Inhibition of L-type amino acid transport with non-physiological amino acids in the Pahenu2 mouse model of phenylketonuria

Phenylketonuria, an autosomal recessive Mendelian disorder, is one of the most common inborn errors of metabolism. Although currently treated by diet, many suboptimal outcomes occur for patients. Neuropathological outcomes include cognitive loss, white matter abnormalities, and hypo- or demyelination, resulting from high concentrations and/or fluctuating levels of phenylalanine. High phenylalanine can also result in competitive exclusion of other large neutral amino acids from the brain, including tyrosine and tryptophan (essential precursors of dopamine and serotonin). This competition occurs at the blood brain barrier, where the L-type amino acid transporter, LAT1, selectively facilitates entry of large neutral amino acids. The hypothesis of these studies is that certain non-physiological amino acids (NPAA; DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), α-aminoisobutyrate (AIB), and α-methyl-aminoisobutyrate (MAIB)) would competitively inhibit LAT1 transport of phenylalanine (Phe) at the blood-brain barrier interface. To test this hypothesis, Pah-/- mice (n=5, mixed gender; Pah+/-(n=5) as controls) were fed either 5% NL, 0.5% NB, 5% AIB or 3% MAIB (w/w 18% protein mouse chow) for 3 weeks. Outcome measurements included food intake, body weight, brain LNAAs, and brain monoamines measured via LCMS/MS or HPLC. Brain Phe values at sacrifice were significantly reduced for NL, NB, and MAIB, verifying the hypothesis that these NPAAs could inhibit Phe trafficking into the brain. However, concomitant reductions in tyrosine and methionine occurred at the concentrations employed. Blood Phe levels were not altered indicating no effect of NPAA competitors in the gut. Brain NL and NB levels, measured with HPLC, verified both uptake and transport of NPAAs. Although believed predominantly unmetabolized, NL feeding significantly increased blood urea nitrogen. Pah-/-disturbances of monoamine metabolism were exacerbated by NPAA intervention, primarily with NB (the prototypical LAT inhibitor). To achieve the overarching goal of using NPAAs to stabilize Phe transport levels into the brain, a specific Phe-reducing combination and concentration of NPAAs must be found. Our studies represent the first in vivo use of NL, NB and MAIB in Pah-/- mice, and provide proof-of-principle for further characterization of these LAT inhibitors. Our data is the first to document an effect of MAIB, a specific system A transport inhibitor, on large neutral amino acid transport.

Carbon in the Peatlands in the Great Lakes Region

Peatlands cover only ~3% of the global land area, but store ~30% of the worlds' soil carbon. There are many different peat types that store different amounts of carbon. Most inventories of carbon storage in northern peatlands have been conducted in the expansive Sphagnum dominated peatlands. Although, northern white cedar peatlands (NW cedar, Thuja occidentalis L.) are also one of the most common peatland types in the Great Lakes Region, occupying more than 2 million hectares. NW cedar swamps are understudied, due in part to the difficulties in collection methods. General lack of rapid and consistent sampling methods has also contributed in a lack of carbon stock quantification for many peatlands. The main objective of this thesis is to quantify: 1) to evaluate peat sampling methods 2) the amount of C-stored and the rates of long-term carbon accumulation in NW cedar peatlands. We sampled 38 peatlands separated into four categories (black ash, NW cedar swamp, sedge, and Sphagnum) during the summers of 2011/2012 across northern MN and the Upper Peninsula of MI. Basal dates of peat indicate that cedar peatlands were between 1970-7790 years old. Cedar peatlands are generally shallower than Sphagnum peat, but due to their higher bulk density, hold similar amounts of carbon with our sites averaging ~800 MgC ha-1. We estimate that NW cedar peatlands store over 1.7 Gt of carbon in the Great Lakes Region. Each of the six methods evaluated had a different level of accuracy and requires varying levels of effort and resources. The depth only method and intermittent sampling method were the most accurate methods of peatland sampling.