow-up period was
221.6 patient years. All patients received metabolic treatment accord-
ing to guideline recommendations [38,39]. Recommendations for
metabolic maintenance treatment are briefly summarized in Suppl.
Table 4. Eight children received products of Milupa Metabolics GmbH
(i.e., Milupa GA1 and GA2 prima) and 26 children received those of
Nutricia GmbH (i.e., SHS LT-AM infant, LT-AM1 and LT-AM2).
3.2. Arginine supplementation via commercially available lysine-free,
tryptophan-reduced AAS
The arginine content of LT-AM infant is 1.5–1.9 fold higher
compared to other AAS used in the study cohort (Table 1). Since LT-
AM infant is recommended for the first year of life, we expected that
arginine supplementation would differ during this time period in
group 1 (Milupa Metabolics) and 2 (Nutricia). As expected, there was
a significant interaction between AAS group and age [F(2,48)=62.18,
pb0.001] regarding arginine supplementation. Planned contrasts
showed that arginine supplementation was higher in group 2 than
in group 1 during the first year of life [t(53)=9.67, pb0.001] but not
during the second [t(55)=−0.83, p=0.413] and third year of life
[t(55)=0.08, p=0.935] (Fig. 1). In analogy, there was a significant
interaction between AAS group and age [F(2,60)=32.84, pb0.001]
regarding the dietary lysine-to-arginineAAS ratio. Planned contrasts
showed that dietary lysine-to-arginineAASratio was lower in group 2
than in group 1 during the first year of life [t(74)=−8.41, pb0.001]
but did not differ in the second [t(75)=0.31, p=0.760] and third year
[t(74)=−1.06, p=0.293] (Fig. 2).
Table 1
Amino acid content (in mg per g protein) in lysine-free amino acid supplements fortified with amino acids, minerals,atrace elementsaand vitamins.a.
CompanyApplied Nutrition Corporation Nutricia GmbHNutricia GmbH Nutricia GmbHMilupa Metabolics GmbH Milupa Metabolics GmbH
Product nameGlutarAde Junior GA-1 drink mixb
LT-AM infantc
LT-AM 1c
LT-AM 2c
GA 1c
GA 2 primac
Lysine
Tryptophan
Arginine
Cystine
Histidine
Isoleucine
Leucine
Methionine
Phenylalanine
Threonine
Tyrosine
Valine
Alanine
Aspartate
Glutamate+glutamine
Glycine
Proline
Serine
0
5
90
n. d.
30
70
115
34
53
53
60
80
n. d.
n. d.
150
n. d.
n. d.
n. d.
0
7
0
7
0
7
0
6
0
6
90
34
52
81
49
31
31
71
124
28
59
60
60
80
51
121
213
31
117
60
59
25
25
60
99
23
45
59
52
77
63
155
225
56
104
65
48
30
30
70
52
34
34
86
138
22
61
68
61
89
52
75
102
81
98
61
118
30
50
56
60
84
50
118
252
30
112
62
142
34
60
68
74
102
58
144
304
34
134
76
n. d., not documented.
aContents of minerals, trace elements, and vitamins are not listed here.
bAccording to Strauss et al. [45].
cAccording to manufacturer information.
3
S. Kölker et al. / Molecular Genetics and Metabolism xxx (2012) xxx–xxx
Please cite this article as: S. Kölker, et al., Complementary dietary treatment using lysine-free, arginine-fortified amino acid supplements in
glutaric aciduria type I — A decade of experience, Mol. Genet. Metab. (2012), doi:10.1016/j.ymgme.2012.03.021

Page 4

3.3. Estimated total arginine intake
For breastfed infants we calculated the total arginine intake as
well as other dietary and anthropometric parameters for the time
period when human milk was the only source of natural protein. The
mean (±SD) total daily arginine intake was 137 (±23) mg per kg
body weight, and the mean (±SD) lysine-to-argininetotalratio was
0.70 (±0.2). These results as well as age, body weight, height, body
mass index, head circumference index, and daily intake of energy
substrates, total protein, natural protein, and L-carnitine showed an
excellent accordance with those of previously published 12 patients
receiving another lysine-free, arginine-fortified AAS [45]. The only
significant difference between both study cohorts was a slightly higher
lysine intake (mean, 93 vs. 77 mg per kg body weight) in our study,
whereas the lysine-to-argininetotalratio was the same (Table 2). In
analogy to the discrepant arginine supplementation via AAS in
group 1 (Milupa Metabolics) and 2 (Nutricia), the total arginine
intake[group1(mean±SD):111±14,group2(mean±SD):145±20;
F(1,22)=15.40, pb0.001] and the lysine-to-argininetotalratio [group 1
(mean±SD): 0.87±0.1, group 2 (mean±SD): 0.65±0.13; F(1,22)=
15.37, pb0.001] differed in both groups, whereas all other parameters
were in the same range.
To estimate the total arginine intake and lysine-to-argininetotal
ratio in older infants and children who received natural foods with
varying arginine content (Suppl. Table 1), we used standard dietary
treatment plans for infants (8 months, Suppl. Table 2) and children
(3 years, Suppl. Table 3). For group 2 (Nutricia), the lysine-to-
argininetotal ratio was similarly low at ages 8 months (lysine-to-
argininetotal ratio: 0.65) and 3 years (lysine-to-argininetotal: 0.64)
compared to breastfed infants. For group 1 (Milupa Metabolics), the
lysine-to-argininetotalratio was lower at 8 months (0.72) and 3 years
(0.70) compared to breastfed children of this group.
3.4. Lysine-to-arginine ratio in plasma
Since arginine supplementation and the dietary lysine-to-arginine
ratio differed in groups 1 and 2 during the first year of life, we next
investigated whether plasma concentrations of these amino acids
showed an analogous discrepancy. Plasma amino acid concentrations
were determined in samples that were taken 3.5–4 h after the last
meal to minimize postprandial plasma fluctuations. Mean plasma
lysine-to-arginine ratios ranged from 1.6 to 2.2 for group 1 and from
1.7 to 2.1 for group 2. Statistical analysis did not reveal differences
between groups 1 and 2 [F(1,23)=0.13, p=0.724] but a significant
age effect [F(2,42)=4.93, p=0.012] due to a significant difference
between ages 1 and 2 years [t(41)=3.12, p=0.003] (Fig. 3).
Dietary arginine intake and plasma lysine-to-arginine ratio did not
correlate (r2=−0.056, p=0.473), nor did dietary lysine intake and
plasma lysine-to-arginine ratio (r2=−0.115, p=0.140) or dietary
and plasma lysine-to-arginine ratios (r2=−0.018, p=0.814). In
Fig. 1. Age-dependent arginine supplementation in patients receiving AAS with
varying arginine contents. Group 1 (n=8 patients) received products of Milupa GA
series including Milupa GA 1 (48 mg arginine/g protein) and GA 2 prima (52 mg
arginine/g protein). Patients of group 2 (n=26 patients) received products of SHS LT
series which include LT-AM infant (90 mg arginine/g protein), LT-AM 1 (49 mg
arginine/g protein) and LT-AM 2 (59 mg arginine/g protein). From 0 to 12 months,
arginine supplementation of group 2 was significantly higher than that of group 2
(pb0.001) but did not differ in older patients.
Fig. 2. Age-dependent dietary lysine-to-arginineAASratio in patients receiving AAS with
varying arginine contents. From 0 to 12 months, lysine-to-arginineAASratio of group 2
(n=26 patients, SHS LT product line) was lower than that of group 2 (n=8 patients,
Milupa GA product line; pb0.001) but did not differ in older patients.
Table 2
Comparison of dietary and anthropometrical parameters in this and a previous study
[45] both using lysine-free, tryptophan-reduced and arginine-fortified AAS.
This study
(n=34)
LYSx group
[45]
(n=12)
ANOVA
p valuea
ParameterMeanSDMeanSD
Age (months)
Body weight (kg)
Body weight (SDS)
Height (cm)
Height (SDS)
BMI (kg/m2)
BMI (SDS)
Head circumference (cm)
Head circumference (SDS)
Head circumference index (cm/m2)
Energy (kcal/kg per day)
Total protein (g/kg per day)
Natural protein (g/kg per day)
Protein in AAS (g/kg per day)
Lysine intake (mg/kg per day)
Total arginine intake (g/kg per day)
Lysine/total arginine
L-Carnitine (mg/kg per day)
4.79
6.95
0.21
64.5
0.14
16.7
0.38
43.2
0.97
123
100
2.2
1.2
0.9
93
137
0.7
99
0.55
0.8
0.68
2.9
0.90
1.3
0.81
1.6
1.4
7
11
0.2
0.2
0.2
8
23
0.2
14
4.7
6.7
n. d.
64
n. d.
16.1
n. d.
n. d.
n. d.
127
91
2.1
1.1
1b
77
119
0.7
85
3
1.9
n. d.
7
n. d.
1.6
n. d.
n. d.
n. d.
17
14
0.3
0.2
n. d.
15
25
0.2
20
0.863
0.541
n/a
0.753
n/a
0.187
n/a
n/a
n/a
0.266
0.035
0.593
0.083
n/a
0.0001*
0.037
0.999
0.016
n/a, not applicable, n. d., not determined.
aSidak adjusted α level for multiple testing (for α=0.05) is 0.004. Accordingly, a p
valueb0.004 was considered as significant (*).
bCalculated value according to Strauss et al. [45].
4
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Please cite this article as: S. Kölker, et al., Complementary dietary treatment using lysine-free, arginine-fortified amino acid supplements in
glutaric aciduria type I — A decade of experience, Mol. Genet. Metab. (2012), doi:10.1016/j.ymgme.2012.03.021

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conclusion, plasma concentrations and ratios of these amino acids did
not reveal a discrepancy between groups 1 and 2 and did not correlate
with dietary parameters.
3.5. Neurological outcome
Since it has been shown that increased dietary arginine intake
reduces the cerebral lysine oxidation and thus the cerebral formation
of putatively neurotoxic metabolites in Gcdh−/−mice [43] and since
dietary arginine fortification has been associated with an improved
outcome in a previous study [45], we evaluated the effect of
discrepant dietary arginine intake in group 1 (Milupa Metabolics)
and group 2 (Nutricia) on neurological outcome parameters. None of
the 34 children died during the course of the study. Three children
developed insidious-onset striatal injury and dystonia which was first
reported at ages 19, 23 and 48 months. Dystonia was rated as mild to
moderate according the Barry Albright dystonia scale (6, 13 and 17
points). The frequency of dystonia, however, did not differ (Fisher's
exact test, p=1) between group 1 (1 of 8 patients, i.e. 12.5%) and
group 2 (2 of 26 patients, i.e. 8%). The age at achievement of gross
motor milestones (i.e. head control, sitting, crawling, standing and
walking) was also not different between both groups [F(1,30)=0.63,
p=0.433] (Fig. 4). In conclusion, study patients of groups 1 and 2 did
not differ in outcome parameters despite significantly different
arginine intake and dietary lysine-to-arginine ratios during the first
year of life (Figs. 1 and 2, Table 1).
3.6. Effect of metabolic treatment on the neurological outcome
Two recently published prospective follow-up studies in neona-
tally diagnosed GA-I patients in Germany [40] and in the Old Order
Amish community of Lancaster county, Pennsylvania [45], indepen-
dently demonstrated that a combination of low lysine diet using
lysine-free, tryptophan-reduced and arginine-fortified AAS, carnitine
supplementation and emergency treatment during intercurrent
illness was associated with the best outcome (i.e. lack of brain injury)
in the majority of patients (92% [Germany, n=38 patients] vs. 100%
[Amish, n=12 patients]) (Table 3). This treatment has been
recommended previously by a guideline [38,39] fulfilling high
standard evidence criteria [50] and was confirmed by both studies
showing excellent accordance in dietary parameters [40,45]. De-
viations from this treatment resulted in a less favorable outcome.
Statistical analysis of the German and Amish cohorts of GA-I patients
did not show a significant difference [χ2(3)=3.23, p=0.357] in
these three treatment groups supporting the notion that both studies
have come to the same conclusion regarding therapeutic strategies
(Table 3).
4. Discussion
The major aim of this study was to investigate the impact of
arginine supplementation using commercially available lysine-free,
tryptophan-reduced and arginine-fortified AAS [Milupa GA product
line (group 1) and SHS LT-AM product line (group 2)] on the outcome
of infants identified to have GA-I by newborn screening in Germany
between 2000 and 2011.
We observed that despite significant differences in the daily total
arginine intake (mean, 111 vs. 145 mg per kg body weight; pb0.001)
and the dietary lysine-to-argininetotalratio (mean, 0.87 vs. 0.65 mg per
kg body weight, pb0.001), there were no measurable differences in
outcome parameters (mortality, dystonia, gross motor milestones)
between the two groups. Furthermore, despite the dietary differences,
theplasmalysine-to-arginineratiosweresimilarinbothgroups,andthe
arginine intake did not correlate with plasma concentrations. Mean
dietary and anthropometrical parameters in the first year of life were
also similar between the two groups, and almost identical to a
previously published study using another lysine-free, arginine-fortified
AAS [45] — except for a slightly higher daily lysine intake in our cohort
(mean, 93 vs. 77 mg lysine per kg body weight, p=0.0001). Since
treatment parameters in this and the previous study by Strauss and
coworkers [45] were very similar, we compared both studies
regarding the neuroprotective effect of emergency and metabolic
maintenance treatment (low lysine diet, carnitine supplementa-
tion). In both studies, a combination of low lysine diet (using lysine-
free, tryptophan-reduced, arginine-fortified AAS), carnitine supple-
mentation and emergency treatment prevented brain injury in
92–100% of patients, confirming the efficacy of published evidence-
based guideline recommendations [38,39].
Fig. 3. Age-dependent plasma lysine-to-arginine ratio in patients receiving AAS with
varying arginine contents. Plasma concentrations were evaluated in blood samples
taken 3.5–4 h after the last meal. Plasma values were matched to corresponding dietary
parameters. Statistical analysis did not reveal a difference between group 1 (n=8,
Milupa GA product line) and group 2 (n=26, SHS LT product line) but showed an age
effect between age groups 0–12 months and 13–24 months (p=0.003).
Fig. 4. Achievement of gross motor milestones in patients receiving AAS with varying
arginine contents. Group 1 (n=8, Milupa GA product line) and group 2 (n=26, SHS LT
product line) did not show any differences in the age at achievement of five gross
motor milestones (p=0.433).
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Please cite this article as: S. Kölker, et al., Complementary dietary treatment using lysine-free, arginine-fortified amino acid supplements in
glutaric aciduria type I — A decade of experience, Mol. Genet. Metab. (2012), doi:10.1016/j.ymgme.2012.03.021

Page 6

4.1. Modulating dietary arginine intake and arginine homeostasis
L-Arginine is a conditionally non-essential amino acid which is
synthesized from L-citrulline by the cytosolic enzymes argininosucci-
nate synthetase and argininosuccinate lyase. These enzymes are
localized in the liver as part of the urea cycle but are also found in
kidneys (proximal tubule cells) and the brain (mostly in astrocytes)
[51]. Quantitatively, arginine synthesis mostly occurs in kidney
proximal tubule cells which extract citrulline from the circulation.
Within the intestine-renal axis of arginine synthesis, citrulline is
produced from glutamine by intestinal epithelial cells using L-Δ1-
pyrroline-5-carboxylate synthetase or as a coproduct of NO synthesis
[51]. In newborns and preterm infants, arginine is considered an
essential amino acid due to the low intestinal expression of enzymes
of the arginine-synthetic pathway. However, the expression of these
enzymes peaks shortly after birth. Arginine is also considered as
essential during severe infectious diseases [52].
Homeostasis of arginine is achieved primarily via its catabolism,
with turnover of body proteins accounting for up to 85% of arginine in
the circulation [53]. Beside the endogenous synthesis and recycling of
arginine, dietary protein is also an important arginine source. It is
thought that about 40% of dietary arginine is metabolized in the
intestine before reaching the circulation [54]. The arginine content of
natural protein varies considerably (Suppl. Table 1) [6] with human
milk (and cow milk) having a quite high lysine-to-arginine ratio of 1.7
(resp.2.15).Asaconsequence,thearginineintakeprimarilydependson
theselectionofnaturalfoodsinchildrenreceivinganormaldiet.InGA-I
patients who receive also lysine-free, tryptophan-reduced, arginine-
fortified AAS, the arginine content of commercially available AAS
products (i.e. 48–90 mg arginine/g protein in this study) and the
amount of AAS protein (i.e. 0.9±0.2 g per kg body weight and day in
infants) within a balanced low lysine diet are important determinants
of the daily arginine intake.
In breastfed infants with GA-I, the partial substitution of human
milk, whichhas a high lysine-to-arginineratio (1.7), withcommercially
available AAS significantly reduces the lysine-to-arginine ratio (0.7
±0.2). The introduction of complementary food with a low lysine
intake and stepwise reduction of human milk in the diet after age
5 months is favorable in terms of lowering the dietary lysine-to-
arginine ratio. Many food items with a low lysine content that are
commonly used for the diet in GA-I patients have a naturally occurring
low lysine-to-arginine ratio (Suppl. Table 1). In principle, the selection
of food with a low lysine-to-arginine ratio could be based on this
nutritional information. Despite these theoretical considerations the
optimalintakeofarginineforhealthyindividualsandpatientswithGA-I
is not known, and dietary recommendations for minimal arginine
requirements do not exist [55]. However, arginine supplementation via
commercially available AAS was below the safe limit for daily intake in
infants which is suggested to be at least 300–700 mg per kg body
weight [56]. In asymptomatic hyperammonemic premature infants,
long-term arginine supplementation (175–350 mg arginine/kg body
weight) did not elicit adverse effects. It increased plasma arginine
concentrationstoapproximatelytwicecomparedtocontrols.Thisisstill
less than one-third of the minimal concentration postulated to result in
neurological effects in hyperargininemia [57]. In analogy, children with
urea cycle defects receiving 210–840 mg/kg body weight did not
develop adverse effects [58]. The arginine dosage for infants and
children with urea cycle defects and MELAS syndrome [59] is much
higherthanthatvialysine-free,arginine-fortifiedAASusedinourstudy.
In contrast to arginine, AAS were not fortified with ornithine.
Theoretically, therapeutical ornithine supplementation would be also
an option in GA-I [42]. However, due to competitive inhibition at
cationic amino acid transporters in proximal tubular cells, increased
plasma ornithine would result in an increased urinary excretion and
concomitantly decreased plasma concentrations of lysine and arginine.
Since the tubular reuptake of lysine and arginine would be similarly
impaired, ornithine supplementation should not relevantly affect the
plasma lysine-to-arginine ratio. Notably, there also might be negative
effects of increased ornithine concentrations, i.e. inhibition of creatine
synthesis [60].
4.2. Prediction of cerebral lysine influx and oxidation based on plasma
lysine and arginine
In our patient cohort, the daily arginine intake did not correlate
with plasma concentrations and the plasma lysine-to-arginine ratio.
Furthermore, the use of AAS with different arginine contents did not
influence the plasma concentration of arginine. However, since
circulating arginine mostly derives from endogenous sources [53], a
linear relationship between the dietary intake of arginine and the
plasma arginine concentration is quite unlikely. Arginine is involved
in protein, urea, creatine, NO, and agmatine synthesis being the
natural substrate of arginases, NO synthases, arginine:glycine amidi-
notransferase and arginine decarboxylase [61]. The net effect of these
actions determines the plasma arginine concentration. Furthermore,it
should be considered that intestinal uptake and plasma kinetics differ
betweenaminoacids deriving from AAS and whole protein [62]. Amino
acids from AAS are absorbed more rapidly and, subsequently, plasma
amino acid concentrations raise and decline more rapidly than after
ingestion of an equivalent amount of whole protein. In GA-I patients
receiving a low lysine diet, dietary lysine only derives from natural
(whole) protein, whereas total arginine intake is the sum of arginine
contents in natural protein and AAS. Therefore, it is quite likely that the
uptake and plasmakinetics of lysine and argininediffer in GA-I patients
receiving a low lysine diet with AAS. As a consequence, a single-point
analysis of plasma amino acid concentrations following standard
procedures cannot accurately reflect this complex kinetics. In addition,
the calculations used to predict brain lysine influx in GA-I patients
Table 3
Effect of metabolic treatment on the prevention of brain injury in GA-I. The analysis was based on previously published prospective studies in (1) the newborn screening cohort in
Germany (n=53, 1999–2011) [40, with modificationsa] and (2) the Old Order Amish community in Pennsylvania (n=60, before 1990–2011) [45].
Brain injury
Heringer et al. [40],
with modificationsa
Strauss et al. [45]Sum
Treatment groupYes NoYesNoYes No
No emergency treatmentb
No low lysine diet (with or without carnitine)c
Emergency treatment, low lysine diet (with lysine-free, tryptophan-reduced, arginine-fortified AAS),
and carnitined
6 (100%)
4 (44%)
3 (8%)
0 (0%)
5 (56%)
35 (92%)
16 (94%)
12 (39%)
0 (0%)
1 (6%)
19 (61%)
12 (100%)
22 (96%)
16 (40%)
3 (6%)
1 (4%)
24 (60%)
47 (94%)
Statistical analysis did not reveal a significant difference between the studies: χ2(3)=3.23, p=0.357.
aAfter the publication of the study, one further patient developed mild dystonia at age 44 months and one additional patient was included into the ongoing study.
bReferring to the groups “non adherence to emergency treatment” [40] and “Pre-1990” [45].
cReferring to the group “non adherence to basic treatment” [40] and merged groups “1990–1994” and “1995–2004” [45].
dReferring to the groups “full treatment” [40] and “2006–2011” [45].
6
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Please cite this article as: S. Kölker, et al., Complementary dietary treatment using lysine-free, arginine-fortified amino acid supplements in
glutaric aciduria type I — A decade of experience, Mol. Genet. Metab. (2012), doi:10.1016/j.ymgme.2012.03.021

Page 7

basedonplasmalevels[45]cannotbeapplicablesincelysinecatabolism
is blocked in GA-I, and this will change the kinetics.
These complex mechanisms leading to arginine plasma fluctua-
tions could explain the range of plasma lysine-to-arginine ratios
(Fig. 3). The wide range of arginine supplementation and dietary
lysine-to-arginine ratios (Figs. 1 and 2) could be explained by the
variation of recommendations for AAS intake (0.8–1.3 g/kg per day,
Suppl. Table 4) during age 0–6 months [38,39]. This variation
becomes smaller with growing age.
Accordingly, it is unlikely that the rate of lysine influx to the brain
compartment across the BBB via CAT1 (and ORNT1) using plasma
lysine and arginine concentrations can be predicted accurately [45]. It
is known that the lysine oxidation rate is influenced by various
dietary parameters [63], and the uptake and metabolism of arginine
via CAT1 in endothelial cells is greatly varied by circulating cytokines
and hormones among others [64]. In addition, although such
mathematical simulations are of theoretical interest [45], the
accuracy of their predictions cannot be tested in patients with GA-I
with available analytical methods.
4.3. Putatively beneficial effects of arginine supplementation
Brain arginine is mostly derived from blood or cerebral protein
breakdown. Furthermore, arginine can be recycled from citrulline
using argininosuccinate synthetase and lyase. In contrast, de novo
arginine synthesis from ammonia and ornithine does not occur in the
brain due to the lack of carbamylphosphate synthetase 1 and
ornithine transcarbamylase [65].
It has been demonstrated in Gcdh−/−mice that oral administra-
tion of arginine, homoarginine and ornithine, which all are substrates
of CAT1, reduces the accumulation of putatively neurotoxic metab-
olites in the brain and limits the manifestation of brain injury and
death in these animals [41–43]. This may be well explained by
competition of lysine and arginine at biological barriers such as the
BBB and the inner mitochondrial membrane [43]. It has been
hypothesized that dietary treatment using arginine supplementation
might be neuroprotective in GA-I patients [44,61]. In line with this, a
recent short-term outcome study in 12 children has provided
evidence that arginine supplementation helps to prevent brain injury
in concert with low lysine diet, carnitine supplementation and
emergency treatment [45]. However, arginine supplementation is
not a ‘new’ principle in the treatment of GA-I. It has been performed
for more than a decade in metabolic centers using commercially
available lysine-free, tryptophan-reduced, arginine-fortified AAS
[18,35,40]. This may explain the more favorable outcome of patients
being treated with a low lysine diet and AAS [38,39] in comparison to
patients receiving a low protein diet [7,13,40,45], although it has still
to be elucidated whether the effect is due to increased arginine or
decreased lysine intake.
Whether reduced cerebral lysine influx is the major neuroprotec-
tive effect of arginine supplementation remains to be elucidated.
Alternative mechanisms of arginine supplementation should also be
considered including the enhanced production of creatine, NO, and
agmatine as well as the stimulation of hormone secretion such as
growth hormone [66–68]. Activation of cerebral creatine metabolism
and thus improving the cerebral ‘energy buffer’ might be of
therapeutic interest since impairment of brain energy metabolism
by accumulating glutaryl-CoA, GA and 3-OH-GA is considered a key
pathomechanism of brain injury in GA-I patients [20,23,24]. Further-
more, exposure of dissociated rat cortex cells with 3-OH-GA was
associated with reduced creatine phosphate levels which were
restored following supplementation with creatine [69,70]. Therefore,
it remains to be elucidated whether therapeutic activation of cerebral
creatine synthesis is neuroprotective for GA-I patients.
Pathologic changes of cerebral hemodynamics have been demon-
strated in GA-I patients using complex neuroradiological techniques
[25]. Perfusionpatternsrevealedchangesthatmightoptimizesubstrate
extraction and energy metabolism at rest but do not allow to
compensate ATP depletion below a critical threshold during catabolism
such as in infectious diseases. These changes may synergize with
neurotoxic effects of accumulating dicarboxylic metabolites resultingin
irreversible injury of vulnerable brain regions such as the striatum [25].
Since cerebral vascular autoregulation relies on close interaction
between local activity of energy-demanding excitatory glutamatergic
neurotransmission, intracellular calcium influx and, in response to this,
calcium-stimulated NO metabolism and NO-mediated dilatation of
blood vessels, it remains to be elucidated whether supplementation of
arginine, the precursor of NO, might improve disturbed cerebral
hemodynamics in GA-I patients. This is of particular interest during
severe infectious diseases which are associated with low plasma
arginine concentrations [71] and precipitate acute striatal injury in
infants with GA-I [7,13,34].
Apart from a specific neuroprotective role of arginine supplemen-
tation, it should also be considered that the use of fortified AAS within
a balanced low lysine diet may have several other positive effects.
This includes adequate supply with essential amino acids, energy
substrates, minerals and micronutrients [38,39]. Adequate supply with
essential (micro-) nutrients is indispensable to prevent malnutrition
and to support normal growth. In contrast, protein restriction alone
may not be suitable to provide sufficient nutrient supply [72].
In conclusion, the present study confirms that the use of commer-
cially available lysine-free, tryptophan-reduced and arginine-fortified
AAS within a balanced low lysine diet according to evidence-based
guideline recommendations results in increased arginine intake and
decreaseddietarylysine-to-arginineratios inGA-Ipatients.Thismaybe
the basis for the development of optimized, i.e. complementary, dietary
treatmentstrategies. Morestudies are required to elucidate the kinetics
of intestinal uptake and plasma concentrations of arginine and to
understand the dietary modulation of cerebral lysine influx and
oxidation as well as the mechanisms underlying the neuroprotective
effect of applied fortified AAS.
Conflict of interest
There are no conflicts to disclose. The applied AAS were prescribed
independently by each metabolic center. The costs for commercially
available AAS were covered by German health insurance companies
for all patients.
Acknowledgments
This work was funded by the Kindness-for-Kids Foundation to
Stefan Kölker and by the European Commission via DG Sanco (for the
action “European registry and network for intoxication type meta-
bolic diseases”). We thank all patients and their families for their
valuable contribution to this study and their trust. We thank the
German patient organization “Glutarazidurie e.V.” for excellent
collaboration.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.ymgme.2012.03.021.
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