Correlations of blood and brain biochemistry in phenylketonuria: Results from the Pah-enu2 PKU mouse.

BACKGROUND: In phenylketonuria (PKU), treatment monitoring is based on frequent blood phenylalanine (Phe) measurements, as this is the predictor of neurocognitive and behavioural outcome by reflecting brain Phe concentrations and brain biochemical changes. Despite clinical studies describing the relevance of blood Phe to outcome in PKU patients, blood Phe does not explain the variance in neurocognitive and behavioural outcome completely. METHODS: In a PKU mouse model we investigated 1) the relationship between plasma Phe and brain biochemistry (Brain Phe and monoaminergic neurotransmitter concentrations), and 2) whether blood non-Phe Large Neutral Amino Acids (LNAA) would be of additional value to blood Phe concentrations to explain brain biochemistry. To this purpose, we assessed blood amino acid concentrations and brain Phe as well as monoaminergic neurotransmitter levels in in 114 Pah-Enu2 mice on both B6 and BTBR backgrounds using (multiple) linear regression analyses. RESULTS: Plasma Phe concentrations were strongly correlated to brain Phe concentrations, significantly negatively correlated to brain serotonin and norepinephrine concentrations and only weakly correlated to brain dopamine concentrations. From all blood markers, Phe showed the strongest correlation to brain biochemistry in PKU mice. Including non-Phe LNAA concentrations to the multiple regression model, in addition to plasma Phe, did not help explain brain biochemistry. CONCLUSION: This study showed that blood Phe is still the best amino acid predictor of brain biochemistry in PKU. Nevertheless, neurocognitive and behavioural outcome cannot fully be explained by blood or brain Phe concentrations, necessitating a search for other additional parameters. TAKE-HOME MESSAGE: Blood Phe is still the best amino acid predictor of brain biochemistry in PKU. Nevertheless, neurocognitive and behavioural outcome cannot fully be explained by blood or brain Phe concentrations, necessitating a search for other additional parameters.

Predictability and inconsistencies of cognitive outcome in patients with phenylketonuria and personalised therapy: the challenge for the future guidelines.

Phenylketonuria (PKU) is a prototypical model of a neurodevelopmental metabolic disease that follows a cascade of pathological events affecting brain maturation and functioning. Neonatal screening and early treatment have eradicated the classical PKU phenotype in patients with early and continuously treated phenylketonuria (ECTPKU). However, effort is required to optimise the treatment of the disease to minimise the risk of lifelong neurological, cognitive and behavioural impairment, and to solve issues on the variability in clinical outcome that are rather not understood and has yet hampered a more personalised approach to its treatment. The aim of the present review is to focus on the inconsistencies in the clinical outcome of adult patients with ECTPKU unexplained by the biochemical markers adopted for the monitoring of the disease to date. The interindividual variability of clinical outcome in late as well as in early treated patients under similar biochemical control suggests the existence of disease-independent determinants influencing the individual vulnerability to the neurotoxic effect of phenylalanine. This is further supported by the low predictive power of blood phenylalanine on the clinical outcome from the second decade of life onwards. In conclusion, individual vulnerability to the metabolic alterations of PKU contributes to the prognosis of PKU, also in patients with ECTPKU. The biological factors constitutive of this vulnerability are unknown (but have not been the object of many studies so far) and should be the target of further research as prerequisite for a personalised treatment aimed at avoiding burden and costs of overtreatment and clinical consequences and risks of undertreatment in patients with PKU.

Tetrahydrobiopterin treatment in phenylketonuria: A repurposing approach.

In phenylketonuria (PKU) patients, early diagnosis by neonatal screening and immediate institution of a phenylalanine-restricted diet can prevent severe intellectual impairment. Nevertheless, outcome remains suboptimal in some patients asking for additional treatment strategies. Tetrahydrobiopterin (BH4 ) could be one of those treatment options, as it may not only increase residual phenylalanine hydroxylase activity in BH4 -responsive PKU patients, but possibly also directly improves neurocognitive functioning in both BH4 -responsive and BH4 -unresponsive PKU patients. In the present review, we aim to further define the theoretical working mechanisms by which BH4 might directly influence neurocognitive functioning in PKU having passed the blood-brain barrier. Further research should investigate which of these mechanisms are actually involved, and should contribute to the development of an optimal BH4 treatment regimen to directly improve neurocognitive functioning in PKU. Such possible repurposing approach of BH4 treatment in PKU may improve neuropsychological outcome and mental health in both BH4 -responsive and BH4 -unresponsive PKU patients.

The first European guidelines on phenylketonuria: Usefulness and implications for BH4 responsiveness testing.

OBJECTIVE: This study aimed to investigate and improve the usefulness of the 48-hour BH4 loading test and to assess genotype for BH4 responsiveness prediction, using the new definition of BH4 responsiveness from the European guidelines, as well as an amended definition. METHOD: Applying the definition of the European guidelines (≥100% increase in natural protein tolerance) and an amended definition (≥100% increase in natural protein tolerance or tolerating a safe natural protein intake) to a previous dataset, we first assessed the positive predictive value (PPV) of the 48-hour BH4 loading test using a cutoff value of 30%. Then, we tried to improve this PPV by using different cutoff values and separate time points. Last, using the BIOPKU database, we compared predicted BH4 responsiveness (according to genotype) and genotypic phenotype values (GPVs) in BH4 -responsive and BH4 -unresponsive patients. RESULTS: The PPV of the 48-hour loading test was 50.0% using the definition of the European guidelines, and 69.4% when applying the amended definition of BH4 responsiveness. Higher cutoff values led to a higher PPV, but resulted in an increase in false-negative tests. Parameters for genotype overlapped between BH4 -responsive and BH4 -unresponsive patients, although BH4 responsiveness was not observed in patients with a GPV below 2.4. CONCLUSION: The 48-hour BH4 loading test is not as useful as previously considered and cannot be improved easily, whereas genotype seems mainly helpful in excluding BH4 responsiveness. Overall, the definition of BH4 responsiveness and BH4 responsiveness testing require further attention.

BH4 treatment in BH4-responsive PKU patients: preliminary data on blood prolactin concentrations suggest increased cerebral dopamine concentrations.

In phenylketonuria (PKU), cerebral neurotransmitter deficiencies have been suggested to contribute to brain dysfunction. Present treatment aims to reduce blood phenylalanine concentrations by a phenylalanine-restricted diet, while in some patients blood phenylalanine concentrations also respond to cofactor treatment with tetrahydrobiopterin (BH4). Recently, a repurposing approach of BH4 was suggested to increase cerebral neurotransmitter synthesis. To investigate whether BH4 may improve cerebral dopamine concentrations in PKU patients beyond its effect through lowering blood phenylalanine concentrations, we investigated blood prolactin concentrations-as a parameter of brain dopamine availability. We retrospectively compared blood prolactin in relation to blood phenylalanine concentrations of nine (male) BH4-responsive PKU patients, when being treated without and with BH4. Blood prolactin concentrations positively correlated to blood phenylalanine concentrations (p=0.002), being significantly lower with than without BH4 treatment (p=0.047). In addition, even in this small number of male patients, blood prolactin concentrations tended to be lower at increasing BH4 dose (p=0.054), while taking blood phenylalanine concentrations into account (p=0.002). In individual BH4-responsive patients, median blood prolactin concentrations were significantly lower while using BH4 than before using BH4 treatment (p=0.024), whereas median blood phenylalanine concentrations tended to be lower, but this did not reach statistical significance (p=0.107). Therefore, these data show that high blood phenylalanine in BH4-responsive PKU male patients seems to be associated with increased blood prolactin concentrations, suggesting reduced cerebral dopamine availability. Moreover, these data suggest that BH4 treatment in itself could decrease blood prolactin concentrations in a dose-responsive way, independent of blood phenylalanine concentrations. We conclude that these preliminary data indicate that BH4 treatment may improve cerebral dopamine concentrations in PKU patients beyond its effect through lowering blood phenylalanine concentrations, possibly in a dose-dependent manner, but further research would be warranted.

Gut-Microbiome Composition in Response to Phenylketonuria Depends on Dietary Phenylalanine in BTBR Pahenu2 Mice.

Phenylketonuria (PKU) is a metabolic disorder caused by a hepatic enzyme deficiency causing high blood and brain levels of the amino acid Phenylalanine (Phe), leading to severe cognitive and psychological deficits that can be prevented, but not completely, by dietary treatment. The behavioral outcome of PKU could be affected by the gut-microbiome-brain axis, as diet is one of the major drivers of the gut microbiome composition. Gut-microbiome alterations have been reported in treated patients with PKU, although the question remains whether this is due to PKU, the dietary treatment, or their interaction. We, therefore, examined the effects of dietary Phe restriction on gut-microbiome composition and relationships with behavioral outcome in mice. Male and female BTBR Pahenu2 mice received either a control diet (normal protein, "high" Phe), liberalized Phe-restricted (33% natural protein restriction), or severe Phe-restricted (75% natural protein restriction) diet with protein substitutes for 10 weeks (n = 14 per group). Their behavioral performance was examined in an open field test, novel and spatial object location tests, and a balance beam. Fecal samples were collected and sequenced for the bacterial 16S ribosomal RNA (rRNA) region. Results indicated that PKU on a high Phe diet reduced Shannon diversity significantly and altered the microbiome composition compared with wild-type animals. Phe-restriction prevented this loss in Shannon diversity but changed community composition even more than the high-Phe diet, depending on the severity of the restriction. Moreover, on a taxonomic level, we observed the highest number of differentially abundant genera in animals that received 75% Phe-restriction. Based on correlation analyses with differentially abundant taxa, the families Entereococacceae, Erysipelotrichaceae, Porphyromonadaceae, and the genus Alloprevotella showed interesting relationships with either plasma Phe levels and/or object memory. According to our results, these bacterial taxa could be good candidates to start examining the microbial metabolic potential and probiotic properties in the context of PKU. We conclude that PKU leads to an altered gut microbiome composition in mice, which is least severe on a liberalized Phe-restricted diet. This may suggest that the current Phe-restricted diet for PKU patients could be optimized by taking dietary effects on the microbiome into account.

Blood and Brain Biochemistry and Behaviour in NTBC and Dietary Treated Tyrosinemia Type 1 Mice.

Tyrosinemia type 1 (TT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Neurocognitive deficiencies have been described in TT1 patients, that have, among others, been related to changes in plasma large neutral amino acids (LNAA) that could result in changes in brain LNAA and neurotransmitter concentrations. Therefore, this project aimed to investigate plasma and brain LNAA, brain neurotransmitter concentrations and behavior in C57 Bl/6 fumarylacetoacetate hydrolase deficient (FAH-/-) mice treated with 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and/or diet and wild-type mice. Plasma and brain tyrosine concentrations were clearly increased in all NTBC treated animals, even with diet (p < 0.001). Plasma and brain phenylalanine concentrations tended to be lower in all FAH-/- mice. Other brain LNAA, were often slightly lower in NTBC treated FAH-/- mice. Brain neurotransmitter concentrations were usually within a normal range, although serotonin was negatively correlated with brain tyrosine concentrations (p < 0.001). No clear behavioral differences between the different groups of mice could be found. To conclude, this is the first study measuring plasma and brain biochemistry in FAH-/- mice. Clear changes in plasma and brain LNAA have been shown. Further research should be done to relate the biochemical changes to neurocognitive impairments in TT1 patients.

Long-term dietary intervention with low Phe and/or a specific nutrient combination improve certain aspects of brain functioning in phenylketonuria (PKU).

INTRODUCTION: In phenylketonuria (PKU), a gene mutation in the phenylalanine metabolic pathway causes accumulation of phenylalanine (Phe) in blood and brain. Although early introduction of a Phe-restricted diet can prevent severe symptoms from developing, patients who are diagnosed and treated early still experience deficits in cognitive functioning indicating shortcomings of current treatment. In the search for new and/or additional treatment strategies, a specific nutrient combination (SNC) was postulated to improve brain function in PKU. In this study, a long-term dietary intervention with a low-Phe diet, a specific combination of nutrients designed to improve brain function, or both concepts together was investigated in male and female BTBR PKU and WT mice. MATERIAL & METHODS: 48 homozygous wild-types (WT, +/+) and 96 PKU BTBRPah2 (-/-) male and female mice received dietary interventions from postnatal day 31 till 10 months of age and were distributed in the following six groups: high Phe diet (WT C-HP, PKU C-HP), high Phe plus specific nutrient combination (WT SNC-HP, PKU SNC-HP), PKU low-Phe diet (PKU C-LP), and PKU low-Phe diet plus specific nutrient combination (PKU SNC- LP). Memory and motor function were tested at time points 3, 6, and 9 months after treatment initiation in the open field (OF), novel object recognition test (NOR), spatial object recognition test (SOR), and the balance beam (BB). At the end of the experiments, brain neurotransmitter concentrations were determined. RESULTS: In the NOR, we found that PKU mice, despite being subjected to high Phe conditions, could master the task on all three time points when supplemented with SNC. Under low Phe conditions, PKU mice on control diet could master the NOR at all three time points, while PKU mice on the SNC supplemented diet could master the task at time points 6 and 9 months. SNC supplementation did not consistently influence the performance in the OF, SOR or BB in PKU mice. The low Phe diet was able to normalize concentrations of norepinephrine and serotonin; however, these neurotransmitters were not influenced by SNC supplementation. CONCLUSION: This study demonstrates that both a long-lasting low Phe diet, the diet enriched with SNC, as well as the combined diet was able to ameliorate some, but not all of these PKU-induced abnormalities. Specifically, this study is the first long-term intervention study in BTBR PKU mice that shows that SNC supplementation can specifically improve novel object recognition.

The Benefit of Large Neutral Amino Acid Supplementation to a Liberalized Phenylalanine-Restricted Diet in Adult Phenylketonuria Patients: Evidence from Adult Pah-Enu2 Mice.

Many phenylketonuria (PKU) patients cannot adhere to the severe dietary restrictions as advised by the European PKU guidelines, which can be accompanied by aggravated neuropsychological impairments that, at least in part, have been attributed to brain monoaminergic neurotransmitter deficiencies. Supplementation of large neutral amino acids (LNAA) to an unrestricted diet has previously been shown to effectively improve brain monoamines in PKU mice of various ages. To determine the additive value of LNAA supplementation to a liberalized phenylalanine-restricted diet, brain and plasma monoamine and amino acid concentrations in 10 to 16-month-old adult C57Bl/6 PKU mice on a less severe phenylalanine-restricted diet with LNAA supplementation were compared to those on a non-supplemented severe or less severe phenylalanine-restricted diet. LNAA supplementation to a less severe phenylalanine-restricted diet was found to improve both brain monoamine and phenylalanine concentrations. Compared to a severe phenylalanine-restricted diet, it was equally effective to restore brain norepinephrine and serotonin even though being less effective to reduce brain phenylalanine concentrations. These results in adult PKU mice support the idea that LNAA supplementation may enhance the effect of a less severe phenylalanine-restricted diet and suggest that cerebral outcome of PKU patients treated with a less severe phenylalanine-restricted diet may be helped by additional LNAA treatment.

Untreated PKU Patients without Intellectual Disability: What Do They Teach Us?

Phenylketonuria (PKU) management is aimed at preventing neurocognitive and psychosocial dysfunction by keeping plasma phenylalanine concentrations within the recommended target range. It can be questioned, however, whether universal plasma phenylalanine target levels would result in optimal neurocognitive outcomes for all patients, as similar plasma phenylalanine concentrations do not seem to have the same consequences to the brain for each PKU individual. To better understand the inter-individual differences in brain vulnerability to high plasma phenylalanine concentrations, we aimed to identify untreated and/or late-diagnosed PKU patients with near-normal outcome, despite high plasma phenylalanine concentrations, who are still alive. In total, we identified 16 such cases. While intellectual functioning in these patients was relatively unaffected, they often did present other neurological, psychological, and behavioral problems. Thereby, these "unusual" PKU patients show that the classical symptomatology of untreated or late-treated PKU may have to be rewritten. Moreover, these cases show that a lack of intellectual dysfunction despite high plasma phenylalanine concentrations does not necessarily imply that these high phenylalanine concentrations have not been toxic to the brain. Also, these cases may suggest that different mechanisms are involved in PKU pathophysiology, of which the relative importance seems to differ between patients and possibly also with increasing age. Further research should aim to better distinguish PKU patients with respect to their cerebral effects to high plasma phenylalanine concentrations.

Large neutral amino acid supplementation as an alternative to the phenylalanine-restricted diet in adults with phenylketonuria: evidence from adult Pah-enu2 mice.

Phenylketonuria treatment mainly consists of a phenylalanine-restricted diet but still results in suboptimal neuropsychological outcome, which is at least partly based on cerebral monoamine deficiencies, while, after childhood, treatment compliance decreases. Supplementation of large neutral amino acids (LNAAs) was previously demonstrated in young phenylketonuria mice to target all three biochemical disturbances underlying brain dysfunction in phenylketonuria. However, both its potential in adult phenylketonuria and the comparison with the phenylalanine-restricted diet remain to be established. To this purpose, several LNAA supplements were compared with a severe phenylalanine-restricted diet with respect to brain monoamine and amino acid concentrations in adult C57Bl/6 Pah-enu2 mice. Adult phenylketonuria mice received a phenylalanine-restricted diet, unrestricted diet supplemented with several combinations of LNAAs or AIN-93M control diet for 6 weeks. In addition, adult wild-type mice on AIN-93M diet served as controls. The severe phenylalanine-restricted diet in adult phenylketonuria mice significantly reduced plasma and brain phenylalanine and restored brain monoamine concentrations, while brain concentrations of most nonphenylalanine LNAAs remained subnormal. Supplementation of eight LNAAs was similarly effective as the severe phenylalanine-restricted diet to restore brain monoamines, while brain and plasma phenylalanine concentrations remained markedly elevated. These results provide biochemical support for the effectiveness of the severe phenylalanine-restricted diet and showed the possibilities of LNAA supplementation being equally effective to restore brain monoamines in adult phenylketonuria mice. Therefore, LNAA supplementation is a promising alternative treatment to phenylalanine restriction in adult phenylketonuria patients to further optimize neuropsychological functioning.

Can untreated PKU patients escape from intellectual disability? A systematic review.

BACKGROUND: Phenylketonuria (PKU) is often considered as the classical example of a genetic disorder in which severe symptoms can nowadays successfully be prevented by early diagnosis and treatment. In contrast, untreated or late-treated PKU is known to result in severe intellectual disability, seizures, and behavioral disturbances. Rarely, however, untreated or late-diagnosed PKU patients with high plasma phenylalanine concentrations have been reported to escape from intellectual disability. The present study aimed to review published cases of such PKU patients. METHODS: To this purpose, we conducted a literature search in PubMed and EMBASE up to 8th of September 2017 to identify cases with 1) PKU diagnosis and start of treatment after 7 years of age; 2) untreated plasma phenylalanine concentrations ≥1200 µmol/l; and 3) IQ ≥80. Literature search, checking reference lists, selection of articles, and extraction of data were performed by two independent researchers. RESULTS: In total, we identified 59 published cases of patients with late-diagnosed PKU and unexpected favorable outcome who met the inclusion criteria. Although all investigated patients had intellectual functioning within the normal range, at least 19 showed other neurological, psychological, and/or behavioral symptoms. CONCLUSIONS: Based on the present findings, the classical symptomatology of untreated or late-treated PKU may need to be rewritten, not only in the sense that intellectual dysfunction is not obligatory, but also in the sense that intellectual functioning does not (re)present the full picture of brain damage due to high plasma phenylalanine concentrations. Further identification of such patients and additional analyses are necessary to better understand these differences between PKU patients.

Presumptive brain influx of large neutral amino acids and the effect of phenylalanine supplementation in patients with Tyrosinemia type 1.

INTRODUCTION: Hereditary Tyrosinemia type 1 (HT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Current treatment consists of 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and a tyrosine and phenylalanine restricted diet. Recently, neuropsychological deficits have been seen in HT1 patients. These deficits are possibly associated with low blood phenylalanine concentrations and/or high blood tyrosine concentrations. Therefore, the aim of the present study was threefold. Firstly, we aimed to calculate how the plasma amino acid profile in HT1 patients may influence the presumptive brain influx of all large neutral amino acids (LNAA). Secondly, we aimed to investigate the effect of phenylalanine supplementation on presumptive brain phenylalanine and tyrosine influx. Thirdly, we aimed to theoretically determine minimal target plasma phenylalanine concentrations in HT1 patient to ensure adequate presumptive brain phenylalanine influx. METHODS: Data of plasma LNAA concentrations were obtained. In total, 239 samples of 9 HT1 children, treated with NTBC, diet, and partly with phenylalanine supplementation were collected together with 596 samples of independent control children. Presumptive brain influx of all LNAA was calculated, using Michaelis-Menten parameters (Km) and Vmax-values obtained from earlier articles. RESULTS: In HT1 patients, plasma concentrations and presumptive brain influx of tyrosine were higher. However, plasma and especially brain influx of phenylalanine were lower in HT1 patients. Phenylalanine supplementation did not only tend to increase plasma phenylalanine concentrations, but also presumptive brain phenylalanine influx, despite increased plasma tyrosine concentrations. However, to ensure sufficient brain phenylalanine influx in HT1 patients, minimal plasma phenylalanine concentrations may need to be higher than considered thus far. CONCLUSION: This study clearly suggests a role for disturbed brain LNAA biochemistry, which is not well reflected by plasma LNAA concentrations. This could play a role in the pathophysiology of the neuropsychological impairments in HT1 patients and may have therapeutic implications.

Voluntary Exercise Prevents Oxidative Stress in the Brain of Phenylketonuria Mice.

BACKGROUND: High phenylalanine levels in phenylketonuria (PKU) have been associated with brain oxidative stress and amino acid imbalance. Exercise has been shown to improve brain function in hyperphenylalaninemia and neurodegenerative diseases. This study aimed to verify the effects of exercise on coordination and balance, plasma and brain amino acid levels, and brain oxidative stress markers in PKU mice. METHODS: Twenty wild-type (WT) and 20 PAH(enu2) (PKU) C57BL/6 mice were placed in cages with (exercise, Exe) or without (sedentary, Sed) running wheels during 53 days. At day 43, a balance beam test was performed. Plasma and brain were collected for analyses of amino acid levels and the oxidative stress parameters superoxide dismutase (SOD) activity, sulfhydryl and reduced glutathione (GSH) contents, total radical-trapping antioxidant potential (TRAP), and total antioxidant reactivity (TAR). RESULTS: SedPKU showed poor coordination (p < 0.001) and balance (p < 0.001), higher plasma and brain phenylalanine (p < 0.001), and increased brain oxidative stress (p < 0.05) in comparison to SedWT. ExePKU animals ran less than ExeWT (p = 0.018). Although no improvement was seen in motor coordination and balance, exercise in PKU restored SOD, sulfhydryl content, and TRAP levels to controls. TAR levels were increased in ExePKU in comparison to SedPKU (p = 0.012). Exercise decreased plasma and brain glucogenic amino acids in ExePKU, but did not change plasma and brain phenylalanine in both WT and PKU. CONCLUSIONS: Exercise prevents oxidative stress in the brain of PKU mice without modifying phenylalanine levels. Hence, exercise positively affects the brain, demonstrating its value as an intervention to improve brain quality in PKU.

A Specific Nutrient Combination Attenuates the Reduced Expression of PSD-95 in the Proximal Dendrites of Hippocampal Cell Body Layers in a Mouse Model of Phenylketonuria.

The inherited metabolic disease phenylketonuria (PKU) is characterized by increased concentrations of phenylalanine in the blood and brain, and as a consequence neurotransmitter metabolism, white matter, and synapse functioning are affected. A specific nutrient combination (SNC) has been shown to improve synapse formation, morphology and function. This could become an interesting new nutritional approach for PKU. To assess whether treatment with SNC can affect synapses, we treated PKU mice with SNC or an isocaloric control diet and wild-type (WT) mice with an isocaloric control for 12 weeks, starting at postnatal day 31. Immunostaining for post-synaptic density protein 95 (PSD-95), a post-synaptic density marker, was carried out in the hippocampus, striatum and prefrontal cortex. Compared to WT mice on normal chow without SNC, PKU mice on the isocaloric control showed a significant reduction in PSD-95 expression in the hippocampus, specifically in the granular cell layer of the dentate gyrus, with a similar trend seen in the cornus ammonis 1 (CA1) and cornus ammonis 3 (CA3) pyramidal cell layer. No differences were found in the striatum or prefrontal cortex. PKU mice on a diet supplemented with SNC showed improved expression of PSD-95 in the hippocampus. This study gives the first indication that SNC supplementation has a positive effect on hippocampal synaptic deficits in PKU mice.

The Behavioral Consequence of Phenylketonuria in Mice Depends on the Genetic Background.

To unravel the role of gene mutations in the healthy and the diseased state, countless studies have tried to link genotype with phenotype. However, over the years, it became clear that the strain of mice can influence these results. Nevertheless, identical gene mutations in different strains are often still considered equals. An example of this, is the research done in phenylketonuria (PKU), an inheritable metabolic disorder. In this field, a PKU mouse model (either on a BTBR or C57Bl/6 background) is often used to examine underlying mechanisms of the disease and/or new treatment strategies. Both strains have a point mutation in the gene coding for the enzyme phenylalanine hydroxylase which causes toxic concentrations of the amino acid phenylalanine in blood and brain, as found in PKU patients. Although the mutation is identical and therefore assumed to equally affect physiology and behavior in both strains, no studies directly compared the two genetic backgrounds to test this assumption. Therefore, this study compared the BTBR and C57Bl/6 wild-type and PKU mice on PKU-relevant amino acid- and neurotransmitter-levels and at a behavioral level. The behavioral paradigms were selected from previous literature on the PKU mouse model and address four domains, namely (1) activity levels, (2) motor performance, (3) anxiety and/or depression-like behavior, and (4) learning and memory. The results of this study showed comparable biochemical changes in phenylalanine and neurotransmitter concentrations. In contrast, clear differences in behavioral outcome between the strains in all four above-mentioned domains were found, most notably in the learning and memory domain. The outcome in this domain seem to be primarily due to factors inherent to the genetic background of the mouse and much less by differences in PKU-specific biochemical parameters in blood and brain. The difference in behavioral outcome between PKU of both strains emphasizes that the consequence of the PAH mutation is influenced by other factors than Phe levels alone. Therefore, future research should consider these differences when choosing one of the genetic strains to investigate the pathophysiological mechanism underlying PKU-related behavior, especially when combined with new treatment strategies.

Therapeutic brain modulation with targeted large neutral amino acid supplements in the Pah-enu2 phenylketonuria mouse model.

BACKGROUND: Phenylketonuria treatment consists mainly of a Phe-restricted diet, which leads to suboptimal neurocognitive and psychosocial outcomes. Supplementation of large neutral amino acids (LNAAs) has been suggested as an alternative dietary treatment strategy to optimize neurocognitive outcome in phenylketonuria and has been shown to influence 3 brain pathobiochemical mechanisms in phenylketonuria, but its optimal composition has not been established. OBJECTIVE: In order to provide additional pathobiochemical insight and develop optimal LNAA treatment, several targeted LNAA supplements were investigated with respect to all 3 biochemical disturbances underlying brain dysfunction in phenylketonuria. DESIGN: Pah-enu2 (PKU) mice received 1 of 5 different LNAA-supplemented diets beginning at postnatal day 45. Control groups included phenylketonuria mice receiving an isonitrogenic and isocaloric high-protein diet or the AIN-93M diet, and wild-type mice receiving the AIN-93M diet. After 6 wk, brain and plasma amino acid profiles and brain monoaminergic neurotransmitter concentrations were measured. RESULTS: Brain Phe concentrations were most effectively reduced by supplementation of LNAAs, such as Leu and Ile, with a strong affinity for the LNAA transporter type 1. Brain non-Phe LNAAs could be restored on supplementation, but unbalanced LNAA supplementation further reduced brain concentrations of those LNAAs that were not (sufficiently) included in the LNAA supplement. To optimally ameliorate brain monoaminergic neurotransmitter concentrations, LNAA supplementation should include Tyr and Trp together with LNAAs that effectively reduce brain Phe concentrations. The requirement for Tyr supplementation is higher than it is for Trp, and the relative effect of brain Phe reduction is higher for serotonin than it is for dopamine and norepinephrine. CONCLUSION: The study shows that all 3 biochemical disturbances underlying brain dysfunction in phenylketonuria can be targeted by specific LNAA supplements. The study thus provides essential information for the development of optimal LNAA supplementation as an alternative dietary treatment strategy to optimize neurocognitive outcome in patients with phenylketonuria.

Infants with Tyrosinemia Type 1: Should phenylalanine be supplemented?

Tyrosinemia type 1 (HT1) is an inborn error of tyrosine catabolism caused by fumarylacetoacetase deficiency. Biochemically, this results in accumulation of toxic metabolites including succinylacetone. Clinically, HT1 is characterized by severe liver, kidney, and neurological problems. Treatment with NTBC and dietary restriction of tyrosine and phenylalanine have strongly improved outcome, but impaired neurocognitive development has been reported. Whether impaired neurocognitive outcome results from high blood tyrosine or low blood phenylalanine concentrations is currently unknown. In this report, two HT1 newborns, diagnosed by neonatal screening, are presented. The first patient showed low phenylalanine concentrations, growth retardation, neurological impairments, and skin problems, clearly improving after institution of phenylalanine supplementation (~30 mg/kg/day) at age 6 months, while both blood phenylalanine and tyrosine concentrations increased. In the second patient, phenylalanine supplementation (~20 mg/kg/day) was initiated as soon as low phenylalanine concentrations were observed at age 19 days. On this regimen, blood phenylalanine concentrations increased, and hypophenylalaninemia was less frequently observed than in the first patient, whereas blood tyrosine concentrations tended to increase. Clinically, no growth, neurological, or skin problems have been observed. The combination of knowledge obtained from these cases suggests that hypophenylalaninemia rather than hypertyrosinemia during the first months of life may impair neurocognitive development in young HT1 infants. Phenylalanine supplementation should really be considered in HT1 patients with consistently low blood phenylalanine concentrations during the first months of life. However, the minimal phenylalanine concentrations acceptable and the optimal phenylalanine supplementation regimen require further investigation.

Large Neutral Amino Acid Supplementation Exerts Its Effect through Three Synergistic Mechanisms: Proof of Principle in Phenylketonuria Mice.

BACKGROUND: Phenylketonuria (PKU) was the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. However, despite this effect, neuropsychological outcome in PKU still remains suboptimal and the phenylalanine-restricted diet is very demanding. To improve neuropsychological outcome and relieve the dietary restrictions for PKU patients, supplementation of large neutral amino acids (LNAA) is suggested as alternative treatment strategy that might correct all brain biochemical disturbances caused by high blood phenylalanine, and thereby improve neurocognitive functioning. OBJECTIVE: As a proof-of-principle, this study aimed to investigate all hypothesized biochemical treatment objectives of LNAA supplementation (normalizing brain phenylalanine, non-phenylalanine LNAA, and monoaminergic neurotransmitter concentrations) in PKU mice. METHODS: C57Bl/6 Pah-enu2 (PKU) mice and wild-type mice received a LNAA supplemented diet, an isonitrogenic/isocaloric high-protein control diet, or normal chow. After six weeks of dietary treatment, blood and brain amino acid and monoaminergic neurotransmitter concentrations were assessed. RESULTS: In PKU mice, the investigated LNAA supplementation regimen significantly reduced blood and brain phenylalanine concentrations by 33% and 26%, respectively, compared to normal chow (p<0.01), while alleviating brain deficiencies of some but not all supplemented LNAA. Moreover, LNAA supplementation in PKU mice significantly increased brain serotonin and norepinephrine concentrations from 35% to 71% and from 57% to 86% of wild-type concentrations (p<0.01), respectively, but not brain dopamine concentrations (p = 0.307). CONCLUSIONS: This study shows that LNAA supplementation without dietary phenylalanine restriction in PKU mice improves brain biochemistry through all three hypothesized biochemical mechanisms. Thereby, these data provide proof-of-concept for LNAA supplementation as a valuable alternative dietary treatment strategy in PKU. Based on these results, LNAA treatment should be further optimized for clinical application with regard to the composition and dose of the LNAA supplement, taking into account all three working mechanisms of LNAA treatment.

Single amino acid supplementation in aminoacidopathies: a systematic review.

Aminoacidopathies are a group of rare and diverse disorders, caused by the deficiency of an enzyme or transporter involved in amino acid metabolism. For most aminoacidopathies, dietary management is the mainstay of treatment. Such treatment includes severe natural protein restriction, combined with protein substitution with all amino acids except the amino acids prior to the metabolic block and enriched with the amino acid that has become essential by the enzymatic defect. For some aminoacidopathies, supplementation of one or two amino acids, that have not become essential by the enzymatic defect, has been suggested. This so-called single amino acid supplementation can serve different treatment objectives, but evidence is limited. The aim of the present article is to provide a systematic review on the reasons for applications of single amino acid supplementation in aminoacidopathies treated with natural protein restriction and synthetic amino acid mixtures.