Complementary Dietary Treatment Using Lysine-Free, Arginine-Fortified

Molecular Genetics and Metabolism 107 (2012) 72–80

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Molecular Genetics and Metabolism

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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 Kölker a,⁎, S.P. Nikolas Boy a, Jana Heringer a, Edith Müller a, Esther M. Maier b, Regina Ensenauer b, Chris Mühlhausen c, Andrea Schlune d, Cheryl R. Greenberg e, David M. Koeller f,g, Georg F. Hoffmann a, Gisela Haege a,1, Peter Burgard a,1 a Department of General Pediatrics, Division of Inherited Metabolic Diseases, University Hospital Heidelberg, Heidelberg, Germany b Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, Munich, Germany c Department of Pediatrics, University Medical Centre, Hamburg, Germany d Department of General Pediatrics and Neonatology, University Children's Hospital, Heinrich-Heine University, Düsseldorf, Germany e Department of Biochemical and Medical Genetics, Winnipeg Children's Hospital, University of Manitoba, Winnipeg, MB, Canada R3A 1R9 f Department of Pediatrics, Doernbecher Children's Hospital, Oregon Health and Science University, Portland, OR 97239, USA g Department of Molecular and Medical Genetics, Doernbecher Children's Hospital, Oregon Health and Science University, Portland, OR 97239, USA article info abstract

Article history: The cerebral formation and entrapment of neurotoxic dicarboxylic metabolites (glutaryl-CoA, glutaric and 3- Received 28 March 2012 hydroxyglutaric acid) are considered to be important pathomechanisms of striatal injury in glutaric aciduria Accepted 28 March 2012 type I (GA-I). The quantitatively most important precursor of these metabolites is lysine. Recommended Available online 4 April 2012 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 Keywords: −/− fl Glutaric aciduria type I (carnitine supplementation). It has been recently shown in Gcdh mice that cerebral lysine in ux and – Arginine oxidation can be modulated by arginine which competes with lysine for transport at the blood brain barrier Lysine and the inner mitochondrial membrane [Sauer et al., Brain 134 (2011) 157–170]. Furthermore, short-term Diet outcome of 12 children receiving arginine-fortified diet showed very promising results [Strauss et al., Mol. Guideline Genet. Metab. 104 (2011) 93–106]. Since lysine-free, arginine-fortified amino acid supplements (AAS) are Dystonia 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.

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: [email protected] (S. Kölker). 1 Both authors contributed equally to this study.

1. Introduction 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 Glutaric aciduria type I (GA-I) is a rare cerebral organic acid disorder fortified with arginine supports this notion [45]. The major aim of this first described in 1975 [1]. The disease is caused by inherited deficiency study is to specifically evaluate the effect of variation of dietary arginine of the homotetrameric mitochondrial flavoprotein glutaryl-CoA dehy- intake on neurological outcome and concentrations of plasma amino drogenase (GCDH; EC 1.3.99.7), which is encoded by the GCDH gene acids in a prospectively followed newborn screening cohort. mapping to human chromosome locus 19p13.2 [2]. More than 200 disease-causing mutations have been identified so far [3,4]. GCDH 2. Patients and methods catalyzes the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA in the final degradative pathways of L-lysine, L-hydroxylysine, and L- 2.1. Study population tryptophan [5].Deficiency of this enzyme results in accumulation of glutaryl-CoA, glutaric acid (GA), 3-hydroxyglutaric acid (3-OH-GA), and Thirty-eight children with confirmed diagnosis of GA-I who were glutarylcarnitine. Natural protein consists of 2–9% lysine and 0.6–2% identified by newborn screening between 2000 and 2011 and received tryptophan and thus L-lysine is considered the quantitatively most metabolic treatment according to a published guideline [38,39] were important precursor for GA, 3-OH-GA, and glutarylcarnitine [6].The evaluated. Their neurological outcome has been published previously concentrations of these compounds determined in body fluids of [40]. Four children of this cohort who had not received either the amino patients and body fluids and tissues of Gcdh-deficient mice, and fruit- acid mixture manufactured by Milupa Metabolics or Nutricia AAS were eating bats showed a positive correlation to protein and lysine intake excluded from the analysis. Dietary, biochemical, anthropometrical, and [1,7–11]. The initial clinical presentation of affected neonates is non- neurological parameters were evaluated in the remaining 34 study specific. The majority of untreated patients develop striatal injury patients. The study was approved by the local ethics committees of all acutely during catabolic conditions or insidiously between 3 and study centers. Written informed consent was given by all parents of 36 months of age [7,12–14]. Striatal injury results in a complex study patients. movement disorder with predominant dystonia [15,16]. Antidystonic treatment is often unsatisfactory, and life expectancy is limited in 2.2. Calculation of dietary arginine intake and lysine-to-arginine ratio severely disabled children [17,18]. The major aim of all therapeutic strategies is to prevent brain injury. The arginine content of commercially available lysine-free, Several studies highlight the role of GA, 3-OH-GA, and glutaryl-CoA tryptophan-reduced, arginine-fortified AAS given to study patients in the pathogenesis of this disease. Precipitation of excitotoxicity and was determined according to the manufacturer information (Table 1). oxidative stress, and impairment of cerebral energy metabolism via SHS LT-AM infant had the same high arginine content (i.e. 90 mg inhibition of the tricarboxylic acid cycle and the dicarboxylic acid arginine/g protein) as GlutarAde Junior GA-1 drink mix, an AAS shuttle between astrocytes and neurons are considered mechanisms previously investigated [45]. All other AAS applied to study patients [19–24]. In addition, disturbance of cerebral hemodynamics is thought had a lower arginine content (i.e. 48–59 mg arginine/g protein) which is to synergize with these mechanisms [25]. We have recently identified similar to that of human milk (i.e. 51 mg arginine/g protein) [6]. AAS did the blood–brain barrier (BBB) to play a central role by trapping not contain ornithine. The amount of AAS consumed was prospectively intracerebrally produced neurotoxic dicarboxylates due to low BBB documented for all study patients from which the daily arginineAAS permeability [21,26–28]. Cerebral GA and 3-OH-GA concentrations of supplementation and the nutritional lysine-to-arginineAAS ratio were untreated patients are 100–1000fold higher than those in plasma. calculated. The effect of dietary treatment on biochemical and anthro- Strikingly, patients with complete loss of GCDH activity (i.e. high pometrical parameters was analyzed between ages 0 and 36 months, i.e. excreters) and those with residual enzyme activity (i.e. low excreters) during the most vulnerable period for the development of striatal injury. reveal similar brain concentrations of GA and 3-OH-GA and share the For breastfed infants (i.e. as long as human milk or adapted infant same risk for brain injury [13,29–32]. Gcdh−/− mice, an animal model formula was the only natural protein source), we also calculated the with complete loss of Gcdh activity [33], demonstrate the same steep total daily arginine intake along with other dietary and anthropo- plasma-to-brain gradient of neurotoxic dicarboxylates [21].Basedon metric parameters in analogy to a previous study [45].When these findings it has been suggested that lowering the cerebral lysine supplementary food is added, the amount of total arginine intake is influx and lysine oxidation is a potential neuroprotective strategy. dependent on the varying arginine content of natural foods (Suppl. In neonatally diagnosed patients, the frequency of brain injury Table 1). To estimate the total arginine intake and the nutritional was reduced to 0–36% compared to 90–95% in historical cohorts lysine-to-argininetotal ratio in infants and children receiving more [7,12,18,34–37]. Evidence-based treatment recommendations in- than one natural protein source, we used standard dietary protocols clude (1) a low L-lysine diet (including the use of lysine-free, at ages 8 months and 3 years for our calculations (Suppl. Tables 2 and tryptophan-reduced amino acid supplements [AAS]), (2) L-carnitine 3). In addition, dietary intake was regularly adjusted every 3–6months supplementation, and (3) emergency treatment during intercurrent by specialized metabolic dietitians. Average lysine and arginine contents illness [38,39]. Recently, we demonstrated that treatment according of natural foods were taken from “Bundeslebensmittelschlüssel” [6],a to the published guideline was associated with a favorable outcome nutritional database provided by the German Ministry of Nutrition, (5% dystonia, n=37 patients) in a newborn screening cohort [40].In Agriculture and Consumer Protection containing average contents of contrast, deviations from emergency treatment (100% dystonia, n=6 approximately 15,000 food items. patients) and metabolic maintenance treatment (i.e. low lysine diet and carnitine supplementation; 44% dystonia, n=9 patients) were 2.3. Calculation of standard deviation scores for anthropometrical associated with a poor outcome. parameters In Gcdh−/− mice, the application of arginine, ornithine, and homoarginine all lowered the cerebral concentrations of GA and 3-OH- Standard deviation scores (SDS) of anthropometrical data (height, GA [41–43]. In analogy, the effect of low lysine diet was amplified by weight, head circumference) were calculated using the LMS method add-on therapy with arginine. This can be explained by competition of [46]. This method provides a way of obtaining normalized growth lysine and arginine at the BBB (cationic amino acid transporter 1, CAT1, centile standards values. Skewed distributions of the measurements which is one of three CATs that are called system y+) and the inner are approximated to normal distributions by power transformation. mitochondrial membrane (mitochondrial ornithine carriers, ORNT1 and The distribution of each covariate is summarized by three parameters, ORNT2) [43]. We have hypothesized that arginine supplementation the Box–Cox power λ, the mean μ, and the coefficient of variation σ. 74 S. Kölker et al. / Molecular Genetics and Metabolism 107 (2012) 72–80

Table 1 Amino acid content (in mg per g protein) in lysine-free amino acid supplements fortiﬁed with amino acids, minerals,a trace elementsa and vitamins.a.

Company Applied Nutrition Corporation Nutricia GmbH Nutricia GmbH Nutricia GmbH Milupa Metabolics GmbH Milupa Metabolics GmbH

Product name GlutarAde Junior GA-1 drink mixb LT-AM infantc LT-AM 1c LT-AM 2c GA 1c GA 2 primac

Lysine 0 0 0 0 0 0 Tryptophan 5 7 7 7 6 6 Arginine 90 90 49 59 48 52 Cystine n. d. 34 31 25 30 34 Histidine 30 52 31 25 30 34 Isoleucine 70 81 71 60 70 86 Leucine 115 138 124 99 118 142 Methionine 34 22 28 23 30 34 Phenylalanine 53 61 59 45 50 60 Threonine 53 68 60 59 56 68 Tyrosine 60 61 60 52 60 74 Valine 80 89 80 77 84 102 Alanine n. d. 52 51 63 50 58 Aspartate n. d. 75 121 155 118 144 Glutamate+glutamine 150 102 213 225 252 304 Glycine n. d. 81 31 56 30 34 Proline n. d. 98 117 104 112 134 Serine n. d. 61 60 65 62 76 n. d., not documented. a Contents of minerals, trace elements, and vitamins are not listed here. b According to Strauss et al. [45]. c According to manufacturer information.

The calculation of SDS for anthropometrical parameters was based on “non adherence to emergency treatment” distribution in the data by previous studies from Germany and Switzerland [47,48]. study 2 [40] was shifted from 6 vs. 0 to 5 vs. 1.

2.4. Assessment of neurological outcome 3. Results

Neurological outcome was assessed by the frequency, age at onset 3.1. Study population and severity of dystonia. Severity of dystonia was rated according to the Barry-Albright dystonia scale, a 5-point ordinal scale for 8 body We studied 34 children (21 girls, 13 boys) with confirmed diagnosis regions (minimum: 0 point, maximum: 32 points) [49]. Furthermore, of GA-I who were identified by newborn screening in Germany we determined the age at achievement of five gross motor milestones between 2000 and 2011. The median age was 7.43 years (range, including head control, sitting without support, hands-and-knees 0.7–10.9 years). At time point of analysis, 29 children (85%) were older crawling, standing alone and walking alone. than 3 years of age, whereas two patients were younger than two years (8 and 9 months), and three patients were between two and three years 2.5. Statistical analysis of age (2.48, 2.82 and 2.95 years). The cumulative follow-up period was 221.6 patient years. All patients received metabolic treatment accord- SAS 9.2 (www.sas.com) for MS Windows and SPSS 18.0.1 were ing to guideline recommendations [38,39]. Recommendations for fl used for the analyses. P valuesb0.05 were considered statistically metabolic maintenance treatment are brie y summarized in Suppl. significant. For multiple testing, Sidak adjusted α levels were used. Table 4. Eight children received products of Milupa Metabolics GmbH For all parameters, descriptive statistical analysis was performed (i.e., Milupa GA1 and GA2 prima) and 26 children received those of including mean and standard deviation or median and range. For the Nutricia GmbH (i.e., SHS LT-AM infant, LT-AM1 and LT-AM2). comparison of biochemical, anthropometrical and neurological outcome parameters in patients receiving AAS from Milupa Meta- 3.2. Arginine supplementation via commercially available lysine-free, bolics GmbH (group 1) or Nutricia GmbH (group 2), we used SAS tryptophan-reduced AAS linear mixed models for repeated measures with the denominator degrees of freedom approximated by the Kenward–Roger procedure, The arginine content of LT-AM infant is 1.5–1.9 fold higher a one-factorial repeated measures analysis of variance (ANOVA), and compared to other AAS used in the study cohort (Table 1). Since LT- Fisher's exact test. Pearson correlation was used to measure the AM infant is recommended for the first year of life, we expected that association between the lysine and arginineAAS contents of the diet arginine supplementation would differ during this time period in with corresponding plasma lysine and arginine concentrations. group 1 (Milupa Metabolics) and 2 (Nutricia). As expected, there was Therapeutic and anthropometrical parameters obtained in breastfed a significant interaction between AAS group and age [F(2,48)=62.18, infants of the study cohort were compared to a previous study [45] pb0.001] regarding arginine supplementation. Planned contrasts using ANOVA. showed that arginine supplementation was higher in group 2 than Therapy-related effects on the prevention of brain injury in two in group 1 during the first year of life [t(53)=9.67, pb0.001] but not recently published studies [40,45] were compared by a Chi-square during the second [t(55) = −0.83, p=0.413] and third year of life procedure. Expected frequencies for the data of study 1 [45] were [t(55)=0.08, p=0.935] (Fig. 1). In analogy, there was a significant calculated by applying the row percentage distributions of study 2 interaction between AAS group and age [F(2,60)=32.84, pb0.001]

[40] to the observed data representing the treatment periods regarding the dietary lysine-to-arginineAAS ratio. Planned contrasts reported in study 1. As the distributions of the sample sizes of the showed that dietary lysine-to-arginineAAS ratio was lower in group 2 three treatment types were different between the two publications, than in group 1 during the ﬁrst year of life [t(74)=−8.41, pb0.001] expected frequencies were calculated row-wise, thereby increasing but did not differ in the second [t(75)=0.31, p=0.760] and third year the degrees of freedom by one. In order to avoid division by zero the [t(74)=−1.06, p=0.293] (Fig. 2). S. Kölker et al. / Molecular Genetics and Metabolism 107 (2012) 72–80 75

receiving another lysine-free, arginine-fortiﬁed AAS [45].Theonly 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-argininetotal ratio 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 [group 1 (mean±SD): 111±14, group 2 (mean±SD): 145±20;

F(1,22)=15.40, pb0.001] and the lysine-to-argininetotal ratio [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-argininetotal ratio was lower at 8 months (0.72) and 3 years (0.70) compared to breastfed children of this group. Fig. 1. Age-dependent arginine supplementation in patients receiving AAS with varying arginine contents. Group 1 (n=8 patients) received products of Milupa GA 3.4. Lysine-to-arginine ratio in plasma 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 Since arginine supplementation and the dietary lysine-to-arginine arginine/g protein) and LT-AM 2 (59 mg arginine/g protein). From 0 to 12 months, ratio differed in groups 1 and 2 during the first year of life, we next arginine supplementation of group 2 was significantly higher than that of group 2 investigated whether plasma concentrations of these amino acids b (p 0.001) but did not differ in older patients. showed an analogous discrepancy. Plasma amino acid concentrations were determined in samples that were taken 3.5–4 h after the last 3.3. Estimated total arginine intake meal to minimize postprandial plasma fluctuations. Mean plasma lysine-to-arginine ratios ranged from 1.6 to 2.2 for group 1 and from For breastfed infants we calculated the total arginine intake as 1.7 to 2.1 for group 2. Statistical analysis did not reveal differences well as other dietary and anthropometric parameters for the time between groups 1 and 2 [F(1,23)=0.13, p=0.724] but a significant period when human milk was the only source of natural protein. The age effect [F(2,42)=4.93, p=0.012] due to a significant difference mean (±SD) total daily arginine intake was 137 (±23) mg per kg between ages 1 and 2 years [t(41)=3.12, p=0.003] (Fig. 3). body weight, and the mean (±SD) lysine-to-argininetotal ratio was Dietary arginine intake and plasma lysine-to-arginine ratio did not 0.70 (±0.2). These results as well as age, body weight, height, body correlate (r2 =−0.056, p=0.473), nor did dietary lysine intake and mass index, head circumference index, and daily intake of energy plasma lysine-to-arginine ratio (r2 =−0.115, p=0.140) or dietary 2 substrates, total protein, natural protein, and L-carnitine showed an and plasma lysine-to-arginine ratios (r =−0.018, p=0.814). In excellent accordance with those of previously published 12 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 LYSx group ANOVA (n=34) [45] p valuea (n=12)

Parameter Mean SD Mean SD

Age (months) 4.79 0.55 4.7 3 0.863 Body weight (kg) 6.95 0.8 6.7 1.9 0.541 Body weight (SDS) 0.21 0.68 n. d. n. d. n/a Height (cm) 64.5 2.9 64 7 0.753 Height (SDS) 0.14 0.90 n. d. n. d. n/a BMI (kg/m2) 16.7 1.3 16.1 1.6 0.187 BMI (SDS) 0.38 0.81 n. d. n. d. n/a Head circumference (cm) 43.2 1.6 n. d. n. d. n/a Head circumference (SDS) 0.97 1.4 n. d. n. d. n/a Head circumference index (cm/m2) 123 7 127 17 0.266 Energy (kcal/kg per day) 100 11 91 14 0.035 Total protein (g/kg per day) 2.2 0.2 2.1 0.3 0.593 Natural protein (g/kg per day) 1.2 0.2 1.1 0.2 0.083 Protein in AAS (g/kg per day) 0.9 0.2 1b n. d. n/a Lysine intake (mg/kg per day) 93 8 77 15 0.0001* Total arginine intake (g/kg per day) 137 23 119 25 0.037 Lysine/total arginine 0.7 0.2 0.7 0.2 0.999 L-Carnitine (mg/kg per day) 99 14 85 20 0.016

Fig. 2. Age-dependent dietary lysine-to-arginineAAS ratio in patients receiving AAS with n/a, not applicable, n. d., not determined. a varying arginine contents. From 0 to 12 months, lysine-to-arginineAAS ratio of group 2 Sidak adjusted α level for multiple testing (for α=0.05) is 0.004. Accordingly, a p (n=26 patients, SHS LT product line) was lower than that of group 2 (n=8 patients, valueb0.004 was considered as signiﬁcant (*). Milupa GA product line; pb0.001) but did not differ in older patients. b Calculated value according to Strauss et al. [45]. 76 S. Kölker et al. / Molecular Genetics and Metabolism 107 (2012) 72–80 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 Fig. 4. Achievement of gross motor milestones in patients receiving AAS with varying walking) was also not different between both groups [F(1,30)=0.63, 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 p=0.433] (Fig. 4). In conclusion, study patients of groups 1 and 2 did motor milestones (p=0.433). 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). recommended previously by a guideline [38,39] fulfilling high standard evidence criteria [50] and was confirmed by both studies 3.6. Effect of metabolic treatment on the neurological outcome showing excellent accordance in dietary parameters [40,45]. De- viations from this treatment resulted in a less favorable outcome. Two recently published prospective follow-up studies in neona- Statistical analysis of the German and Amish cohorts of GA-I patients tally diagnosed GA-I patients in Germany [40] and in the Old Order did not show a significant difference [χ2(3)=3.23, p=0.357] in Amish community of Lancaster county, Pennsylvania [45], indepen- these three treatment groups supporting the notion that both studies dently demonstrated that a combination of low lysine diet using have come to the same conclusion regarding therapeutic strategies lysine-free, tryptophan-reduced and arginine-fortified AAS, carnitine (Table 3). 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% 4. Discussion [Amish, n=12 patients]) (Table 3). This treatment has been 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-argininetotal ratio (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, the plasma lysine-to-arginine ratios were similar in both groups, and the 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- Fig. 3. Age-dependent plasma lysine-to-arginine ratio in patients receiving AAS with tion). In both studies, a combination of low lysine diet (using lysine- varying arginine contents. Plasma concentrations were evaluated in blood samples free, tryptophan-reduced, arginine-fortified AAS), carnitine supple- taken 3.5–4 h after the last meal. Plasma values were matched to corresponding dietary mentation and emergency treatment prevented brain injury in parameters. Statistical analysis did not reveal a difference between group 1 (n=8, – fi fi Milupa GA product line) and group 2 (n=26, SHS LT product line) but showed an age 92 100% of patients, con rming the ef cacy of published evidence- effect between age groups 0–12 months and 13–24 months (p=0.003). based guideline recommendations [38,39]. S. Kölker et al. / Molecular Genetics and Metabolism 107 (2012) 72–80 77

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], Strauss et al. [45] Sum with modiﬁcationsa

Treatment group Yes No Yes No Yes No

No emergency treatmentb 6 (100%) 0 (0%) 16 (94%) 1 (6%) 22 (96%) 1 (4%) No low lysine diet (with or without carnitine)c 4 (44%) 5 (56%) 12 (39%) 19 (61%) 16 (40%) 24 (60%) Emergency treatment, low lysine diet (with lysine-free, tryptophan-reduced, arginine-fortiﬁed AAS), 3 (8%) 35 (92%) 0 (0%) 12 (100%) 3 (6%) 47 (94%) and carnitine d

Statistical analysis did not reveal a signiﬁcant difference between the studies: χ2(3)=3.23, p=0.357. a After 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. b Referring to the groups “non adherence to emergency treatment” [40] and “Pre-1990” [45]. c Referring to the group “non adherence to basic treatment” [40] and merged groups “1990–1994” and “1995–2004” [45]. d Referring to the groups “full treatment” [40] and “2006–2011” [45].

4.1. Modulating dietary arginine intake and arginine homeostasis weight [56]. In asymptomatic hyperammonemic premature infants, long-term arginine supplementation (175–350 mg arginine/kg body

L-Arginine is a conditionally non-essential amino acid which is weight) did not elicit adverse effects. It increased plasma arginine synthesized from L-citrulline by the cytosolic enzymes argininosucci- concentrations to approximately twice compared to controls. This is still nate synthetase and argininosuccinate lyase. These enzymes are less than one-third of the minimal concentration postulated to result in localized in the liver as part of the urea cycle but are also found in neurological effects in hyperargininemia [57]. In analogy, children with – kidneys (proximal tubule cells) and the brain (mostly in astrocytes) urea cycle defects receiving 210 840 mg/kg body weight did not [51]. Quantitatively, arginine synthesis mostly occurs in kidney develop adverse effects [58]. The arginine dosage for infants and proximal tubule cells which extract citrulline from the circulation. children with urea cycle defects and MELAS syndrome [59] is much fi Within the intestine-renal axis of arginine synthesis, citrulline is higher than that via lysine-free, arginine-forti ed AAS used in our study. 1 fi produced from glutamine by intestinal epithelial cells using L-Δ - In contrast to arginine, AAS were not forti ed with ornithine. pyrroline-5-carboxylate synthetase or as a coproduct of NO synthesis Theoretically, therapeutical ornithine supplementation would be also [51]. In newborns and preterm infants, arginine is considered an an option in GA-I [42]. However, due to competitive inhibition at essential amino acid due to the low intestinal expression of enzymes cationic amino acid transporters in proximal tubular cells, increased of the arginine-synthetic pathway. However, the expression of these plasma ornithine would result in an increased urinary excretion and enzymes peaks shortly after birth. Arginine is also considered as concomitantly decreased plasma concentrations of lysine and arginine. essential during severe infectious diseases [52]. Since the tubular reuptake of lysine and arginine would be similarly Homeostasis of arginine is achieved primarily via its catabolism, impaired, ornithine supplementation should not relevantly affect the with turnover of body proteins accounting for up to 85% of arginine in plasma lysine-to-arginine ratio. Notably, there also might be negative the circulation [53]. Beside the endogenous synthesis and recycling of effects of increased ornithine concentrations, i.e. inhibition of creatine arginine, dietary protein is also an important arginine source. It is synthesis [60]. thought that about 40% of dietary arginine is metabolized in the intestine before reaching the circulation [54]. The arginine content of 4.2. Prediction of cerebral lysine influx and oxidation based on plasma natural protein varies considerably (Suppl. Table 1) [6] with human lysine and arginine milk (and cow milk) having a quite high lysine-to-arginine ratio of 1.7 (resp. 2.15). As a consequence, the arginine intake primarily depends on In our patient cohort, the daily arginine intake did not correlate the selection of natural foods in children receiving a normal diet. In GA-I with plasma concentrations and the plasma lysine-to-arginine ratio. patients who receive also lysine-free, tryptophan-reduced, arginine- Furthermore, the use of AAS with different arginine contents did not fortified AAS, the arginine content of commercially available AAS influence the plasma concentration of arginine. However, since products (i.e. 48–90 mg arginine/g protein in this study) and the circulating arginine mostly derives from endogenous sources [53],a amount of AAS protein (i.e. 0.9±0.2 g per kg body weight and day in linear relationship between the dietary intake of arginine and the infants) within a balanced low lysine diet are important determinants plasma arginine concentration is quite unlikely. Arginine is involved of the daily arginine intake. in protein, urea, creatine, NO, and agmatine synthesis being the In breastfed infants with GA-I, the partial substitution of human natural substrate of arginases, NO synthases, arginine:glycine amidi- milk, which has a high lysine-to-arginine ratio (1.7), with commercially notransferase and arginine decarboxylase [61]. The net effect of these available AAS significantly reduces the lysine-to-arginine ratio (0.7 actions determines the plasma arginine concentration. Furthermore, it ±0.2). The introduction of complementary food with a low lysine should be considered that intestinal uptake and plasma kinetics differ intake and stepwise reduction of human milk in the diet after age between amino acids deriving from AAS and whole protein [62].Amino 5 months is favorable in terms of lowering the dietary lysine-to- acids from AAS are absorbed more rapidly and, subsequently, plasma arginine ratio. Many food items with a low lysine content that are amino acid concentrations raise and decline more rapidly than after commonly used for the diet in GA-I patients have a naturally occurring ingestion of an equivalent amount of whole protein. In GA-I patients low lysine-to-arginine ratio (Suppl. Table 1). In principle, the selection receiving a low lysine diet, dietary lysine only derives from natural of food with a low lysine-to-arginine ratio could be based on this (whole) protein, whereas total arginine intake is the sum of arginine nutritional information. Despite these theoretical considerations the contents in natural protein and AAS. Therefore, it is quite likely that the optimal intake of arginine for healthy individuals and patients with GA-I uptake and plasma kinetics of lysine and arginine differ in GA-I patients is not known, and dietary recommendations for minimal arginine receiving a low lysine diet with AAS. As a consequence, a single-point requirements do not exist [55]. However, arginine supplementation via analysis of plasma amino acid concentrations following standard commercially available AAS was below the safe limit for daily intake in procedures cannot accurately reflect this complex kinetics. In addition, infants which is suggested to be at least 300–700 mg per kg body the calculations used to predict brain lysine influx in GA-I patients 78 S. Kölker et al. / Molecular Genetics and Metabolism 107 (2012) 72–80 based on plasma levels [45] cannot be applicable since lysine catabolism [25]. Perfusion patterns revealed changes that might optimize substrate is blocked in GA-I, and this will change the kinetics. extraction and energy metabolism at rest but do not allow to These complex mechanisms leading to arginine plasma fluctua- compensate ATP depletion below a critical threshold during catabolism tions could explain the range of plasma lysine-to-arginine ratios such as in infectious diseases. These changes may synergize with (Fig. 3). The wide range of arginine supplementation and dietary neurotoxic effects of accumulating dicarboxylic metabolites resulting in lysine-to-arginine ratios (Figs. 1 and 2) could be explained by the irreversible injury of vulnerable brain regions such as the striatum [25]. variation of recommendations for AAS intake (0.8–1.3 g/kg per day, Since cerebral vascular autoregulation relies on close interaction Suppl. Table 4) during age 0–6 months [38,39]. This variation between local activity of energy-demanding excitatory glutamatergic becomes smaller with growing age. neurotransmission, intracellular calcium influx and, in response to this, Accordingly, it is unlikely that the rate of lysine influx to the brain calcium-stimulated NO metabolism and NO-mediated dilatation of compartment across the BBB via CAT1 (and ORNT1) using plasma blood vessels, it remains to be elucidated whether supplementation of lysine and arginine concentrations can be predicted accurately [45].It arginine, the precursor of NO, might improve disturbed cerebral is known that the lysine oxidation rate is influenced by various hemodynamics in GA-I patients. This is of particular interest during dietary parameters [63], and the uptake and metabolism of arginine severe infectious diseases which are associated with low plasma via CAT1 in endothelial cells is greatly varied by circulating cytokines arginine concentrations [71] and precipitate acute striatal injury in and hormones among others [64]. In addition, although such infants with GA-I [7,13,34]. mathematical simulations are of theoretical interest [45], the Apart from a specific neuroprotective role of arginine supplemen- accuracy of their predictions cannot be tested in patients with GA-I tation, it should also be considered that the use of fortified AAS within with available analytical methods. a balanced low lysine diet may have several other positive effects. This includes adequate supply with essential amino acids, energy 4.3. Putatively beneficial effects of arginine supplementation substrates, minerals and micronutrients [38,39]. Adequate supply with essential (micro-) nutrients is indispensable to prevent malnutrition Brain arginine is mostly derived from blood or cerebral protein and to support normal growth. In contrast, protein restriction alone breakdown. Furthermore, arginine can be recycled from citrulline may not be suitable to provide sufficient nutrient supply [72]. using argininosuccinate synthetase and lyase. In contrast, de novo In conclusion, the present study confirms that the use of commer- arginine synthesis from ammonia and ornithine does not occur in the cially available lysine-free, tryptophan-reduced and arginine-fortified brain due to the lack of carbamylphosphate synthetase 1 and AAS within a balanced low lysine diet according to evidence-based ornithine transcarbamylase [65]. guideline recommendations results in increased arginine intake and It has been demonstrated in Gcdh−/− mice that oral administra- decreased dietary lysine-to-arginine ratios in GA-I patients. This may be tion of arginine, homoarginine and ornithine, which all are substrates the basis for the development of optimized, i.e. complementary, dietary of CAT1, reduces the accumulation of putatively neurotoxic metab- treatment strategies. More studies are required to elucidate the kinetics olites in the brain and limits the manifestation of brain injury and of intestinal uptake and plasma concentrations of arginine and to death in these animals [41–43]. This may be well explained by understand the dietary modulation of cerebral lysine influx and competition of lysine and arginine at biological barriers such as the oxidation as well as the mechanisms underlying the neuroprotective BBB and the inner mitochondrial membrane [43]. It has been effect of applied fortified AAS. hypothesized that dietary treatment using arginine supplementation might be neuroprotective in GA-I patients [44,61]. In line with this, a Conflict of interest recent short-term outcome study in 12 children has provided evidence that arginine supplementation helps to prevent brain injury There are no conflicts to disclose. The applied AAS were prescribed in concert with low lysine diet, carnitine supplementation and independently by each metabolic center. The costs for commercially emergency treatment [45]. However, arginine supplementation is available AAS were covered by German health insurance companies not a ‘new’ principle in the treatment of GA-I. It has been performed for all patients. for more than a decade in metabolic centers using commercially available lysine-free, tryptophan-reduced, arginine-fortified AAS Acknowledgments [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 This work was funded by the Kindness-for-Kids Foundation to patients receiving a low protein diet [7,13,40,45], although it has still Stefan Kölker and by the European Commission via DG Sanco (for the to be elucidated whether the effect is due to increased arginine or action “European registry and network for intoxication type meta- decreased lysine intake. bolic diseases”). We thank all patients and their families for their Whether reduced cerebral lysine influx is the major neuroprotec- valuable contribution to this study and their trust. We thank the tive effect of arginine supplementation remains to be elucidated. German patient organization “Glutarazidurie e.V.” for excellent Alternative mechanisms of arginine supplementation should also be collaboration. considered including the enhanced production of creatine, NO, and agmatine as well as the stimulation of hormone secretion such as Appendix A. Supplementary data growth hormone [66–68]. Activation of cerebral creatine metabolism and thus improving the cerebral ‘energy buffer’ might be of Supplementary data to this article can be found online at therapeutic interest since impairment of brain energy metabolism doi:10.1016/j.ymgme.2012.03.021. 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- References more, exposure of dissociated rat cortex cells with 3-OH-GA was fi associated with reduced creatine phosphate levels which were [1] S.I. Goodman, J.G. Kohlhoff, Glutaric aciduria: inherited de ciency of glutaryl-CoA dehydrogenase activity, Biochem. 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