Recent Studies of Phenylketonuria By: Jennifer Gastelum Dr. Koni Stone Copyright 2014

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1 Recent Studies of Phenylketonuria By: Jennifer Gastelum Dr. Koni Stone Copyright 2014 Phenylketonuria (PKU) is an autosomal recessive genetic disorder that causes an accumulation of toxic metabolites in the blood. This disorder is characterized by a deficiency of the phenylalanine hydroxylase enzyme (PAH). The mutation causes a buildup of phenylalanine in brain tissue and cerebrospinal fluid. If left untreated, due to the gradual accretion of L-phenylalanine and its metabolites results in various degrees of neurological damage. In this paper, recent research studies conducted on PKU will be explained. In normal functionality, the PAH enzyme catalyzes the conversion of phenylalanine (Phe) to tyrosine (Tyr). PAH is expressed in the kidneys and liver. Specifically, PAH is located on chromosome 12 between positions 22 and More precisely, the gene for PAH is located from base pair 102,838,325 to base pair 102,917,602 on chromosome 12. Using X-ray crystallography, the structure is found to have three domains: N-terminal, catalytic domain, and a C-terminal. To begin the conversion of Phe to Tyr, PAH catalyzes the hydroxylation of Phe by incorporating one oxygen atom from molecular oxygen into the Phe aromatic ring via electrophilic aromatic substitution. In addition, PAH requires a catalytic non-heme iron and tetrahydrobiopterin (BH4) to further proceed. BH4 supplies the reaction with two electrons that are needed to reduce the oxygen atom to water. Thus, the mechanism leaves Tyr and water as products. In addition, when BH4 is oxidized by PAH it forms a pterin 4a-carbinolamine product. Pterin 4a-carbinolamine dehydratase produces quinoid dihydropterin. Dihydropterin reductase then converts the intermediate back to BH4 to restart the conversion of another Phe (1).

2 When there is a malfunction of the PAH enzyme, the effect of high concentrations of Phe triggers neuronal apoptosis. Recent studies found that RhoA/ROCK pathway and apoptosis are linked. This raised the question if RhoA/ROCK signaling is involved with Phe induced apoptosis. RhoA is known to regulate neuronal development and initiates cellular process by acting on Rho-associated kinase (ROCK). ROCK is a serine/threonine kinase that phosphorylates the myosin light chain (MLC) to regulate cell growth (2). Thus, researchers further studied how Phe induces RhoA activation and MLC phosphorylation and whether it may be a result from ROCK activation. In an experiment, GST-RBD pull down assay was utilized. Neurons were treated with 0.9 mm Phe and as the cell lysates they were incubated with GST-RBS. The amount of active RhoA was detected using Western blotting. Results from the experiment showed that RhoA activity was up regulated after Phe treatment. In addition, to test if Phe activates ROCK, MLC phosphorylation levels were examined. By using Western Blotting, it showed an increase in MLC phosphorylation in Phe treated cells. As a control, neurons were treated with Phe in the presence of a ROCK inhibitor. The presence of the ROCK inhibitor nearly ended the increase of phosphorylated MLC. Thus, it emphasizes that MLC phosphorylation triggered by Phe is mediated by ROCK. Both experiments indicate that Phe activates the RhoA/ROCK pathway (2). Studies found that the mitochondria apoptotic pathways are involved in Phe-induced apoptosis in cortical neurons therefore;; researchers conducted an experiment to examine whether the ER stress-initiated pathway could contribute to phenylalanine-induced apoptosis (3). Apoptosis was measured by TdT-mediated dutp nick end labeling (TUNEL) assay using a cell death fluorescence detection kit. TUNEL assay is a common method in detecting DNA fragmentation that results from apoptosis. TUNEL assay works by using a TdT enzyme that can locate cuts in the DNA and labels the

3 terminal end of nucleic acids (3). The intensity of the TUNEL staining was examined by fluorescence microscopy. DAPI, a nuclear counterstain was used as a contrast to make the stains more visible to account for the total neurons in the sample (3). Researchers conducted four separate experimental controls consisting of four samples of cortical neuronal cells. ElF4 blocker salubrinal (SAL) is a blocker that was used as a control in attempt to inhibit the cell from undergoing apoptosis. One trial consisted of cells with elevated levels of Phe, second with cells mixed with SAL, third with cells with high Phe and SAL, and a fourth with neither present. Apoptotic cells were labeled TUNEL-positive, which meant that the cells came up on the fluorescent microscopy as a dark brown with blue-like nuclei. Cells that were treated and did not undergo apoptosis were labeled negative and showed up only as a bright blue color. In the photos, the experiments that lacked high Phe had a few blue spots, which means there was a negative result for apoptosis. In addition, in the photos with Phe, there were a significant amount of TUNNEL- positive dark blue-brown spots. Even in the presence of the SAL blocker the Phe still induced apoptosis (3). The impact of Phe accumulation is directly linked to cell apoptosis. Therefore, to prevent severe cases of PKU it is essential to detect the disorder as quickly. PKU patients undergo life-long treatment regulation;; therefore, it is essential to provide a low invasive technique. An experimental design was developed to test whether the detection of Phe and Tyr concentration levels may be possible. Blood samples of patients with PKU were analyzed using HPLC. To maintain quality control researchers ran healthy blood samples in the HPLC in order to construct a calibration curve. Amino acid concentrations were examined from the calibration curve via retention time and absorption spectra. Using the control as guidance, researchers used PKU samples to identify Phe and Tyr by quantification of their retention time on the HPLC and their absorption ratio in comparison to the ratio of authentic compounds in the

4 calibrated solution. Since, PKU patients have an elevated amount of Phe and lower level of Tyr, their absorption ratios were expected to change and show a significant difference between the control and experimental data (4) The concentrations of Phe and Tyr were determined for all PKU patients both in serum and dried blood samples (DBS). The results from both specimens for each specific amino acid were plotted in a graph of serum concentration versus DBS concentration. A linear trendline in both graphs showed a good correlation between the analyte concentrations. By using trendline analysis, the equation of both trendlines showed similar slopes of This value accounts as a correction factor between concentrations in both specimens. Thus, using the calibration curve with known amino acid concentrations allowed to determine exact concentrations of Phe and Tyr in both specimens. Since the PKU cases differ in severity knowing the approximate concentrations of both amino acid levels allow doctors to develop an effective patient specific treatment. (4). This method proves to be invasive because it allows patients to mail in their samples into the laboratory and DBS lasts a long time in the right cool conditions. Currently, maintaining a healthy diet is the most effective treatment for PKU patients. Specifically, maintaining low Phe intake is important. In order to lower and regulate Phe levels scientists are testing whether increasing tetrahyrobiopterin (BH4) intake may provide an effective treatment option. BH4 is recycled using vitamin K, Folate, Niacin, and others essential nutrients;; therefore, in an attempt to increase the levels of BH4 nutrients must be consumed (5). The experimental design included hundreds of samples of patients with PKU. To test whether BH4 provided a significant effect, blood Phe levels were measured by using an amino acid analyzer or tandem-mass spectrometry before

5 treatment. These methods were utilized again to determine the blood phenylalanine levels when challenged with BH4 at 4, 8, and 24 hours after administering the sample with BH4(5). Utilizing statistical analysis, researchers found that patients with severe elevated Phe concentrations between ( ) nmol/l responded with up to 50% decrease of their initial phenylalanine levels. Depending on the severity of Phe concentration, patients with the most severe case of PKU known as classical PKU had a 20% decrease compared to the patients with less severe PKU had a 30% decrease in Phe at the 8 hr mark. At the 24-hr mark, the percentage decrease increased to an additional 5-10% Phe decrease (5). These findings concluded that the role of BH4 provided a way with decreasing Phe concentrations. Thus, developing a treatment with increasing BH4 in PKU patients can aid with Phe regulation. Overall, research studies to fully understand PKU are well under way. Developing methods to effectively detect and regulate the disorder aids in lowering the rate of its negative side effects. The published research is a stepping-stone to further comprehend and sets up other hypothesis for further studies. It is known that the elevated levels of Phe is toxic and causes significant damage to neuron cells. For instance, the increasing the levels of Phe trigger the apoptotic pathway which kills the neuron cells and rendering them not functional. Therefore, it is prominent to provide effective treatment option in terms of regulating the amino acid levels and increasing BH4 recycling to aid with the conversion of Phe to Tyr. References

6 1. Kenneth M. Roberts, Jorge Alex Pavon, and Paul F. Fitzpatrick (2013). Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single-Turnover Experiments Biochemistry (6), Zhang, Y., Gu, X., & Yuan, X. (2007). Phenylalanine activates the mitochondria-mediated apoptosis through the RhoA/Rho-associated kinase pathway in cortical neurons. European Journal Of Neuroscience, 25(5), doi: /j x 3. Huang, X., Lu, Z., Lv, Z., Yu, T., Yang, P., Shen, Y., &... Yu, Y. (2013). The Fas/Fas Ligand Death Receptor Pathway Contributes to Phenylalanine-Induced Apoptosis in Cortical Neurons. Plos ONE, 8(8), 1-7. doi: /journal.pone Pecce, R., Scolamiero, E., Ingenito, L., Parenti, G., & Ruoppolo, M. (2013). Optimization of an HPLC method for phenylalanine and tyrosine quantization in dried blood spot. Clinical Biochemistry, 46(18), doi: /j.clinbiochem Fiege, B & Blau, N. (2007). Assessment of tetrahydrobiopterin (BH4) Responsiveness in Phenylketonuria. The journal of pediatrics, 150(6),