Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis, whereas L-arginine (Arg) and L-homoarginine (hArg) serve as substrates for NO synthesis. ADMA and other methylated arginines are generally believed to exclusively derive from guanidine (N (G))-methylated arginine residues in proteins by protein arginine methyltransferases (PRMTs) that use S-adenosylmethionine (SAM) as the methyl donor. L-Lysine is known for decades as a precursor for hArg, but only recent studies indicate that arginine:glycine amidinotransferase (AGAT) is responsible for the synthesis of hArg. AGAT catalyzes the formation of guanidinoacetate (GAA) that is methylated to creatine by guanidinoacetate methyltransferase (GAMT) which also uses SAM. The aim of the present study was to learn more about the mechanisms of ADMA and hArg formation in humans. Especially, we hypothesized that ADMA is produced by N (G)-methylation of free Arg in addition to the known PRMTs-involving mechanism. In knockout mouse models of AGAT- and GAMT-deficiency, we investigated the contribution of these enzymes to hArg synthesis. Arg infusion (0.5 g/kg, 30 min) in children (n = 11) and ingestion of high-fat protein meals by overweight men (n = 10) were used to study acute effects on ADMA and hArg synthesis. Daily Arg ingestion (10 g) or placebo for 3 or 6 months by patients suffering from peripheral arterial occlusive disease (PAOD, n = 20) or coronary artery disease (CAD, n = 30) was used to study chronic effects of Arg on ADMA synthesis. Mass spectrometric methods were used to measure all biochemical parameters in plasma and urine samples. In mice, AGAT but not GAMT was found to contribute to plasma hArg, while ADMA synthesis was independent of AGAT and GAMT. Arg infusion acutely increased plasma Arg, hArg and ADMA concentrations, but decreased the plasma hArg/ADMA ratio. High-fat protein meals acutely increased plasma Arg, hArg, ADMA concentrations, as well as the plasma hArg/ADMA ratio. In the PAOD and CAD studies, plasma Arg concentration increased in the verum compared to the placebo groups. Plasma ADMA concentration increased only in the PAOD patients who received Arg. Our study suggests that in humans a minor fraction of free Arg is rapidly metabolized to ADMA and hArg. In mice, GAMT and N (G)-methyltransferases contribute to ADMA and hArg synthesis from Arg, whereas AGAT is involved in the synthesis of hArg but not of ADMA. The underlying biochemical mechanisms remain still elusive.

The L-arginine/nitric oxide (L-Arg/NO) pathway regulates endothelial function and may play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). Yet, this pathway is poorly investigated in children suffering from DMD. Endothelial dysfunction can affect the perfusion of contracting muscles, thus leading to ischemia and hypoxia. In the present study, we tested the hypothesis that reduced NO production due to elevated synthesis of N (G),N (G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of NO synthesis, is a possible pathophysiological mechanism for progressive intramuscular muscle ischemia and disturbed endothelial function in children with DMD. Given the possible antagonistic action of homoarginine (hArg) on ADMA, we also analyzed this amino acid. We investigated 55 male patients with DMD and 54 healthy male controls (HC; aged 11.9 ± 4.8 vs. 11.1 ± 4.9 years, mean ± SD). Urinary creatinine and metabolites of the L-Arg/NO pathway were measured in plasma and urine by GC-MS or GC-MS/MS. Urine levels of ADMA and its major urinary metabolite dimethylamine (DMA), nitrite and nitrate (P < 0.001 for all) and hArg (P = 0.002) were significantly higher in DMD patients compared to HC, while the urinary DMA/ADMA molar ratio was lower (P = 0.002). In plasma, nitrate (P < 0.001), hArg (P = 0.002) and the hArg/ADMA ratio (P < 0.001) were lower in DMD than in HC. In plasma, ADMA (631 ± 119 vs. 595 ± 129 nM, P = 0.149), arginine and nitrite did not differ between DMD and HC. In DMD, positive correlations between ADMA, DMA or nitrate excretion and the stage of disease (according to Vignos and Thompson) were found. In DMD patients on steroid medication, lower concentrations of ADMA in plasma, and of DMA, ADMA, nitrate and hArg in urine were observed compared to non-treated patients. The L-Arg/NO pathway is impaired in DMD patients, with the disease progression being clinically negatively correlated with the extent of impairment. One of the underlying mechanisms in DMD may involve insufficient antagonism of ADMA by hArg. Steroids, but not creatine supplementation, seems to improve the L-Arg/NO pathway in DMD.

Knowledge on pediatric herpes simplex virus encephalitis is limited. Here we summarize 6 neonates and 32 children diagnosed by polymerase chain reaction (n = 37) or serological studies (n = 1), respectively. Diagnosis was difficult, as only 15 patients presented neurologic symptoms. Moreover, cerebrospinal fluid glucose, protein, and leukocytes were normal in 6 patients. Subsequently, all but 2 showed neurologic symptoms. Diffusion-weighted neuroimaging was the most sensitive early imaging method. Despite acyclovir treatment, 8 patients experienced early relapses, showing movement abnormalities, impaired vigilance, and seizures. Diffuse white matter changes, found in 3 of 5 relapse patients on neuroimaging, and a negative cerebrospinal fluid herpes simplex virus polymerase chain reaction suggested inflammatory processes. All relapse patients were again treated with acyclovir, and 3 responded to additional corticosteroid treatment. Whereas outcome after relapses was poor, overall outcome was good. No child died; 14 were asymptomatic at discharge, and neuroimaging remained normal in 7 of 30 patients studied.

This study investigated the effect of hippotherapy on gross motor function (Gross Motor Function Measure [GMFM]-66, GMFM dimension E and D) and quality of life (Child Health Questionnaire [CHQ 28], KIDSCREEN-27 parental versions) in children with bilateral spastic cerebral palsy. Seventy-three children (age: 9.1 ± 3.3 years; male = 44; GMFCS levels II = 27; III = 17; IV = 29) were randomized to an early ( = 35) or late ( = 38) treatment group. Data from 66 probands were available for further analysis. Probands received hippotherapy once to twice weekly during a period of 16 to 20 weeks (mean: 17 treatments) in a crossover approach. Whereas no significant changes were found for total GMFM scores and quality of life parameters, a significant increase in GMFM dimension E was found. Children terminating the study early showed lower mean psychosocial quality of life scores than children who completed the whole study (CHQ-28 "psychosocial dimension"; KIDSCREEN-27 "mood and emotional dimension"). Our data are in line with previous reports and suggest that hippotherapy shows distinct therapeutic strengths with regard to promoting upright stand and gait in children with cerebral palsy. Children with higher psychosocial burden of disease may need special support to get access to and benefit from intensified physiotherapy programs.

High circulating levels of asymmetric dimethylarginine (ADMA) and low circulating levels of homoarginine (hArg) are known cardiovascular risk factors in adults. While in adults with type 1 diabetes mellitus (T1DM) circulating ADMA is significantly elevated, in children and adolescents the reported ADMA data are contradictory. In 102 children with T1DM and 95 healthy controls (HC) serving as controls, we investigated the L-arginine (Arg)/nitric oxide (NO) pathway. Children with T1DM were divided into two groups, i.e., in children with newly diagnosed diabetes mellitus [T1DM-ND; n = 10; age, 8.8 (4.4-11.2) years; HbA1c, 13 (8.9-13.9) %] and in those with long-term treatment [T1DM-T; n = 92; age, 12.5 (10.5-15.4) years; HbA1c, 8.0 (7.2-8.6) %]. The age of the HC was 11.3 (8-13.3) years. Amino acids and NO metabolites of the Arg/NO pathway, creatinine and the oxidative stress biomarker malondialdehyde (MDA) were measured by GC-MS or GC-MS/MS. Plasma hArg, ADMA and the hArg/ADMA molar ratio did not differ between the T1DM and HC groups. There was a significant difference between T1DM-T and HC with regard to plasma nitrite [0.53 (0.48-0.61) vs 2.05 (0.86-2.36) µM, P < 0.0001] as well as to urinary nitrite [0.09 (0.06-0.17) vs 0.22 (0.13-0.37) μmol/mmol creatinine, P < 0.0001]. Plasma, but not urinary nitrite, differed between T1DM-ND and HC [0.55 (0.50-0.66) vs 2.05 (0.86-2.36) µM, P < 0.0001]. Plasma MDA did not differ between the groups. The urinary nitrate-to-nitrite molar ratio (UNOXR), a measure of nitrite-dependent renal carbonic anhydrase (CA) activity, was higher in T1DM-T [1173 (738-1481), P < 0.0001] and T1DM-ND [1341 (1117-1615), P = 0.0007] compared to HC [540 (324-962)], but did not differ between T1DM-T and T1DM-ND (P = 0.272). The lower nitrite excretion in the children with T1DM may indicate enhanced renal CA-dependent nitrite reabsorption compared with healthy children. Yet, lower plasma nitrite concentration in the T1DM patients may have also contributed to the higher UNOXR. Patients' age correlated positively with plasma hArg and hArg/ADMA and urinary DMA/ADMA. Plasma ADMA and urinary ADMA, DMA, nitrite and nitrate correlated negatively with age of the T1DM-T children. Significant correlations were found between plasma hArg and plasma Arg (r = 0.468, P < 0.0001), and urinary DMA (r = -0.426, P = 0.0001), ADMA (r = -0.266, P = 0.021) and nitrate (r = -0.234, P = 0.043). Plasma hArg correlated positively with age at diagnosis (r = +0.337, P = 0.002). ADMA, but not hArg, correlated with HbA1c in T1DM-T (r = -0.418, P < 0.0001) and T1DM-ND (r = +0.879, P = 0.0016). The greatest differences between T1DM-T and T1DM-ND were observed for urinary ADMA, DMA/ADMA ratio, nitrite and nitrate. The Arg/NO pathway is altered in T1DM in childhood and adolescence, yet the role and the importance of hArg and ADMA in T1DM remain to be elucidated. In young T1DM patients, oxidative stress (lipid peroxidation) is not elevated.

Adult subjects with growth hormone (GH) deficiency (GHD) are known to have reduced life expectancy due to increased cardiovascular and cerebrovascular events. In adults, these events are associated with elevated circulating concentrations of asymmetric dimethylarginine (ADMA) which is an endogenous inhibitor of L-arginine (Arg)-derived nitric oxide (NO). Low circulating concentrations of homoarginine (hArg) emerged as a cardiovascular risk factor. In adults, hArg seems to antagonize ADMA. In the present work, we tested the hypothesis that children with short stature without or with GHD have altered Arg/NO pathway as compared to children with normal growth. We studied 66 short stature children (38 boys, 28 girls) aged 3.5-17.3 years, who underwent the routine L-Arginine Test to diagnose presence of GHD. GHD was confirmed in 47 children (GHD group; 30 boys, 17 girls) and was absent in the remaining 19 children (non-GHD group; 8 boys, 11 girls). In addition, we investigated 24 healthy age- and gender-matched children (10 boys, 14 girls) with normal growth. In EDTA plasma samples of all children, we determined by mass spectrometry-based methods the concentrations of Arg, hArg and ADMA, and calculated the Arg/ADMA and hArg/ADMA molar ratios. With respect to these biochemical parameters, we did not find statistically significant differences between the GHD and non-GHD groups. Comparing short with normal stature children, we found small differences regarding plasma hArg concentrations [mean ± SD; median (25th-75th percentile)]: 2.06 ± 0.52 µM; 2.12 (1.74-2.36) µM vs. 1.7 ± 0.5 µM; 1.6 (1.4-1.8) µM, P < 0.001. Compared to normal stature children, short stature children had considerably higher plasma concentrations of ADMA [0.77 ± 0.15 µM; 0.77 (0.66-0.85) µM vs. 0.57 ± 0.09 µM; 0.58 (0.50-0.63) µM, P < 0.001], but not of Arg [83.3 ± 19.2 µM; 82.2 (71.9-90.3) µM vs. 86.5 ± 17.8 µM; 84.8 (77.2-94.8) µM, P = 0.336], or the hArg/ADMA ratio [2.74 ± 0.76; 2.7 (2.2-3.1) vs. 3.1 ± 1.2; 2.85 (2.42-3.66), P = 0.161. hArg in the GHD group (r = 0.41, P = 0.004) and the hArg/ADMA ratio in both groups (r = 0.44, P = 0.002 in GHD; r = 0.55, P = 0.01 in non-GHD)], but not ADMA were positively correlated with insulin-like growth factor-1 (IGF-1). hArg and hArg/ADMA differed between girls and boys in the GHD and non-GHD groups but in the normal growth group. The hArg/ADMA ratio increased with age in all groups. Our study suggests that hArg and ADMA are involved in growth in the childhood, presumably in an antagonistic manner, with ADMA slowing and hArg accelerating growth.

Leigh syndrome (MIM 25600), also known as infantile subacute necrotizing encephalomyelopathy, is a neurodegenerative disorder with characteristic bilateral symmetric lesions in basal ganglia and subcortical brain regions. It is commonly associated with systemic cytochrome c oxidase (COX) deficiency and mutations in the SURF1 gene (MIM 185620), encoding a putative assembly or maintenance factor of COX. The clinical course is dominated by neurodevelopmental regression, brain stem, and basal ganglia involvement (e.g., dystonia, apnea) with death often occurring before the age of 10 years. Herein, we present three sisters carrying a previously reported homozygous SURF1 mutation (c.868_869insT) that is predicted to result in a truncated protein with loss of function. Our patients show heterogeneous clinical findings with different distribution patterns of metabolic lesions in brain magnetic resonance imaging (MRI) as well as a Chiari malformation with hydrocephalus in one patient. However, all three siblings show an unusual long survival (12 years and>16 years). COX activity was not detectable in one patient and strongly reduced in the other two. We discuss these findings with respect to a review of the literature. A total of 15 additional patients with survival>14 years have been reported so far. Overall, no clear genotype-phenotype correlations are detectable among these patients.

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