Molecular genetics of PKU in Poland and potential impact of mutations on BH4 responsiveness.

Tetrahydrobiopterin (BH4) has been recently approved as a treatment of patients with phenylketonuria. However, as a confirmation of BH4-responsiveness, it might require a very expensive trial treatment with BH4 or prolonged BH4-loading procedures. The selection of patients eligible for BH4-therapy by means of genotyping of the PAH gene mutations may be recommended as a complementary approach. A population-wide genotyping study was carried out in 1286 Polish phenyloketonuria-patients. The aim was to estimate the BH4 demand and to cover prospectively the treatment by a National Health Fund. A total of 95 types of mutations were identified. Genetic variants corresponding with probable BH4-responsiveness were found in 28.2% of cases. However, patients with mild or classical phenylketonuria who require continuous treatment accounted for 11.4% of the studied population only. Analysis of the published data shows similar percentage of the "BH4-responsive" variants of a PAH gene in patients from other countries of Eastern Europe. Therefore, it can be concluded, that the proportion of phenylketonuria-patients who could benefit from the use of BH4 reaches approximately 10% in the entire region.

Main issues in micronutrient supplementation in phenylketonuria.

For almost all patients with PKU, a low phenylalanine diet is the basis of the treatment despite a widely varying natural protein tolerance. A vitamin and mineral supplement is essential and it is commonly added to a phenylalanine-free (phe-free) source of L-amino acids. In PKU, many phe-free L-amino acid supplements have age-specific vitamin and mineral profiles to meet individual requirements. The main micronutrient sources are chemically derived and their delivery dosage is usually advised in three or more doses throughout the day. Within the EU, the composition of VM (vitamin and mineral) phe-free L-amino acid supplements is governed by the Foods for Special Medical Purposes (FSMP) directive (European Commission Directive number 1999/21/EC and amended by Directive 2006/141/EC). However the micronutrient composition of the majority fails to remain within FSMP micronutrient maximum limits per 100 kcal due to their low energy content and so compositional exceptions to the FSMP directive have to be granted for each supplement. All patients with PKU require an annual nutritional follow-up, until it has been proven that they are not at risk of any vitamin and mineral imbalances. When non-dietary treatments are used to either replace or act as an adjunct to diet therapy, the quality of micronutrient intake should still be considered important and monitored systematically. European guidelines are required about which micronutrients should be measured and the conditions (fasting status) for monitoring.

Dopamine precursor depletion impairs timing in healthy volunteers by attenuating activity in putamen and supplementary motor area.

Neuropsychological investigations of patients with Parkinson's disease, schizophrenia, or attention deficit disorder converge with psychopharmacological studies in animals and healthy volunteers to implicate dopamine (DA) pathways in timing. In parallel, single-cell recording and functional neuroimaging studies have highlighted the importance of basal ganglia, prefrontal cortex, and supplementary motor area (SMA) for timing. In a placebo-controlled, within-subject design, we combined event-related functional magnetic resonance imaging with a DA manipulation (acute phenylalanine/tyrosine depletion; APTD) in healthy volunteers to pinpoint the neuroanatomical and functional substrates of the DA modulation of timing. Behaviorally, APTD selectively impaired accuracy of perceptual timing, with no effect on performance of a color-control task matched for difficulty, working memory (WM), and attentional demands. Neurally, APTD attenuated timing-specific activity in the putamen and SMA. Notably, APTD-induced decreases in brain activity were directly correlated to APTD-induced impairments in timing performance. Moreover, APTD modulated timing-specific activity selectively during initial storage of the sample duration, but had no effect during its subsequent retrieval or comparison to a probe. Our results do not simply reflect DA modulation of WM since the color task controlled for the WM updating process necessary for timing of durations in the seconds range. Moreover, preliminary evidence indicated APTD effects on putamen and SMA were greater for subsecond (540 ms) than suprasecond (1080 ms) durations, when WM demands would actually be lower. Instead, we show for the first time in healthy humans that DA manipulation perturbs timing by attenuating the activity in putamen and SMA that mediates initial storage of temporal information into WM.

Effects of selective and combined serotonin and dopamine depletion on the loudness dependence of the auditory evoked potential (LDAEP) in humans.

BACKGROUND: The loudness dependence of the auditory evoked potential (LDAEP) has been suggested as a possible in vivo measure of central serotonin function. However, more recent studies suggest that the LDAEP may be modulated by multiple neuromodulatory systems in addition to the serotonergic system. Accordingly we further examined the effects of selective serotonin, dopamine and simultaneous serotonin and dopamine depletion on the LDAEP in healthy subjects. METHODS: The study employed a placebo-controlled, double-blind, cross over design. Fourteen subjects were tested under four acute treatment conditions: placebo (balanced amino acid drink), tryptophan (serotonin) depletion (ATD), tyrosine/phenylalanine (dopamine) depletion (ATPD) and combined tryptophan/tyrosine/phenylalanine (serotonin and dopamine) depletion (CMD). Testing was conducted 5.5 h post-depletion and changes in the amplitude of the N1/P2 at varying intensities (60, 70, 80, 90, 100 dB) were examined at C(Z). RESULTS: Greater than 80% plasma precursor depletion was achieved across all conditions. Despite significant depletion of monoamine precursors, ATD, (p = 0.318), ATPD (p = 0.061) and CMD (p = 0.104) had no effects on the LDAEP (60-100 dB). CONCLUSION: Acute serotonin and dopamine depletion did not modulate the LDAEP. This finding adds support to growing evidence that the LDAEP is insensitive to acute changes in serotonin and dopamine neurotransmission.

Parenteral glycyl-L-tyrosine maintains tyrosine pools and supports growth and nitrogen balance in phenylalanine-deficient rats.

Poor solubility hampers the addition of sufficient amounts of free tyrosine to parenteral amino solutions. We investigated the use of a highly soluble synthetic dipeptide, glycyl-L-tyrosine, as a parenteral tyrosine source in 18 male Wistar rats (body weight 180-200 g). The animals were randomized into three equal groups and catheterized to facilitate isoenergetic (1.2 MJ.kg-1.d-1) and isonitrogenous (1.25 g nitrogen.kg-1.d-1) total parenteral nutrition for 7 d. Controls (Group 1) received a complete amino acid solution, Group 2 received the same solution deficient in phenylalanine (nitrogen replaced with glycine), and group 3 received the phenylalanine-deficient solution supplemented with glycyl-L-tyrosine. Between d 4 and 7, weight gain and nitrogen retention were lower in Group 2 and in Group 1 or 3. In plasma and organ samples obtained at the end of the study, amino acids and dipeptides were analyzed by means of reversed phase-HPLC. In Group 2, phenylalanine and tyrosine concentrations were lower than in controls in plasma, muscle and kidney; in liver, only the tyrosine concentration was lower compared with controls. With glycyl-L-tyrosine supplementation, plasma, liver and kidney tyrosine concentrations and the phenylalanine:tyrosine ratio were normal. Intact glycyl-L-tyrosine was not detectable, suggesting a virtually quantitative elimination or utilization of the infused dipeptide. The results indicate that in phenylalanine-deficient rats, parenteral glycyl-L-tyrosine rapidly provides free tyrosine to facilitate normal growth, promote nitrogen metabolism and maintain intra- and extracellular tyrosine pools.

Modulation of B16-BL6 murine melanoma metastatic phenotype by tyrosine and phenylalanine restriction in the absence of host selection pressures.

We previously showed that restriction of tyrosine (Tyr) and phenylalanine (Phe) in vivo dramatically suppresses the metastatic phenotype of B16-BL6 (BL6) murine melanoma. Present results indicate a direct effect of Tyr and Phe restriction on the tumor in the absence of host selection pressures. Lung colonizing ability of BL6 is dramatically suppressed after one passage in vitro in media containing low levels of Tyr and Phe. This antimetastatic effect is immediate, stable for at least 5 in vitro passages in Tyr and Phe restricted media, and evident event after levels of Tyr and Phe are restored to normal. Heterogeneity for lung colonizing ability is suppressed, as evidence by fewer tumor colonies formed by clones following i.v. inoculation into mice fed normal diet. This suppression of BL6 metastatic phenotype is not due to differential clearance and retention in the lung or to decreased growth, but is specific for these two amino acids. As the mechanism(s) for the antitumor effects of Tyr and Phe restriction are detailed, the relevance of Tyr and Phe restriction as an early adjuvant to effective cancer treatment can be explored.

Primary hypothyroidism in an adult patient with protein-calorie malnutrition: a study of its mechanism and the effect of amino acid deficiency.

A man with diabetes mellitus, chronic hepatitis, chronic pancreatitis, and blind loop syndrome but without any previous thyroid disease developed three episodes of transient primary hypothyroidism associated with protein-calorie malnutrition (PCM). Clinical examinations suggested that this primary hypothyroidism was not caused by chronic thyroiditis, iodine deficiency, or iodine excess. Since the three times association of primary hypothyroidism with PCM suggested the possibility that the primary hypothyroidism was caused by PCM, we have tried to clarify its mechanism. For this purpose we have investigated the change of thyroid functions during protein-calorie repletion and the effect of amino acid deficiency. Total parenteral nutrition with full supplementation of amino acids resulted in a rapid increase in serum thyroxine (T4), triiodothyronine (T3), free T4, and reverse T3, and subsequently, a rapid decrease in TSH in several days after the nutrition was begun. When amino acid solution was changed to that depleted of phenylalanine and tyrosine after the restoration of thyroid functions, serum T4 and T3 showed a gradual decrease, but serum free T4 and TSH remained within normal range. However, resupplementation of phenylalanine and tyrosine after 8 weeks of depletion gave a rapid increase in serum T4, T3, free T4, and reverse T3. These results suggested that the primary hypothyroidism was caused by an impaired T4 production and that the deficiency of amino acids in PCM partly contributed to the impairment of T4 production.

Selective killing of transformed cells by methotrexate with histidine deprivation or with alpha-amino alcohols.

The effects of amino acid deprivation and treatment with amino alcohols upon the growth, viability, and susceptibility to methotrexate (MTX) cytotoxicity were examined in BALB/3T3 cells and their virally transformed counterparts, SV-T2 cells. Cells were deprived of either histidine or tyrosine plus phenylalanine, or they were treated with amino alcohol analogues of histidine and tyrosine (histidinol and tyrosinol). When incubated in medium lacking histidine and supplemented with dialyzed serum (histidine-deficient medium), the BALB/3T3 cells remained viable for at least 3 days, but they ceased logarithmic growth, and the cell number reached an early plateau. In contrast, the SV-T2 cells continued to divide in histidine-deficient medium. Neither cell line ceased division in medium deficient in both phenylalanine and tyrosine. Incubation of the BALB/3T3 cells with 1.5 mM histidinol or 1.0 mM tyrosinol caused an early plateau similar to the effect of histidine deprivation. SV-T2 cells were not affected by these concentrations of histidinol or tyrosinol, but growth was arrested at higher concentrations. Any of the conditions used which caused a plateau in the number of BALB/3T3 cells substantially protected the treated cells from cell death caused by MTX. Therefore, pretreatment of BALB/3T3 cells with histidine deprivation, 1.5 mM histidinol, or 1.0 mM tyrosinol protected this cell line against MTX-induced cell death, while the same pretreatment conditions failed to protect SV-T2 cells. (SV-T2 cells were protected by 4.0 mM histidinol.) Thus, the ability of MTX to kill cells in vitro can be selectively modified by conditions which protect cells which retain normal growth control characteristics, but which do not protect virally transformed cells.

Phenylalanine-tyrosine deficiency syndrome as a complication of the management of hereditary tyrosinemia.

A male infant with type I hereditary tyrosinemia developed a phenylalanine-tyrosine deficiency syndrome after receiving a synthetic diet which was low in these amino acids. The syndrome was characterized by growth failure, anorexia, lethargy, and hypotonia. Hypophenylalaninemia and hypotyrosinemia were discovered. The blood concentration of most other amino acids were increased. Supplementation of the patient's diet with phenylalanine and tyrosine resulted in a prompt and dramatic reversal of both clinical and biochemical abnormalities. Dietary therapy had no effect on the child's hepatic cirrhosis.

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16. II. The isolation and characterization of mutants auxotrophic for phenylalanine and tyrosine.

1. Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H 16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the "pin-point" isolation method. Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase. Both activities were regained by reversion to prototrophy. The auxotrophic strains accumulated chorismic acid. (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion. Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold. Mutants grown in minimal medium excreted prephenic acid. (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate. The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present. Some of them were even more active than in the wild-type. 2. Mutants accumulating chorismic acid or prepheric acid were able to grow on minimal medium when incubated long enough. The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth. 3. A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A.eutrophus H 16. Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 21 of medium.