Journal of Inherited Metabolic Disease

, Volume 36, Issue 3, pp 513–523 | Cite as

Non-physiological amino acid (NPAA) therapy targeting brain phenylalanine reduction: pilot studies in PAHENU2 mice

  • Kara R. Vogel
  • Erland Arning
  • Brandi L. Wasek
  • Teodoro Bottiglieri
  • K. Michael Gibson
Original Article


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 Pahenu2 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 Pahenu2 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.





5-hydroxyindoleacetic acid






Aminoisobutyric acid


Blood brain barrier




3,4-dihydroxyphenylacetic acid


Homovanillic acid

Ile; I





L-type amino acid transporter

Leu; L



Large neutral amino acid




Methyl-aminoisobutyric acid

Met; M



2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (2-aminonorbornane)


Non-physiological amino acid


Phenylalanine hydroxylase

Phe; F








Trp; W


Tyr; Y


Val; V



  1. Aaslestad HG, Larson AD (1964) Bacterial metabolism of 2-methylalanine. J Bacteriol 88:1296–1303PubMedGoogle Scholar
  2. Arning E, Bottiglieri T, Sun Q et al (2009) Metabolic profiling in phenylalanine hydroxylase-deficient (Pah-/-) mouse brain reveals decreased amino acid neurotransmitters and preferential alterations of the serotoninergic system. Mol Genet Metab 98:21Google Scholar
  3. Babu E, Kanai Y, Chairoungdua A et al (2003) Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters. J Biol Chem 278:43838–43845PubMedCrossRefGoogle Scholar
  4. Bodoy S, Martín L, Zorzano A, Palacín M, Estévez R, Bertran J (2005) Identification of LAT4, a novel amino acid transporter with system L activity. J Biol Chem 280:12002–12011PubMedCrossRefGoogle Scholar
  5. Bröer S, Brookes N (2001) Transfer of glutamine between astrocytes and neurons. J Neurochem 77:705–719PubMedCrossRefGoogle Scholar
  6. Choi TB, Pardridge WM (1986) Phenylalanine transport at the human blood–brain barrier. Studies with isolated human brain capillaries. J Biol Chem 261:6536–6541PubMedGoogle Scholar
  7. Christensen HN, Streicher JA, Elbinger RL (1948) Effects of feeding individual amino acids upon the distribution of other amino acids between cells and extracellular fluid. J Biol Chem 172:515–524PubMedGoogle Scholar
  8. Christensen HN (1990) Role of amino acid transport and countertransport in nutrition and metabolism. Physiol Rev 70:43–77PubMedGoogle Scholar
  9. Chrostowski MK, McGonnigal BG, Stabila JP, Padbury JF (2009) LAT-1 expression in pre- and post-implantation embryos and placenta. Placenta 30:270–276PubMedCrossRefGoogle Scholar
  10. Crawhall JC, Purkiss P (1973) Transport of methionine and proline by rat liver slices and the effect of certain hormones. Biochem J 136:15–24PubMedGoogle Scholar
  11. Der Garabedian PA, Vermeersch JJ (1987) Candida L-norleucine, leucine:2-oxoglutarate aminotransferase. Purification and properties. Eur J Biochem 167:141–147CrossRefGoogle Scholar
  12. Dotremont H, Francois B, Diels M, Gillis P (1995) Nutritional value of essential amino acids in the treatment of adults with phenylketonuria. J Inherit Metab Dis 18:127–130PubMedCrossRefGoogle Scholar
  13. Ennis SR, Ren XD, Betz AL (1994) Transport of alpha-aminoisobutyric acid across the blood–brain barrier studied with in situ perfusion of rat brain. Brain Res 643:100–107PubMedCrossRefGoogle Scholar
  14. Enns GM, Koch R, Brumm V, Blakely E, Suter R, Jurecki E (2010) Suboptimal outcomes in patients with PKU treated early with diet alone: revisiting the evidence. Mol Genet Metab 101:99–109PubMedCrossRefGoogle Scholar
  15. Geldenhuys WJ, Van der Schyf CJ (2011) Role of serotonin in Alzheimer’s disease: a new therapeutic target. CNS Drugs 25:765–781PubMedCrossRefGoogle Scholar
  16. Han SJ, Choi S-E, Yi S-A et al (2012) β-Cell-protective effect of 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid as a glutamate dehydrogenase activator in db/db mice. J Endocrinol 212:307–315Google Scholar
  17. Kanai Y, Segawa H, Miyamoto K, Uchino H, Takeda E, Endou H (1998) Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem 273:23629–23632PubMedCrossRefGoogle Scholar
  18. Knudsen GM, Hasselbalch S, Toft PB, Christensen E, Paulson OB, Lou H (1995) Blood–brain barrier transport of amino acids in healthy controls and in patients with phenylketonuria. J Inherit Metab Dis 18:653–664PubMedCrossRefGoogle Scholar
  19. Kurian MA, Gissen P, Smith M, Heales S Jr, Clayton PT (2011) The monoamine neurotransmitter disorders: an expanding range of neurological syndromes. Lancet Neurol 10:721–733PubMedCrossRefGoogle Scholar
  20. Lepley PR, Mukkada AJ (1983) Characteristics of an uptake system for alpha-aminoisobutyric acid in Leishmania tropica promastigotes. J Protozool 30:41–46PubMedGoogle Scholar
  21. Lin J, Raoof DA, Thomas DG et al (2004) L-type amino acid transporter-1 overexpression and melphalan sensitivity in Barrett’s adenocarcinoma. Neoplasia 6:74–84PubMedCrossRefGoogle Scholar
  22. Matalon R, Michals-Matalon K, Bhatia G et al (2006) Large neutral amino acids in the treatment of phenylketonuria (PKU). J Inherit Metab Dis 29:732–738PubMedCrossRefGoogle Scholar
  23. McKean CM, Boggs DE, Peterson NA (1968) The influence of high phenylalanine and tyrosine on the concentrations of essential amino acids in brain. J Neurochem 15:235–241PubMedCrossRefGoogle Scholar
  24. Michals-Matalon K, Bhatia G, Guttler F, Tyring SK, Matalon R (2007) Response of phenylketonuria to tetrahydrobiopterin. J Nutr 137:1564S–1567SPubMedGoogle Scholar
  25. Ogburn KD, Bottiglieri T, Wang Z, Figueiredo-Pereira ME (2006) Prostaglandin J2 reduces catechol-O-methyltransferase activity and enhances dopamine toxicity in neuronal cells. Neurobiol Dis 22:294–301PubMedCrossRefGoogle Scholar
  26. Pietz J, Kreis R, Rupp A, Mayatepek E, Rating D, Boesch C, Bremer HJ (1999) Large neutral amino acids block phenylalanine transport into brain tissue in patients with phenylketonuria. J Clin Invest 103:1169–1178PubMedCrossRefGoogle Scholar
  27. Pisoni RL, Flickinger KS, Thoene JG, Christensen HN (1987) Characterization of carrier-mediated transport systems for small neutral amino acids in human fibroblast lysosomes. J Biol Chem 262:6010–6017PubMedGoogle Scholar
  28. Segawa H, Fukasawa Y, Miyamoto K, Takeda E, Endou H, Kanai Y (1999) Identification and functional characterization of a Na + -independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem 274:19745–19751PubMedCrossRefGoogle Scholar
  29. Shennan DB, McNeillie SA (1994) Characteristics of alpha-aminoisobutyric acid transport by lactating rat mammary gland. J Dairy Res 61:9–19PubMedCrossRefGoogle Scholar
  30. Skvorak KJ, Hager EJ, Arning E et al (2009) Hepatocyte transplantation (HTx) corrects selected neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD). Biochim Biophys Acta 1792:1004–1010PubMedCrossRefGoogle Scholar
  31. Smith QR, Momma S, Aoyagi M, Rapoport SI (1987) Kinetics of neutral amino acid transport across the blood–brain barrier. J Neurochem 49:1651–1658PubMedCrossRefGoogle Scholar
  32. Tews JK, Harper AE (1986) Tissue amino acids in rats fed norleucine, norvaline, homoarginine or other amino acid analogues. J Nutr 116:1464–1472PubMedGoogle Scholar
  33. Tews JK, Repa JJ, Harper AE (1990) Norleucine: a branched-chain amino acid analog affecting feeding behavior of rats. Pharmacol Biochem Behav 35:911–921PubMedCrossRefGoogle Scholar
  34. Tews JK, Repa JJ, Harper AE (1991) Branched-chain and other amino acids in tissues of rats fed leucine-limiting amino acid diets containing norleucine. J Nutr 121:364–378PubMedGoogle Scholar
  35. Tovar A, Tews JK, Torres N, Harper AE (1988) Some characteristics of threonine transport across the blood–brain barrier of the rat. J Neurochem 51:1285–1293PubMedCrossRefGoogle Scholar
  36. van Spronsen FJ, Enns GM (2010) Future treatment strategies in phenylketonuria. Mol Genet Metab 99:S90–S95PubMedCrossRefGoogle Scholar
  37. van Spronsen FJ, de Groot MJ, Hoeksma M, Reijngoud DJ, van Rijn M (2010) Large neutral amino acids in the treatment of PKU: from theory to practice. J Inherit Metab Dis 33:671–676PubMedCrossRefGoogle Scholar
  38. Wadhwani KC, Smith QR, Rapoport SI (1990) Facilitated transport of L-phenylalanine across blood-nerve barrier of rat peripheral nerve. Am J Physiol 258:R1436–R1444PubMedGoogle Scholar
  39. Zagreda L, Goodman J, Druin DP, McDonald D, Diamond A (1999) Cognitive deficits in a genetic mouse model of the most common biochemical cause of human mental retardation. J Neurosci 19:6175–6182PubMedGoogle Scholar
  40. Zinnanti WJ, Lazovic J, Griffin K et al (2009) Dual mechanism of brain injury and novel treatment strategy in maple syrup urine disease. Brain 132:903–918PubMedCrossRefGoogle Scholar

Copyright information

© SSIEM and Springer 2012

Authors and Affiliations

  • Kara R. Vogel
    • 1
  • Erland Arning
    • 2
  • Brandi L. Wasek
    • 2
  • Teodoro Bottiglieri
    • 2
  • K. Michael Gibson
    • 1
  1. 1.Section of Clinical Pharmacology, College of PharmacyWashington State UniversityPullmanUSA
  2. 2.Institute of Metabolic Disease, Baylor Research InstituteBaylor University Medical CenterDallasUSA

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