Hepatic Phenylalanine Hydroxylase Activity Research Articles

The Pah-R261Q mouse reveals oxidative stress associated with amyloid-like hepatic aggregation of mutant phenylalanine hydroxylase

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.

Prise en charge nutritionnelle du jeune patient phénylcétonurique

Phenylketonuria is a hereditary disease induced more often by a defect in hepatic phenylalanine hydroxylase activity, enzyme-converting phenylalanine to tyrosine. This dysfunction leads to a blood phenylalanine accumulation, which induces mentally retard if no treatment. Only a preventive nutritional treatment, with low controled phenylalaline content, allows to avoid the mental after-effects. This paper details the nutritional intervention proposed for young patients with phenylketonuria. The different conversion systems and the available dietary products are presented. The dietary regimen and the clinical, dietetical, psychological and biological survey are described. Difficulties encountered and factors influencing the diet compliance are also approached.

Stimulation of hepatic phenylalanine hydroxylase activity but not Pah-mRNA expression upon oral loading of tetrahydrobiopterin in normal mice

Tetrahydrobiopterin (BH 4) supplementation in patients with BH 4-responsive phenylalanine hydroxylase (PAH) deficiency is an alternative to low-phenylalanine diet. To further investigate hepatic BH 4-responsiveness, oral administration of 50 mg BH 4/kg/day for 5 weeks was performed in wild-type mice. We observed a 2-fold increase in PAH protein by quantitative Western blot analysis and a 1.7-fold increase in enzyme activity, but no change in Pah-mRNA expression by quantitative real-time PCR analysis in treated mice compared to controls. Our findings support the proposed chemical-chaperone effect of BH 4 to protect PAH.

Phenylacetylglycine, a putative biomarker of phospholipidosis: Its origins and relevance to phospholipid accumulation using amiodarone treated rats as a model

Amiodarone was given to male Sprague–Dawley rats at a dose of 150 mg kg−1 day−1 for 7 consecutive days to induce phospholipidosis in the lungs of treated rats. Amiodarone was given alone or concurrently with phenobarbitone. Animals given amiodarone had raised total phospholipid in serum, lung and lymphocytes, and elevated lyso(bis)phosphatidic acid (LBPA) in all tissues. Urinary and plasma phenylacetylglycine (PAG) and hepatic portal:aortal phenylacetate (PA) ratio were increased, whereas hepatic phenylalanine hydroxylase (PAH) activity and plasma phenylalanine:tyrosine ratio were not affected. Phenobarbitone treatment increased hepatic total P450 content and induced 7-pentoxyres-orufin O-dealkylatian (PROD) activity, as expected, but had no effect on any other biochemical parameter. Plasma amiodarone concentration was reduced in rats co-administered both drugs and phospholipid accumulation in target tissues was attenuated compared with rats treated with amiodarone alone. However, phenobarbitone co-administration failed to alter the magnitude of response with regards to urinary PAG excretion and plasma concentration of its precursors after amiodarone treatment. Increased intestinal absorption of PAG precursors probably resulted in the raised urinary PAG after amiodarone treatment. Urinary PAG correlated weakly with serum, lymphocyte and lung phospholipids. However, urinary PAG excretion was similar in rats dosed solely with amiodarone or in combination with phenobarbitone, despite the fact that the degree of phospholipid accumulation was far less in rats given the combined treatment. Nevertheless, urinary PAG was raised only in animals exhibiting abnormal phospholipid accumulation in target tissues and may thus be useful as a surrogate biomarker for phospholipidosis.

Effect of peroxovanadate compound on phenylalanine hydroxylase gene expression.

Vanadium, a trace element in human cells and regarded as an essential nutrient, plays an active role in all tissues. It is known that peroxovanadate-nicotinic acid (POV), a complex compound of vanadium, can decrease hyperglycemia; however, its biochemical mechanism remains unclear. The object of the present study is to explore the hypoglycemia mechanism of POV at gene molecular levels. Rats rendered diabetic with streptozotocin were treated with POV. Total RNA was isolated from rat liver, and phenylalanine hydroxylase (PAH) mRNA abundance was determined by reverse transcriptase-polymerase chain reaction. PAH activity, blood glucose, and lipid levels were measured. Significantly increased hepatic PAH activity and corresponding mRNA with concomitant hyperglycemia and hyperlipemia were found in diabetic rats. These levels returned to normal after POV treatment and accompanied by negative glucosuria, normoglycemia, and normolipemia. The results from the current study indicates one of the mechanisms of POV action is to inhibit PAH gene expression and PAH activity, thus decreasing gluconeogenesis and hyperglycemia. At the same time, POV is able to promote diabetic recovery by lowering hyperlipemia.

Developmental pattern of phenylalanine hydroxylase activity in the chicken

Experiments were conducted to determine the conditions for assay of hepatic phenylalanine hydroxylase (PAH) activity in the chicken and to determine the developmental pattern of PAH activity in liver 25,000 x g supernatant. PAH activity was detected in liver supernatant and (postnuclear) 25,000 x g particulate fraction. Optimum assay conditions differed for the two cell fractions, the most notable difference being a broad pH optimum of 7.7 to 9.2 for the supernatant and 4.7 and 5.6 for the particulate fraction. The PAH activity in the supernatant increased to a maximum as L-phenylalanine concentration in the assay medium increased from 0.02 to 0.5 mM and 1.0 mM. Activity increased in the particulate fraction as the Phe concentration increased to 0.5 mM. Substrate inhibition of PAH activity occurred at Phe concentrations of 3 to 5 mM in the supernatant but not in the particulate fraction. Concentrations of the cofactor, 6(R)-5,6,7,8-tetrahydrobiopterin, ranging from 0.09 to 0.75 mM, resulted in maximal PAH activity. The developmental pattern of PAH in supernatant was determined using a modified assay in which substrate and cofactor concentrations and pH were optimum. The PAH activity in liver supernatant was present at a low level in 11 d chick embryos and increased several fold between Days 15 and 17 to a maximum at Days 17 to 21. Activity declined at hatching to levels that were present in 11 to 15 d embryos and remained at this level in male chicks through 4 wk of age. Mature males had higher PAH activity than mature laying females.

Gene therapy for phenylketonuria.

Classical phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). Limitations of the current dietary treatment for PKU have led to the development of potential treatments based on somatic gene transfer. Three different vector systems have been examined. Vectors derived from a recombinant retrovirus or a DNA/protein complex can efficiently transduce the PAH cDNA into PAH-deficient hepatocytes in vitro, but the application of these vector systems is presently limited by their low transduction efficiency in vivo. In contrast, a vector derived from a recombinant adenovirus can restore 10%-80% of normal hepatic PAH activity into PAH-deficient mice, which completely normalizes serum phenylalanine levels. This treatment is transient and cannot be effectively re-administered due to the presence of neutralizing antibodies directed against the recombinant adenoviral vector. However, these findings suggest that PKU can be completely corrected by somatic gene therapy, and provide some direction for the future development of adenoviral vectors.

Gene therapy for phenylketonuria

Classical phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). Three different vector systems have been developed to examine the potential of somatic gene therapy for the treatment of PKU. Recombinant retroviral vectors and DNA/protein complexes can efficiently transduce PAH-deficient hepatocytes in vitro, but their present application is limited by their low transduction efficiency in vivo. In contrast, infusion of a recombinant adenoviral vector expressing the human PAH cDNA into the portal circulation of PAH-deficient mice restores 10-80% of normal hepatic PAH activity and completely normalizes serum phenylalanine levels. At present, this effect is transient and re-administration has no further effect. However, this result suggests that PKU can be completely corrected by somatic gene therapy as more persistent vectors are developed.

The PKU mouse project: its history, potential and implications

To produce genetic-based animal models for the human disease phenylketonuria (PKU), we treated mice with the powerful germline mutagen ethylnitrosourea and screened the progeny of these animals for the symptom hyperphenylalaninemia (HPH). Six independent mutant strains have been produced to date that exhibit heritable HPH. The first mutation isolated was found to cause a reduced level of GTP-cyclohydrolase I activity and, as such, yields a model for tetrahydrobiopterin-dependent HPH. The next two mutations have yet to be fully characterized but cause syndromes that appear distinct from any PKU or HPH syndromes yet reported for humans and they are allelic. Next we isolated a mutation that caused a marked reduction in hepatic phenylalanine hydroxylase activity levels. The enzyme deficiency was not sufficient to cause a PKU syndrome but instead produced a mild HPH syndrome. This strain played an instrumental role, however, in the identification of two additional mutant strains that appear to model human PKU very accurately in the laboratory mouse. These latter strains have levels of HPH very similar to human PKU patients, exhibit a phenylalanine-dependent hypopigmentation, and have reproductive difficulties that resemble human maternal PKU.

Relation between genotype and phenotype in Swedish phenylketonuria and hyperphenylalaninemia patients.

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are caused mostly by an inherited (autosomal recessive) deficiency in hepatic phenylalanine hydroxylase (PAH) activity. More than 50 PAH mutations have ben reported. The goal of the present study was to examine the molecular basis for the clinical heterogeneity of Swedish PKU and HPA patients. Mutations were identified through allele-specific oligonucleotide hybridization or DNA sequencing on 128 of the 176 mutant alleles (73%). Three mutations (R408W, Y414C and IVS12) together accounted for 56% of all mutant alleles and ten relatively infrequent mutations were found on another 17% of all mutant alleles. Patients from 50 of the 88 families (57%) had identified mutations in both PAH genes and allowed use to compare the clinical effects of different combinations of PAH mutations. The in vitro activity of all of these mutations, including the newly identified G272X and delta L364, have been tested in a eukaryotic expression system. There was a strong relationship between the average in vitro PAH activity of the two mutant enzymes and both the phenylalanine tolerance and the neonatal pretreatment serum phenylalanine concentration. This confirms previous observations in Danish and German PKU patients that disease phenotype is a consequence of the nature of the mutations at the PAH locus and not significantly influenced by other loci. The sample population in the previous study did not, however, include mild HPA patients, and the observed correlation is thus restricted to severe and moderate mutant alleles. Since a comparatively high proportion of the Swedish patients were mildly affected, we have provided additional evidence that this correlation is valid throughout a continuous spectrum of clinical varieties.(ABSTRACT TRUNCATED AT 250 WORDS)

High Protein Diet and Phenylalanine Hydroxylase Activities in Rats

SummaryThe effects of a diet of 85% casein on the activities of the phenylalanine hydroxylases of rat liver and kidney have been compared. Whereas only the tetrahydrobiopterin-dependent activity of rat hepatic phenylalanine hydroxylase is significantly stimulated, both the tetrahydrobiopterin-dependent and the dimethyltetrahydropterin- dependent activities of the renal enzyme are significantly decreased, after five days of feeding a casein diet. The animals fed a high protein diet for seven days have an increased rate of phenylalanine catabolism in vivo, which is also reflected in increased flux of label from phenylalanine into glucose. The regulation of phenylalanine metabolism, under these conditions, is discussed.

In vivo tyrosine hydroxylation rate in retina: effects of phenylalanine and tyrosine administration in rats pretreated with p-chlorophenylalanine

p-Chlorophenylalanine was administered to rats to inhibit hepatic phenylalanine hydroxylase activity. Two days later, phenylalanine injection was noted to produce substantial increases in serum phenylalanine levels, and relatively modest increments in serum tyrosine levels. Rats injected with p-chlorophenylalanine 2 days earlier showed a normal light-induced activation of retinal tyrosine hydroxylase activity in vivo, measured as dihydroxyphenylalanine accumulation following pharmacologic inhibition in vivo of aromatic L-amino acid decarboxylase activity. In addition, tyrosine injection into p-chlorophenylalanine-treated rats in the light produced anticipated increments in retinal tyrosine hydroxylation rate, showing the enzyme to be functionally normal. The acute administration of phenylalanine (62.5–500mg/kg i.p.) to p-chlorophenylalanine-treated rats produced dose-related increments in retinal phenylalanine. In vivo tyrosine hydroxylation rate in retina was normal at all doses below 300mg/kg. However, at the highest dose (500 mg/kg), when retinal phenylalanine levels were almost 5-times normal, tyrosine hydroxylation rate consistently fell (to about half-normal values). These results demonstrate that very large elevations in tissue phenylalanine levels do not stimulate tyrosine hydroxylation in vivo, and that at extremely high levels phenylalanine inhibits tyrosine hydroxylation rate.

Regulation of the activity of hepatic phenylalanine hydroxylase

Rat liver phenylalanine hydroxylase catalyzes the tetrahydropterin-dependent oxidation of phenylalanine to tyrosine, according to equation 1. In addition to the naturally-occurring coenzyme, tetrahydrobiopterin (BH 4), certain synthetic analogs of BH 4 such as 6-methyltetrahydropterin (6MPH 4) have high cofactor activity. l-phenylalanine + tetrahydropterin + O 2 → l-tyrosine + quinonoid-dihydropterin + H 2O The hydroxylase can be activated by a variety of reversible and irreversible modifications, including those caused by partial proteolysis, by interaction with phospholipids such as lysolecithin, by alkylation of a single sulfhydryl group, by phosphorylation catalyzed by cAMP-dependent protein kinase, and by preincubation with its substrate, phenylalanine. All of these modes of activation greatly increase the hydroxylase activity in the presence of BH 4, whereas the activity in the presence of 6MPH 4 is increased only slightly. The ratio of hydroxylase activity in the presence of BH 4 compared to the activity in the presence of 6MPH 4, therefore, is a useful index of the state of activation of the enzyme. Of the various activation mechanisms listed above, only phosphorylation of the enzyme and phenylalanine-activation appear to operate in vivo. The evidence indicates that these two regulatory mechanisms act synergistically. Thus, phosphorylation of the enzyme by cAMP-dependent protein kinase is stimulated by phenylalanine, especially in the presence of BH 4, (which by itself inhibits), whereas phosphorylation sensitizes the enzyme to activation by phenylalanine. One of the consequences of these interlocking control mechanisms is to enhance the responsiveness of the activity of the hydroxylase to alterations in tissue levels of phenylalanine. As a result, elevated concentrations of phenylalanine can be rapidly metabolized, thereby protecting the fetal and neonatal brain from possible damage by excess phenylalanine.

The effects of hyperphenylalaninemia on fetal development: a new animal model of maternal phenylketonuria.

A new model has been developed for the study of maternal phenylketonuria. Beginning on the 12th day of gestation the diet of pregnant rats was supplemented with 0.5% alpha-methylphenylalanine and 3% phenylalanine. This resulted in an 83% reduction of hepatic phenylalanine hydroxylase activity. The maternal plasma phenylalanine was elevated 10-20-fold for two-thirds of the day, but the degree and persistence of the fetal hyperphenylalaninemia may have been even greater. The brain phenylalanine concentrations in the fetus were raised up to 2900 nmole/g brain, whereas the highest level observed in the dam was 382 nmole/g. Experimentally-treated fetuses showed small reductions in both body and brain weight when compared to age-matched controls; however, no differences were seen in crown to rump length, litter size, DNA and protein concentrations per g, or in postnatal survival. Initiation of the diet at conception rather than on the 12th day caused a significantly greater inhibition of fetal growth, and 21% mortality. The fetal cerebral concentrations of methionine and the branched chain amino acids (valine, leucine and isoleucine) were decreased by hyperphenylalaninemia. From the 16th day on, the concentration of the inhibitor neurotransmitter glycine was elevated. Cerebral serotonin showed a 20-30% deficit and its primary metabolite 5-hydroxyindoleacetic acid a 71-77% deficit. Of twelve enzymes quantified in the brains of hyperphenylalaninemic fetuses only phosphoserine phosphatase showed any change. From the 20th to the 22nd day of gestation its activity was 46-67% higher in experimental than in normal fetuses. Measurement on the 22nd day of gestation showed that the increases in phosphoserine phosphatase activity and glycine content were present in brain stem, cerebellum, and forebrain.

Hepatic phenylalanine hydroxylase activity in hyperphenylalaninaemia.

Phenylalanine tolerance in vivo has been compared with the phenylalanine hydroxylase activity of liver biopsy specimens in 14 newly detected cases of hyperphenylalaninaemia. Hepatic phenylalanine hydroxylase activity does not predict the degree of dietary restriction that will be required.

Effect of alpha-methylphenylalanine and phenylalanine on brain polyribosomes and protein synthesis.

A chronic hyperphenylalanemia was effectively produced in developing mice by daily administrations of phenylalanine (2 mg/g body wt) and a phenylalanine hydroxylase inhibitor alpha-methyl-D,L-phenylalanine (0.43 mg/g body wt). The presence of alpha-methylphenylalanine in newborn mice inhibited 65-70% of hepatic phenylalanine hydroxylase activity within 12 h. Since this maximum inhibition persisted for 24 h or longer, decreased enzyme activity was maintained by daily administrations. Whereas concentrations of phenylalanine increased approximately 40-fold in both plasma and brain following injection of alpha-methylphenylalanine and phenylalanine, plasma levels of tyrosine were not altered significantly. Concomitant with changes in phenylalanine concentrations we observed the brain polyribosomes' disaggregation, which reached a maximum 3 h after injection and persisted as long as 18 h. Polyribosomes did not become refractory to as many as 10 daily injections of alpha-methylphenylalanine and phenylalanine. In addition to polyribosome disaggregation, chronic hyperphenylalanemia reduced the rates of polypeptide chain elongation on polyribosomes isolated from brain homogenates.

The mechanism of the irreversible inhibition ofrat liver phenylalanine hydroxylase due to treatment with p-chlorophenylalanine. The lack of effect on turnover of phenylalanine hydroxylase.

The administration of a single dose of p-chlorophenylalanine (360 mg/kg) to rats leads to the irreversible loss of 90% of hepatic phenylalanine hydroxylase activity after 24 h. This loss of activity is not the result of either an alteration in the overall structure of the enzyme, as determined by its antigenicity, or in the total immunologically reactive protein in the liver, as tested with a specific antiserum prepared against native phenylalanine hydroxylase. Neither the rate of synthesis nor the rate of degradation of phenylalanine hydroxylase is changed by p-chlorophenylalanine (pClPhe) treatment. The half-life for the enzyme is about 2 days in control and in pClPhe-treated rats. In addition, there is no detectable incorporation of pClPhe into the phenylalanine hydroxylase molecule itself.

Lowering Brain Phenylalanine Levels by Giving Other Large Neutral Amino Acids

Hepatic phenylalanine hydroxylase activity was inhibitied in F344 rat pups by injection with parachlorophenylalanine on days 7, 8, and 9 after birth. On day 10, animals were divided into control and experimental groups. Controls received an injection of phenylalanine, with the characteristic phenylketonuria (PKU) changes of elevated phenylalanine but normal tyrosine levels. The experimental group received a similar injection of phenylalanine but in addition received either a single amino acid or a combination of amino acids from the large neutral amino acids (LNAA) group. The LNAA group of amino acids--phenylalanine, tyrosine, tryptophan, leucine, isoleucine, and valine--compete with each other for entry into brain by a common transport mechanism. Compared with controls, the experimental group had lower phenylalanine levels in brain, but no change in serum levels, giving a lower brain-serum phenylalanine ratio. Increasing the serum concentrations of amino acids competitive with phenylalanine for transport across the blood brain barrier might form an alternative approach to effective dietary treatment of PKU.

The regulation of phenylalanine hydroxylase in rat tissues in vivo. Substrate- and cortisol-induced elevations in phenylalanine hydroxylase activity.

Injections of phenylalanine increased a 2.5-fold in 9 h the hepatic phenylalanine hydroxylase activity of 6-day-old or adult rats that had been pretreated (24h earlier) with p-chlorophenylalanine; without such pretreatment, phenylalanine did not raise the enzyme concentration. This difference is paralleled by the much greater extent to which the injected phenylalanine accumulated in livers of the pretreated compared with the normal animals. The hormonal induction of hepatic phenylalanine hydroxylase activity obeyed different rules: an injection of cortisol was without effect on adult livers but caused a threefold rise in phenylalanine hydroxylase activity of immature ones, both without and after pretreatment with p-chlorophenylalanine. In the latter instance, the effects of cortisol, and of phenylalanine were additive. Actinomycin inhibited the cortisol- but not the substrate-induced increase of phenylalanine hydroxylase, whereas puromycin inhibited both. The results indicate that substrate and hormone, two potential positive regulators of the amount of the hepatic (but not the renal) phenylalanine hydroxylase, act independently by two different mechanisms. The negative effector, p-chlorophenylalanine, also appears to interact with the synthetic (or degradative) machinery rather than with the existing phenylalanine hydroxylase molecules: 24h were required in vivo for an 85% decrease to ensue, and no inhibition occurred in vitro when incubating the enzyme with p-chlorophenylalanine or with liver extracts from p-chlorophenylalanine-treated rats.

Distribution and metabolism of phenylalanine and tyrosine during tualraemia in the rat.

Tularaemia in rats causes a doubling of the serum phenylalanine/tyrosine ratio and a more than twofold increase in muscle phenylalanine which cannot be accounted for by decreased hepatic phenylalanine hydroxylase (EC 1.14.16.1) activity, but appear to result from a generalized movement of amino acids from muscle to liver during infection.