Hydroxylation Of Phenylalanine Research Articles

Multiplex Snapshot minisequencing for detection of common PAH gene mutations in Iranian patients with Phenylketonuria.

Phenylketonuria (PKU) is one of the most common inborn faults of amino acid metabolism in the world. It is caused by an autosomal recessive insufficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) that catalysis the irreversible hydroxylation of phenylalanine to tyrosine. More than 950 different disease-causing mutations in the PAH gene have been identified. The most prominent complication of PKU if not diagnosed and treated is severe mental retardation. Early diagnosis and initiation of nutritional therapy are the most important measures in preventing this mental retardation. For this reason, we developed a simple and rapid molecular test for detection of the most frequent phenylalanine hydroxylase mutations. We have developed a PCR multiplex SNaPshot reaction that targets all 10 common mutations identified in PAH gene. Multiplex assays based on the SNaPshot minisequencing approach were developed to simultaneous genotype ten mutations at the PAH gene. We have optimized the detection of this mutations in one multiplex polymerase chain reaction (PCR) followed by ten single-nucleotide extension reactions. The different genotypes were determined based on the position and the fluorescent color of the peaks in a single electropherogram. DNA sequencing assay also were used to verify genotyping results obtained by SNaPshot minisequencing, all mutations were concordant. This method can be useful as secondary test in neonatal screening for hyperphenylalaninemia in neonates with a positive screening test and also be suitable for carrier screening. The assay can be easily applied for accurate, time and cost efficient genotyping of the selected SNPs in various populations.

Circulating Metabolites in Relation to the Kidney Allograft Function in Posttransplant Patients.

End-stage kidney disease is preferably treated by kidney transplantation. The suboptimal function of the allograft often results in misbalances in kidney-controlled processes and requires long-term monitoring of allograft function and viability. As the kidneys are organs with a very high metabolomic rate, a metabolomics approach is suitable to describe systematic changes in post-transplant patients and has great potential for monitoring allograft function, which has not been described yet. In this study, we used blood plasma samples from 55 patients after primary kidney transplantation identically treated with immunosuppressants with follow-up 50 months in the mean after surgery and evaluated relative levels of basal plasma metabolites detectable by NMR spectroscopy. We were looking for the correlations between circulating metabolites levels and allograft performance and allograft rejection features. Our results imply a quantitative relationship between restricted renal function, insufficient hydroxylation of phenylalanine to tyrosine, lowered renal glutamine utilization, shifted nitrogen balance, and other alterations that are not related exclusively to the metabolism of the kidney. No link between allograft function and energy metabolism can be concluded, as no changes were found for glucose, glycolytic intermediates, and 3-hydroxybutyrate as a ketone body representative. The observed changes are to be seen as a superposition of changes in the comprehensive inter-organ metabolic exchange, when the restricted function of one organ may induce compensatory effects or cause secondary alterations. Particular differences in plasma metabolite levels in patients with acute cellular and antibody-mediated allograft rejection were considered rather to be related to the loss of kidney function than to the molecular mechanism of graft rejection since they largely follow the alterations observed by restricted allograft function. In the end, we showed using a simple mathematical model, multilinear regression, that the basal plasmatic metabolites correlated with allograft function expressed by the level of glomerular filtration rate (with creatinine: p-value = 4.0 × 10−26 and r = 0.94, without creatinine: p-value = 3.2 × 10−22 and r = 0.91) make the noninvasive estimation of the allograft function feasible.

Structural studies of a novel auxiliary-domain-containing phenylalanine hydroxylase from Bacillus cereus ATCC 14579.

Phenylalanine hydroxylase (PAH), which belongs to the aromatic amino-acid hydroxylase family, is involved in protein synthesis and pyomelanine production through the hydroxylation of phenylalanine to tyrosine. In this study, the crystal structure of PAH from Bacillus cereus ATCC 14579 (BcPAH) with an additional 280 amino acids in the C-terminal region was determined. The structure of BcPAH consists of three distinct domains: a core domain with two additional inserted α-helices and two novel auxiliary domains: BcPAH-AD1 and BcPAH-AD2. Structural homologues of BcPAH-AD1 and BcPAH-AD2 are known to be involved in mRNA regulation and protein-protein interactions, and thus it was speculated that BcPAH might utilize the auxiliary domains for interaction with its partner proteins. Furthermore, phylogenetic tree analysis revealed that the three-domain PAHs, including BcPAH, are completely distinctive from both conventional prokaryotic PAHs and eukaryotic PAHs. Finally, biochemical studies of BcPAH showed that BcPAH-AD1 might be important for the structural integrity of the enzyme and that BcPAH-AD2 is related to enzyme stability and/or activity. Investigations into the intracellular functions of the two auxiliary domains and the relationship between these functions and the activity of PAH are required.

A low postabsorptive whole body protein balance is associated with markers of poor daily physical functioning in Chronic Obstructive Pulmonary Disease

Postabsorptive whole body protein kinetics are related to age, gender, body mass index (BMI), and habitual protein intake level. It is unclear how protein synthesis, breakdown, and postabsorptive protein balance rates are affected in Chronic Obstructive Pulmonary Disease (COPD)) and whether these relate to disease severity, lifestyle characteristics and poor daily functioning. We studied 91 COPD (GOLD 1-4) and 56 age matched control subjects without COPD or other chronic or acute health disease/condition in the postabsorptive state and measured body composition by Dual-energy X-ray Absorptiometry, and disease severity and comorbidities by medical screening, blood analysis and questionnaires. We assessed whole body production rates of phenylalanine and tyrosine by pulse stable isotope tracer infusion to calculate whole body protein breakdown (PB) and hydroxylation of phenylalanine to tyrosine, representative of postabsorptive protein balance. We measured muscle and cognitive function, and physical performance by isokinetic dynamometry, cognitive assessments, and 6-min walk test. We assessed physical activity level, mood and dietary protein intake by questionnaires. We measured plasma enrichments by LC-MS/MS and statistics by Fisher's exact test or analysis of covariance. Data are mean [95% CI]. The COPD patients had moderate to severe airflow obstruction, multiple comorbidities, and elevated values for plasma high sensitivity c-reactive protein (hs-CRP) and glucose. Although PB (3630 [3361, 3900] vs 3504 [3297, 3711] umol/h, p=0.1649) was not different, postabsorptive protein balance was lower in COPD patients (274.2 [242.4, 306.1] vs 212.9 [194.7, 231.0] umol/h, p<0.0001), both compared to control subjects. A lower postabsorptive protein balance was associated with age (p<0.0001) and higher levels for systolic blood pressure (p=0.0051) and hs-CRP (p=0.0046) but not with lung function. Furthermore, a lower postabsorptive protein balance level was associated with a lower intake of total calories and protein (p<0.0001) and lower muscle strength (p=0.0248), while only in COPD with a lower physical performance (p=0.0343). We found no association with cognitive function or mood. For all subjects, a cumulative model that included group, gender, age, BMI, systolic blood pressure, hs-CRP, caloric intake, protein intake, and leg strength was able to explain 55% of the variation in postabsorptive protein balance. These data suggest that systemic inflammation, high blood pressure and low protein intake are risk factors of a lower postabsorptive protein balance in COPD patients. A lower postabsorptive protein balance is associated with markers of poor daily physical functioning.

Phenylalanine hydroxylase (PAH) plays a positive role during WSSV and Vibrio parahaemolyticus infection in Litopenaeus vannamei

Phenylalanine hydroxylase (PAH) is involved in immune defence reactions by providing the starting material, tyrosine, to synthesise catecholamines and melanin. PAH is an important metabolic enzyme of aromatic amino acids and the rate-limiting enzyme in the hydroxylation of amino acid phenylalanine to tyrosine. In the present study, a PAH gene, LvPAH, was cloned and identified from Litopenaeus vannamei. The open reading frame (ORF) of LvPAH was 1383 bp, encoding a protein of 460 amino acids comprised of an ACT domain and a Biopterin_H domain. LvPAH was constitutively expressed in healthy L. vannamei, with the highest expression levels in the eyestalk and the lowest in the hepatopancreas. Both white spot syndrome virus (WSSV) and Vibrio parahaemolyticus infection upregulated LvPAH expression in hemocytes, hepatopancreas and gills of L. vannamei. Inhibition of LvPAH resulted in a significantly lower survival rate of L. vannamei after WSSV infection than the control group, consistent with the observation that WSSV viral load was significantly higher in LvPAH-silenced L. vannamei. After a V. parahaemolyticus challenge, there was no significant difference between the survival rate of LvPAH-silenced and the control L. vannamei. However, the load of V. parahaemolyticus in LvPAH-silenced L. vannamei was significantly higher than the control population for L. vannamei. The effect of LvPAH on L. vannamei from a neuroendocrinological perspective was assessed by measuring l-DOPA, dopamine (DA) and noradrenaline (NE) levels in the hemocytes after the knockdown of LvPAH. The results showed that phenoloxidase (PO), l-DOPA and DA levels in the hemolymph of LvPAH-silenced L. vannamei were significantly decreased starting from 24hpi. In contrast, the NE levels in the hemolymph of shrimp decreased significantly at first and then increased. The results suggest that LvPAH may play an important role in antiviral and bacterial immunity in L. vannamei.

Differences in the sperm metabolomes of smoking and nonsmoking men†.

Currently, spermiogram analysis is the most relevant method used to clarify the potential infertility of a couple. However, in some cases, the reasons for infertility remain obscure. Smoking is among the factors that have been described to adversely affect male fertility. Smoking increases oxidative stress and thus promotes various pathological processes. Comparative studies, particularly those on metabolomic changes in sperm and seminal plasma caused by smoking, have not yet been published. Thus, the present pilot study aimed at the mass spectrometric characterization of the metabolomes of specimens from both smoking and nonsmoking subjects and the comparison of the evaluated data in terms of sperm apoptosis and spermiogram parameters. The results provided evidence that the conventional spermiogram is not altered in smokers compared to nonsmokers. However, a more careful investigation of sperm cells by metabolomic profiling reveals profound effects of smoking on sperm: first, nitrogen oxide synthase, a marker of oxidative stress, is activated. Second, the uptake of fatty acids into sperm mitochondria is reduced, leading to an impaired energy supply. Third, phenylalanine hydroxylation and tryptophan degradation, which are both indications of altered tetrahydrobiopterin biosynthesis, are reduced. Moreover, flow cytometry approaches indicated increased sperm caspase-3 activity, a sign of apoptosis. The present study clearly shows the negative effects of smoking on semen quality. Especially for idiopathic cases, metabolomic profiling can help to shed light on male subfertility or infertility.

The role of phenylalanine hydroxylase in lipogenesis in the oleaginous fungus Mortierella alpina.

Phenylalanine hydroxylase (PAH) catalyses the irreversible hydroxylation of phenylalanine to tyrosine, which is the rate-limiting reaction in phenylalanine metabolism in animals. A variety of polyunsaturated fatty acids can be synthesized by the lipid-producing fungus Mortierella alpina, which has a wide range of industrial applications in the production of arachidonic acid. In this study, RNA interference (RNAi) with the gene PAH was used to explore the role of phenylalanine hydroxylation in lipid biosynthesis in M. alpina. Our results indicated that PAH knockdown decreased the PAH transcript level by approximately 55% and attenuated cellular fatty acid biosynthesis. Furthermore, the level of NADPH, which is a critical reducing agent and the limiting factor in lipogenesis, was decreased in response to PAH RNAi, in addition to the downregulated transcription of other genes involved in NADPH production. Our study indicates that PAH is part of an overall enzymatic and regulatory mechanism supplying NADPH required for lipogenesis in M. alpina.

Contamination profiles and health impact of benzothiazole and its derivatives in PM2.5 in typical Chinese cities

Although benzothiazole and its derivatives (BTHs) are considered emerging contaminants in diverse environments and organisms, little information is available about their contamination profiles and health impact in ambient particles. In this study, an optimized method of ultrasound-assisted extraction coupled with the selected reaction monitoring (SRM) mode of GC-EI-MS/MS was applied to characterize and analyze PM2.5-bound BTHs from three cities of China (Guangzhou, Shanghai, and Taiyuan) during the winter of 2018. The total BTH concentration (ΣBTHs) in PM2.5 samples from the three cities decreased in the order of Guangzhou > Shanghai > Taiyuan, independently of the PM2.5 concentration. Despite the large variation in concentration of ΣBTHs in PM2.5, 2-hydroxybenzothiazole (OTH) was always the predominant compound among the PM2.5-bound BTHs and accounted for 50–80% of total BTHs in the three regions. Results from human exposure assessment and toxicity screening indicated that the outdoor exposure risk of PM2.5-bound BTHs in toddlers was much higher than in adults, especially for OTH. The developmental and reproduction toxicity of OTH was further explored in vivo and in vitro. Exposure of mouse embryonic stem cells (mESCs) to OTH for 48 h significantly increased the intracellular reactive oxygen species (ROS) and induced DNA damage and apoptosis via the functionally activating p53 expression. In addition, the growth and development of zebrafish embryos were found to be severely affected after OTH treatment. An overall metabolomics study was conducted on the exposed zebrafish larvae. The results indicated that exposure to OTH inhibited the phenylalanine hydroxylation reaction, which further increased the accumulation of toxic phenylpyruvate and acetylphenylalanine in zebrafish. These findings provide important insights into the contamination profiles of PM2.5-bound BTHs and emphasize the health risk of OTH.

Indicators of blood biochemical analysis in PKU patients being on amino acid mixtures substitute nutrition

Topicality. Phenylketonuria is a congenital pathology of amino acid metabolism, which is a rare (orphan) disease. Depending on the regions of the world and the ethnic origin of the inhabitants, its prevalence can vary from 1/2600 to 1/200000. Phenylketonuria is caused by a deficiency of liver enzyme phenylalanine hydroxylase, which catalyzes the hydroxylation of phenylalanine, resulting in the formation of tyrosine. Enzyme deficiency leads to hyperphenylalaninemia, and in the absence of adequate therapeutic measures will lead to profound and irreversible disability of the patient.Aim. To conduct a comparative analysis of amino acid mixtures “Nutri-2”, “Nutri-3”, “PAM-2” and “PAM-3”, as well as to determine the biochemical parameters of the blood of four groups of patients on these diets according to their age needs.Materials and methods. On the basis of Kharkiv interregional specialized medical-genetic center of rare (orphan) diseases, treatment of phenylketonuria with the use of amino acid mixtures was introduced and further biochemical analysis of patients’ blood was performed. The results of biochemical parameters of blood of four groups of heterosexual and different-aged patients on different amino acid mixtures were taken for the study. The study involved 16 patients of different sexes aged 4 to 29 years.Results and discussion. The determined values of most biochemical markers in patients with PKU were within the reference values. In some patients taking PAM-2, minor changes were observed, which were reflected in increased urea content and activity of aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatase outside the upper limit of normal.Conclusions. New therapeutic foods have more balanced nutritional composition, developed according to modern dietary recommendations, which is confirmed by stable biochemical parameters of the blood of patients with phenylketonuria.

Role of tetrachloro-1,4-benzoquinone reductase in phenylalanine hydroxylation system and pentachlorophenol degradation in Bacillus cereus AOA-CPS1

This study reports a ≅12.5 kDa protein tetrachloro-1,4-benzoquinone reductase (CpsD) from Bacillus cereus strain AOA-CPS1 (BcAOA). CpsD is purified to homogeneity with a total yield of 35% and specific activity of 160 U·mg−1 of protein. CpsD showed optimal activity at pH 7.5 and 40 °C. The enzyme was found to be functionally stable between pH 7.0–7.5 and temperature between 30 °C and 35 °C. CpsD activity was enhanced by Fe2+ and inhibited by sodium azide and SDS. CpsD followed Michaelis-Menten kinetic exhibiting an apparent vmax, Km, kcat and kcat/Km values of 0.071 μmol·s−1, 94 μmol, 0.029 s−1 and 3.13 × 10−4 s−1·μmol−1, respectively, for substrate tetrachloro-1,4-benzoquinone. The bioinformatics analysis indicated that CpsD belongs to the PCD/DCoH superfamily, with specific conserved protein domains of pterin-4α-carbinolamine dehydratase (PCD). This study proposed that CpsD catalysed the reduction of tetrachloro-1,4-benzoquinone to tetrachloro-p-hydroquinone and released the products found in phenylalanine hydroxylation system (PheOHS) via a Ping-Pong or atypical ternary mechanism; and regulate expression of phenylalanine 4-monooxygenase by blocking reverse flux in BcAOA PheOHS using a probable Yin-Yang mechanism. The study also concluded that CpsD may play a catalytic and regulatory role in BcAOA PheOHS and pentachlorophenol degradation pathway.

Tetrahydrobiopterin Plays a Functionally Significant Role in Lipogenesis in the Oleaginous Fungus Mortierella alpina.

Tetrahydrobiopterin (BH4) is well-known as a cofactor of phenylalanine hydroxylase (PAH) and nitric oxide synthase (NOS), but its exact role in lipogenesis is unclear. In this study, the GTP cyclohydrolase I (GTPCH) gene was overexpressed to investigate the role of BH4 in lipogenesis in oleaginous fungus Mortierella alpina. Transcriptome data analysis reveal that GTPCH expression was upregulated when nitrogen was exhausted, resulting in lipid accumulation. Significant changes were also found in the fatty acid profile of M. alpina grown on medium that contained a GTPCH inhibitor relative to that of M. alpina grown on medium that lacked the inhibitor. GTPCH overexpression in M. alpina (the MA-GTPCH strain) led to a sevenfold increase in BH4 levels and enhanced cell fatty acid synthesis and poly-unsaturation. Increased levels of nicotinamide adenine dinucleotide phosphate (NADPH) and upregulated expression of NADPH-producing genes in response to enhanced BH4 levels were also observed, which indicate a novel aspect of the NADPH regulatory mechanism. Increased BH4 levels also enhanced phenylalanine hydroxylation and nitric oxide synthesis, and the addition of an NOS or a PAH inhibitor in the MA-GTPCH and control strain cultures decreased fatty acid accumulation, NADPH production, and the transcript levels of NADPH-producing genes. Our research suggests an important role of BH4 in lipogenesis and that the phenylalanine catabolism and arginine–nitric oxide pathways play an integrating role in translating the effects of BH4 on lipogenesis by regulating the cellular NADPH pool. Thus, our findings provide novel insights into the mechanisms of efficient lipid biosynthesis regulation in oleaginous microorganisms and lay a foundation for the genetic engineering of these organisms to optimize their dietary fat yield.

6-Pyruvoyltetrahydropterin Synthase Deficiency: Review and Report of 28 Arab Subjects

BackgroundTetrahydrobiopterin is an essential cofactor for the hydroxylation of aromatic amino acids phenylalanine, tyrosine, and tryptophan. Therefore, tetrahydrobiopterin deficiency results in hyperphenylalaninemia as well as dopamine and serotonin depletion in the central nervous system. The enzyme 6-pyruvoyltetrahydropterin synthase catalyzes the second step of de novo synthesis of tetrahydrobiopterin, and its deficiency is the most frequent cause of tetrahydrobiopterin metabolism disorders. MethodWe conducted a retrospective chart review of 28 subjects from 24 families with molecularly confirmed 6-pyruvoyltetrahydropterin synthase deficiency from six centers in three Arab countries. We reviewed clinical, biochemical, and molecular data. We also reviewed previously published cohorts of subjects with 6-pyruvoyltetrahydropterin synthase deficiency. ResultsSimilar to previous observations, we show that early treatment (less than two months) is associated with better outcome. We identify eight PTS variants in 24 independent families. The most common variant is (c.238A>G; p.M80V) with an allele count of 33%. We also identify one novel variant (c.2T>G; p.?). ConclusionThe deficiency of 6-pyruvoyltetrahydropterin synthase is relatively common in the Arab population and should be considered in individuals with hyperphenylalaninemia. More natural history studies with comprehensive biochemical and molecular genetics data are needed for a robust base for the development of future therapy.

Elucidating the Role of Hydroxylated Phenylalanine in the Formation and Structure of Cross-Linked Aβ Oligomers.

Amyloid β-protein (Aβ) oligomers play a seminal role in Alzheimer's disease (AD). Cross-linking (X-linking), which can be used to determine Aβ oligomer size distributions experimentally, was reported to stabilize Aβ oligomers. Aβ oligomers X-linked in the presence of copper and hydrogen peroxide may represent the proximate neurotoxic species in AD. Our previous computational study demonstrated that X-linking of Aβ40 and Aβ42 oligomers via tyrosines alone cannot explain experimental findings. Here, we explore three plausible X-linking mechanisms, which involve, in addition to tyrosine, also lysine (mechanism 1), histidine (mechanism 2), and hydroxylated phenylalanine (mechanism 3). By examining the effect of X-linking on oligomer size distributions, we show that only mechanism 3 is consistent with experimental data. Our findings provide important insights into the two-step X-linking via mechanism 3, which consists of a simple covalent bonding via tyrosines in the presence of hydroxylated phenylalanines, followed by covalent bonding among tyrosines and hydroxylated phenylalanines. Structural analysis of X-linked Aβ oligomers revealed increased solvent exposure at the N-terminal region, which was previously associated with increased oligomer toxicity. Our results elucidate a potentially important role of phenylalanine hydroxylation and increased toxicity of Aβ oligomers induced by X-linking.

Effects of a high-protein intake on metabolic targets for weight loss in children with obesity: a randomized trial.

SummaryObjectiveThe objective of this research is to study effects of a 4‐week high‐protein (HP) diet on energy intake, resting energy expenditure (REE), protein turnover and body composition in children with obesity.MethodsIn this randomized placebo‐controlled single‐blind crossover study, children with obesity (n = 14; mean age: 10.1 years ± 1.2 standard deviation; body mass index–standard deviation score [BMI‐SDS]: 2.8 ± 0.5) received an ad libitum HP (+50 g protein per day) or normal‐protein (NP) diet for 4 weeks with a washout period of ≥2 weeks. Energy intake, REE, protein turnover, weight, BMI‐SDS and body composition were measured.ResultsNo differences were found in energy intake or REE between HP and NP. There was an increased urea production and phenylalanine hydroxylation after HP compared with NP (p < 0.05). There was an increased rise in fat‐free mass after HP compared with NP (∆HP: 0.8 ± 0.8 kg vs. ∆NP: 0.1 ± 0.6 kg, p < 0.05). BMI and BMI‐SDS increased during the study (BMI‐SDS start: 2.8 ± 0.5, end: 2.9 ± 0.5, p < 0.05) without a difference between groups.ConclusionsA 4‐week HP diet with ad libitum food intake did not affect energy intake and energy expenditure in children with obesity. BMI increased, although that could be partly explained by an increase in fat‐free mass.

A critical evaluation of the anabolic response after bolus or continuous feeding in COPD and healthy older adults.

After bolus and continuous enteral feeding of the same protein, different digestion and absorption kinetics and anabolic responses are observed. Establishing which mode of feeding has the highest anabolic potential in patients with chronic obstructive pulmonary disease (COPD) may aid in the prevention of muscle wasting, but an important confounding factor is the duration of assessments after bolus feeding. We hypothesized that the anabolic response to bolus and continuous feeding in COPD patients is comparable when methodological issues are addressed. Twenty-one older adults (12 patients with stage II-IV COPD and 9 healthy controls) were studied after intake of a fast-absorbing hydrolyzed casein protein-carbohydrate mixture either as a single bolus or as small sips (crossover design). Whole body protein synthesis (PS), breakdown (PB), net PS (PS - PB) protein efficiency (netPSPE), net protein balance (phenylalanine (PHE) intake - PHE hydroxylation) protein efficiency (netBalPE), and splanchnic PHE extraction (SPEPHE) were assessed using stable isotope tracer methodology. Bolus feeding assessments were done at 90, 95, and 99% of the calculated duration of the anabolic response. At 99%, netBalPE was higher for sip feeding than bolus feeding in both groups (P<0.0001). Nevertheless, bolus feeding was associated with a lower SPEPHE (P<0.0001) and higher netPSPE (P<0.0001). At 90% compared with 99%, PS and netBalPE after bolus feeding was significantly overestimated. In conclusion, several factors complicate a comparison of the anabolic capacity of bolus and continuous feeding in acute studies, including the critical role of SPE calculation and assumptions, and the duration of postprandial assessments after bolus feeding.

An isotope dilution model for partitioning of phenylalanine and tyrosine uptake by the liver of lactating dairy cows

An isotope dilution model to describe the partitioning of phenylalanine and tyrosine in the bovine liver was developed. The model comprises four intracellular and six extracellular pools and various flows connecting these pools and external blood. Conservation of mass principles were applied to generate the fundamental equations describing the behaviour of the system in the steady state. The model was applied to datasets from multi-catheterised dairy cattle during a constant infusion of [1-13C]phenylalanine and [2,3,5,6-2H]tyrosine tracers. Model solutions described the extraction of phenylalanine and tyrosine from the liver via the portal vein and hepatic artery. In addition, the exchange of free phenylalanine and tyrosine between extracellular and intracellular pools was explained and the hydroxylation of phenylalanine to tyrosine was estimated. The model was effective in providing information about the fates of phenylalanine and tyrosine in the liver and could be used as part of a more complex system describing amino acid metabolism in the whole animal.

Biochemical analysis of Centaurea depressa phenylalanine ammonia lyase (PAL) for biotechnological applications in phenylketonuria (PKU)

Context: Phenylketonuria (PKU) is the most common hereditary defect of phenylalanine hydroxylase (PAH) enzyme achieving the hydroxylation of phenylalanine (Phe). Phenylalanine ammonia lyase (PAL) converts Phe to a harmless metabolite, trans-cinnamic acid (TCA) in plants and PAL enzyme activity is fairly high in plants rich in flavonoids.Objective: The study aimed the biochemical analysis of PAL form Centaurea depressa BIEB. (Asteraceae) a flavonoid rich plant. This study may form the main frame of future research efforts for the development of a plant preparation aimed for oral intake in PKU patients in an attempt to enrich their diet by allowing them to ingest some food stuff containing Phe without being exposed to complications.Materials and methods: PAL was partially purified from the leaves of C. depressa. Enzyme activity was determined in comparison with that of other herbs that reportedly have a high PAL activity. Enzyme optimization was achieved and the PAL protein was detected by western blotting.Results: C. depressa PAL demonstrated high activity (34.9 ± 0.6 U/mg protein). The enzyme was purified by 1.92-fold, which resulted in an activity of 53.30 ± 0.2 U/mg protein. The high-performance liquid chromatography analyzes of the PAL activity both before and after purification were in agreement. Western blot of PAL exhibited a 70 kDa protein band. The optimum pH and temperature are pH 8.8 and 37 °C. The optimum activities under gastric and intestinal digestion conditions were observed at pH 4.0 and pH 8.0, respectively.Discussion and conclusion: PAL activity of C. depressa is high, and does not disappear under different environmental conditions. This enzyme could be used for the development of dietary foods and biotechnological products for patients with PKU.

Neurological Involvement in Tetrahydrobiopterin Deficiency

Tetrahydrobiopterin (BH4) is a natural and essential cofactor for the enzymatic hydroxylation of phenylalanine (Phe) and tyrosine (Tyr), and for two tryptophan hydroxylases, three nitric oxide synthases, and glyceryl-ether monooxygenase. Five separate genetic conditions affecting BH4 synthesis or recycling have been identified so far, including deficiency in (1) 6-pyruvoyltetrahydropterin synthase; (2) dihydropteridine reductase; (3) GTP cyclohydrolase I; (4) sepiapterin reductase; and (5) pterin-4α-carbinolamine dehydratase. These disorders cause hyperphenylalaninemia and impaired synthesis of serotonin and dopamine since tyrosine hydroxylase and neuronal tryptophan hydroxylase require BH4 and serotonin/dopamine products (5-hydroxytryptophan and L-dopa). All these five genetic conditions can be identified by newborn screening procedures due to elevated blood levels of Phe (with the sole exception of sepiapterin reductase deficiency). BH4 loading tests and measurement of neurotransmitter metabolites, pterins, and folates in cerebrospinal fluid can add further important information on disease severity. Untreated patients develop a complex neurological phenotype, which includes Parkinson-like features, brain degeneration, and early death. The gold standard treatment of severe disorders of BH4 metabolism is based on replacement therapy with BH4, 5-hydroxytryptophan, L-dopa, and carbidopa, with the addition, in certain cases, of folinic acid supplements and pramipexole. Dopamine agonists can improve L-dopa therapy, making treatment easier, relieving symptoms, stabilizing clinical course, and possibly ameliorating long-term outcomes. The outcome of patients with disorders of biopterin synthesis can be favorable, with either normal or near-normal cognition, and with some residual neurological symptoms usually manifesting diurnal variation, that is, worst when patients become tired or when the dosage or interval for medications is inadequate.

Short-term amino acid infusion improves protein balance in critically ill patients.

IntroductionEvidence behind the recommendations for protein feeding during critical illness is weak. Mechanistic studies are needed to elucidate the effects of amino acid and/or protein supplementation on protein metabolism before larger clinical trials with higher levels of protein feeding are initiated.MethodsWe studied the effects of parenteral amino acid supplementation (equivalent to 1 g/kg/day) over the course of 3 hours on whole-body protein turnover in critically ill patients in the intensive care unit (ICU) during the first week after admission. Patients were studied at baseline during ongoing nutrition and during extra amino acid supplementation. If the patient was still in the ICU 2 to 4 days later, these measurements were repeated. Protein kinetics were measured using continuous stable isotope-labeled phenylalanine and tyrosine infusions.ResultsThirteen patients were studied on the first study occasion only, and seven were studied twice. Parenteral amino acid supplementation significantly improved protein balance on both occasions, from a median of −4 to +7 μmol phenylalanine/kg/hr (P =0.001) on the first study day and from a median of 0 to +12 μmol phenylalanine/kg/hr (P =0.018) on the second study day. The more positive protein balance was attributed to an increased protein synthesis rate, which reached statistical significance during the first measurement (from 58 to 65 μmol phenylalanine/kg/hr; n =13; P =0.007), but not during the second measurement (from 58 to 69 μmol phenylalanine/kg/hr; n =7; P =0.09). Amino acid oxidation rates, estimated by phenylalanine hydroxylation, did not increase during the 3-hour amino acid infusion. A positive correlation (r =0.80; P <0.0001) was observed between total amino acids and/or protein given to the patient and whole-body protein balance.ConclusionExtra parenteral amino acids infused over a 3-hour period improved whole-body protein balance and did not increase amino acid oxidation rates in critically ill patients during the early phase (first week) of critical illness.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0844-6) contains supplementary material, which is available to authorized users.

Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

BackgroundBased on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates.ObjectiveTo assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake.MethodsA randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans.ResultsAfter 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001). Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03), synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01) and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001) were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042±0.01 vs 0.045±0.01%/h;P = 0.620).ConclusionsIn the overnight fasted state, adaptation to a low-protein intake (0.4 g/kg/d) does not result in a more negative whole-body protein balance and does not lower basal muscle protein synthesis rates when compared to a high-protein intake.Trial RegistrationClinicaltrials.gov NCT01551238.