ABSTRACT
BACKGROUND: Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain. METHODS: To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses. RESULTS: LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 µM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU. CONCLUSIONS: Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders.
Subject(s)
Amino Acids/metabolism , Brain/metabolism , Dietary Proteins/administration & dosage , Disease Models, Animal , Maple Syrup Urine Disease/diet therapy , Amino Acids/blood , Animals , Maple Syrup Urine Disease/metabolism , Maple Syrup Urine Disease/physiopathology , Mice , Polymerase Chain ReactionABSTRACT
BACKGROUND: Our laboratory seeks a pharmacotherapeutic intervention for PKU that utilizes non-physiological amino acids (NPAAs) to block the accumulation of phenylalanine (Phe) in the brain. In previous studies (Vogel et al. 2013), methylation of the amino group of 2-aminoisobutyrate (AIB) provided an enhanced degree of selectivity for Phe restriction into the brain of Pah(enu2) mice in comparison to unmethylated AIB, leading to the hypothesis that 2-(methylamino)alkanoic acid analogs of AIB might represent targeted inhibitors of Phe accretion into the brain. METHODS: Pah(enu2) and control mice were intraperitoneally administered (500-750 mg/kg body weight, once daily; standard 19% protein diet) AIB, methyl AIB (MAIB), isovaline, and two MAIB analogs, 2-methyl-2-(methylamino)butanoic (MeVal) and 3-methyl-2-(methylamino)pentanoic (MePent) acids for one week, followed by brain and blood isolation for amino acid analyses using UPLC. RESULTS: In the brain, AIB significantly reduced Phe accretion in Pah(enu2) mice, while MeVal significantly improved glutamine and aspartic acids. Four of five test compounds improved brain threonine and arginine levels. AIB, MAIB and IsoVal significantly reduced blood Phe, with no effect of any drug intervention on other sera amino acids. CONCLUSIONS: Further evaluation of AIB and the 2-(methylamino)alkanoic acids as inhibitors of brain Phe accumulation in Pah(enu2) mice is warranted, with more detailed evaluations of route of administration, combinatorial intervention, and detailed toxicity studies.
Subject(s)
Acids, Acyclic/pharmacology , Aminoisobutyric Acids/pharmacology , Blood-Brain Barrier/metabolism , Brain/metabolism , Isoleucine/analogs & derivatives , Phenylalanine/metabolism , Phenylketonurias/drug therapy , Valine/analogs & derivatives , Acids, Acyclic/administration & dosage , Aminoisobutyric Acids/administration & dosage , Animals , Disease Models, Animal , Humans , Isoleucine/administration & dosage , Isoleucine/pharmacology , Large Neutral Amino Acid-Transporter 1/chemistry , Large Neutral Amino Acid-Transporter 1/metabolism , Methylation , Mice , Mice, Transgenic , Molecular Targeted Therapy , Organ Specificity , Phenylalanine/blood , Protein Conformation , Protein Folding , Valine/administration & dosage , Valine/pharmacologyABSTRACT
Transport of large neutral amino acids (LNAA) across the blood brain barrier (BBB) is facilitated by the L-type amino acid transporter, LAT1. Peripheral accumulation of one LNAA (e.g., phenylalanine (phe) in PKU) is predicted to increase uptake of the offending amino acid to the detriment of others, resulting in disruption of brain amino acid homeostasis. We hypothesized that selected non-physiological amino acids (NPAAs) such as DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), 2-aminoisobutyrate (AIB), and N-methyl-aminoisobutyrate (MAIB), acting as competitive inhibitors of various brain amino acid transporters, could reduce brain phe in Pah (enu2) mice, a relevant murine model of PKU. Oral feeding of 5 % NL, 5 % AIB, 0.5 % NB and 3 % MAIB reduced brain phe by 56 % (p < 0.01), -1 % (p = NS), 27 % (p < 0.05) and 14 % (p < 0.01), respectively, compared to untreated subjects. Significant effects on other LNAAs (tyrosine, methionine, branched chain amino acids) were also observed, however, with MAIB displaying the mildest effects. Of interest, MAIB represents an inhibitor of the system A (alanine) transporter that primarily traffics small amino acids and not LNAAs. Our studies represent the first in vivo use of these NPAAs in Pah (enu2) mice, and provide proof-of-principle for their further preclinical development, with the long-term objective of identifying NPAA combinations and concentrations that selectively restrict brain phe transport while minimally impacting other LNAAs and downstream intermediates.
