Dietary Phenylalanine Research Articles

Individualized long-term outcomes in blood phenylalanine concentrations and dietary phenylalanine tolerance in 11 patients with primary phenylalanine hydroxylase (PAH) deficiency treated with Sapropterin-dihydrochloride.

We analyzed long-term sustainability of improved blood Phenylalanine (Phe) control and changes to dietary Phe tolerance in 11 patients (1 month to 16 years), with various forms of primary PAH deficiency (classic, moderate, severe phenylketonuria [PKU], mild hyperphenylalaninemia [HPA]), who were treated with 15-20mg/kg/d Sapropterin-dihydrochloride during a period of 13-44 months. 7/11 patients had a sustainable, significant reduction of baseline blood Phe concentrations and 6 of them also had an increase in mg/kg/day Phe tolerance. In 2 patients with mild HPA, blood Phe concentrations remained in the physiologic range even after a 22 and 36% increase in mg/kg/day Phe tolerance and an achieved Phe intake at 105% and 268% of the dietary reference intake (DRI) for protein. 2 of these responders had classic PKU. 1 patient with mild HPA who started treatment at 2 months of life, had a significant and sustainable reduction in pretreatment blood Phe concentrations, but no increase in the mg/kg/day Phe tolerance. An increase in Phe tolerance could only be demonstrated when expressing the patient's daily Phe tolerance with the DRI for protein showing an increase from 58% at baseline to 78% of normal DRI at the end of the observation. Long-term follow-up of patients with an initial response to treatment with Sapropterin is essential to determine clinically meaningful outcomes. Phenylalanine tolerance should be expressed in mg/kg/day and/or % of normal DRI to differentiate medical therapy related from physiologic growth related increase in daily Phe intake.

Adherence to tetrahydrobiopterin therapy in patients with phenylketonuria

Phenylketonuria (PKU) is an inborn error in phenylalanine metabolism due to deficiency of the enzyme, phenylalanine hydroxylase (PAH). Treatment includes restriction of dietary phenylalanine, and in some individuals, supplementation with the PAH cofactor, tetrahydrobiopterin (sapropterin dihydrochloride). A survey was conducted among patients with PKU who had been prescribed sapropterin to assess reasons for continuing or discontinuing the drug. The primary reason that sapropterin responders discontinued the drug was because of side effects, followed by insufficient reduction of blood phenylalanine and insurance issues. Conversely, those who remained on therapy cited increased tolerance for dietary protein as the main reason for continuation, along with lower blood phenylalanine concentrations and feeling better. This study suggests that adherence to sapropterin therapy is mainly dependent upon the increase in dietary protein allowed when on the drug.

O-112 The Indicators In The Biochemical Phenotype Of 58 Infants With Hepatorenal Tyrosinemia

Hepatorenal Tyrosinemia (HRT, OMIM 276700) is an autosomal recessive inborn error of metabolism which mainlyaffects the liver, kidneys and peripheral nerve. The primary defect has been attributed to the last enzyme in the catabolic pathway of tyrosine: fumarylacetoacetate hydrolase (FAH: E. C. 3.7.1.2). Early diagnosis and timely treatment offers an improved prognosis of the tyrosinemic patients. This treatment would require an administration of 2-(2-nitoro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC, OrfadinO), a low phenylalanine and tyrosine diet, and liver transplantation. Since several years, our laboratory carry out the diagnosis and the biological follow-up of tyrosinemic of all the own territory. In this report, we summarize the biochemical phenotype of 58 infants with HRT, focusing on the laboratory findings. The 58 infants (30 boys and 28 girls and 16 boys) came from 54 different families. The age at onset of the first symptoms of the disease varied from one week to four months, Thirty three patients died in the same month that the diagnosis was established, and twenty five are actually followed up after NTBC therapy and restriction diet. Elevated concentration of succinyl acetone in the urine, hypertyrosinemia and raised a-fetoprotein level were the most common indicators at the diagnosis. Urinalysis revealed no proteinuria, no cetonuria but sometimes glycosuria. Galactose and ferric chloride tests were negative. Positive reactions with 2, 4-dinitrophenyl hydrazine and cyanide-nitroprusside were observed occasionally. Only five urine specimens presented peculiar odour. All patients showed hyperbilirubinemia (both direct and indirect bilirubin) and deterioration of liver function (elevated ASAT, ALAT and slightly increazed PAL and GGT). Increased plasma ferritin and b2-microglobulin levels were seen intwenty four and five infants respectively. However, fast plasma glucose, total cholesterol, triglycerides, albumin and proteins concentrations were either normal or slightly decreased. HRT did not affect serum creatinine, urea, calcium, phosphorus and blood ammonia levels.

Blood phenylalanine concentrations and dietary phenylalanine tolerance in patients with PKU in response to treatment with sapropterin hydrochloride: A multidimensional approach to determine clinically meaningful outcomes

Blood phenylalanine concentrations and dietary phenylalanine tolerance in patients with PKU in response to treatment with sapropterin hydrochloride: A multidimensional approach to determine clinically meaningful outcomes

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

Dietary phenylalanine requirement and tyrosine replacement value for phenylalanine for fingerling Catla catla (Hamilton)

Abstract Two 12-week experiments were conducted to determine the dietary phenylalanine requirement and tyrosine replacement value for phenylalanine for fingerling Catla catla. In experiment I, phenylalanine requirement was determined by feeding six casein–gelatin based amino acid test diets (330 g kg− 1 CP; 16.72 kJ g− 1 GE) with graded levels of phenylalanine (3.9, 6.4, 8.7, 11.2, 13.8, 16.2 g kg− 1 dry diet) at a constant level (10 g kg− 1) of dietary tyrosine to triplicate groups of fish (3.95 ± 0.24 cm; 0.68 ± 0.19 g) near to satiation. Live weight gain (LWG%), specific growth rate (SGR % day− 1), feed conversion ratio (FCR), protein retention efficiency (PRE%), phenylalanine retention efficiency (PHRE%) and RNA/DNA ratio responded positively with the increasing concentrations of phenylalanine reaching the highest values at 11.2 g kg− 1 of dry diet. Quadratic regression analysis of LWG, SGR, PRE, PHRE and RNA/DNA ratio at 95% of maximum response against varying levels of dietary phenylalanine exhibited the requirement at 10.3, 10.1, 9.9, 9.7 and 10.6 g kg− 1 dry diet, respectively. The above analysis revealed that inclusion of phenylalanine at 10.1 g kg− 1 of dry diet, corresponding to 594 g kg− 1 of lysine is optimum. In experiment II, six diets with different levels of l -tyrosine (1.9, 3.8, 5.9, 8.1, 9.8, 11.8 g kg− 1 dry diet) with 10.1 g kg− 1 phenylalanine (determined in experiment I) fixed in all the test diets were fed to fish (3.85 ± 0.25 cm; 0.66 ± 0.16 g) to determine the tyrosine requirement under identical conditions. Quadratic regression analysis of LWG, SGR, PRE, PHRE and RNA/DNA ratio at 95% of maximum response against dietary tyrosine concentrations indicated the requirement at 6.3, 6.5, 6.5, 7.1 and 7.3 g kg− 1 dry diet, respectively. Hence, inclusion of tyrosine at 6.8 g kg− 1 of dry diet, corresponding to 378 g kg− 1 of lysine is taken as the tyrosine required for optimum utilization of phenylalanine. Based on above data, a total requirement of phenylalanine and tyrosine for fingerling C. catla was found to be 16.9 g kg− 1 (10.1 g kg− 1 phenylalanine + 6.8 g kg− 1 tyrosine) of dry diet, corresponding to 939 g kg− 1 of lysine. Tyrosine replacement value for phenylalanine was computed to be 37% on molar basis.

Strategies Used in Production of Phenylalanine-Free Foods for PKU Management.

Phenylketonuria (PKU) is one of the most widespread dysfunctions caused by an inheritable problem in the phenylalanine metabolism. In this metabolic disease, gene mutations in phenylalanine hydroxylase (PAH) result in phenylalanine accumulation that causes varying degrees of mental retardation. The most effective treatment is restriction of phenylalanine in diet provided through different strategies including combination of low-protein foods, hydrolyzing of protein-rich foods, or use of protein substitutes. Meat and meat products, sea foods, milk and infant formula, cereal products, and beans are among the modified foods for PKU management. Elimination of phenylalanine from food presents major technological, nutritional, and organolleptic challenges because protein, as an essential structure-building and nutritional element, is removed. In addition, following this program is laborious, boring, and restrictive for both patients and their families. This paper reviews the current findings about PKU and the recent developments in the production of phenylalanine-free foods. Also, the nutritional requirements and challenges encountered by PKU individuals and food technologists are finally discussed.

Progressive neuropsychiatric manifestations of phenylketonuria in adulthood

IntroductionNeuropsychiatric signs and MRI abnormalities can occur in patients with phenylketonuria in adulthood. We describe clinical and radiological features of phenylketonuric patients and we discuss the advantage of continuing diet in adulthood. MethodWe report late onset neuropsychiatric symptoms of four phenylketonuric patients (33–45years) diagnosed in infancy and report the case of a patient (33years) diagnosed with phenylketonuria because of late onset neurological signs. We describe clinical and radiological features of these 5 patients, and their evolution under diet and propose a review of the literature. ResultsThe main neurological abnormalities in phenylketonuric patients diagnosed in infancy are: brisk reflexes, spastic paraparesis, psychiatric signs that appear 10.5years after the diet arrest. A leukoencephalopathy was present in 93% of cases and 91.7% improve clinically after poor phenylalanine diet reintroduction. In 4 patients, neurological abnormalities (spastic paraparesis, dementia, Parkinsonism) led to the late diagnosis. Two of them had a leukoencephalopathy on brain MRI. Patients had high levels of phenylalanine (above 1500μmol/L) when neuropsychiatric signs occurred. Improvement after diet suggests that hyperphenylalaninemia has a direct toxic effect on the brain. Discussion/ConclusionThe long-term follow-up of phenylketonuric patients is mandatory to depict and treat neurological complications in time. Diet reintroduction is efficacious in most cases.

Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular‐scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron-10 ((10)B) atoms to individual tumour cells. Cell killing results from the (10)B (n, α)(7) Li neutron capture and fission reactions that occur if a sufficient number of (10)B atoms are localized in the tumour cells. Intranuclear (10)B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of (10)B atoms reflects both bound and free pools of boron in individual tumour cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular-scale resolution by clinically applicable techniques such as positron emission tomography and magnetic resonance imaging. In this study, a secondary ion mass spectrometry based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high-grade gliomas, recurrent tumours of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumour cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols.

Carbohydrate Coingestion Delays Dietary Protein Digestion and Absorption but Does Not Modulate Postprandial Muscle Protein Accretion

Dietary protein digestion and absorption is an important factor modulating muscle protein accretion. However, there are few data available on the effects of coingesting other macronutrients with protein on digestion and absorption kinetics and the subsequent muscle protein synthetic response. The objective of the study was to determine the impact of carbohydrate coingestion with protein on dietary protein digestion and absorption and muscle protein accretion in healthy young and older men. Twenty-four healthy young (aged 21± 1 y, body mass index 21.8 ±0.5 kg/m(2)) and 25 older (aged 75 ± 1 y, body mass index 25.4 ± 0.6 kg/m(2)) men received a primed continuous L-[ring-(2)H5]-phenylalanine and L-[ring-3,5-(2)H2]-tyrosine infusion and ingested 20 g intrinsically L-[1-(13)C]-phenylalanine-labeled protein with (Pro+CHO) or without (Pro) 60 g carbohydrate. Plasma samples and muscle biopsies were collected in a postabsorptive and postprandial state. Carbohydrate coingestion delayed the appearance of exogenous phenylalanine in the circulation (P = .001). Dietary protein-derived phenylalanine availability over the 5-hour postprandial period was lower in the older (62 ± 2%) when compared with the young subjects (74 ± 2%; P = .007), with no differences between conditions (P = .20). Carbohydrate coingestion did not modulate postprandial muscle protein synthesis rates (0.035 ± 0.003 vs 0.043 ± 0.004 and 0.033 ± 0.002 vs 0.035 ± 0.003%/h after Pro vs Pro+CHO in the young and older group, respectively). In accordance, no differences in muscle protein-bound L-[1-(13)C]-phenylalanine enrichments were observed between conditions (0.020 ± 0.002 vs 0.020 ± 0.002 and 0.019 ± 0.003 vs 0.022 ± 0.004 mole percent excess after Pro vs Pro+CHO in the young and older subjects, respectively). Carbohydrate coingestion with protein delays dietary protein digestion and absorption but does not modulate postprandial muscle protein accretion in healthy young or older men.

Evaluation of effectiveness and outcome of PKU screening and management in the State of Sergipe, Brazil.

Phenylketonuria (PKU) was the first inherited metabolic disease known to cause mental retardation for which a newborn screening program (NBS) was developed. The objective of this study was to evaluate the effectiveness of PKU NBS and the management of cases in the northeastern Brazilian state of Sergipe (SE). We reviewed the phenylalanine concentrations in filter-paper collected from the heel (PKUneo) of 43,449 newborns; blood concentrations obtained by venipuncture in the subjects with abnormal PKUneo; the children's age at several phases of the program, the incidence of the disease from January 2007 to June 2008; and metabolic control of the patients. The coverage of NBS/SE was 78.93%. The children's age was 10 ± 7 days at PKUneo collection. Twelve children were recalled based on the PKUneo cutoff value at 28 ± 13 days. From these, the concentrations of phenylalanine collected by venipuncture were normal in five children. The incidence of hyperphenylalaninemia was 1/43,449, and of PKU was 1/8,690 (5 cases). One suspected subject died. Another death occurred in the cohort, in a confirmed PKU case. PKU treatment began within 51 ± 12 days of life. In the four patients under dietary phenylalanine restriction, metabolic control was often difficult. PKU NBS/SE has satisfactory coverage and adequate cutoff for recalling patients and diagnosis, but the onset of treatment is delayed, and follow-up metabolic control is frequently inadequate.

A questionnaire survey on the usage of low protein staple foods by people with phenylketonuria in Scotland

There is scarce evidence available with respect to an evaluation of the role of low protein staple foods (LPSF) in the management of phenylketonuria (PKU). The present study explored beliefs, acceptability and issues around the use of LPSF by people with PKU or their carers. A semi-anonymous questionnaire was posted to 178 people with PKU in Scotland (104 children, aged 2-17years, and 74 adults). Questions explored were: the type and amount of LPSF ordered; perceptions on use and usefulness of LPSF; acceptability of the LPSF sensory properties (i.e. taste, smell, texture, appearance); support for the supply and use of LPSF; and comments from primary healthcare professionals regarding dispensing and prescription. Eighty-two individuals responded (46% response rate): 97% perceived that LPSF were useful for PKU management; more than 85% reported that LPSF were important for phenylalanine control, satisfying appetite, and diet variety. The most common LPSF ordered were pasta/rice/cous cous, flour, biscuits and bread. Fifty percent of respondents ordered <51% of the recommended unit allowance of LPSF. The sensory properties of LPSF were well perceived. Forty-nine percent (n=39) had received a comment from primary healthcare staff regarding the prescription or dispensing of LPSF; 59% (n=23) received negative comments, the majority of which came within general practitioner surgeries. There is a positive attitude and perception on the use and usefulness of LPSF in the management of PKU. Issues with respect to the supply and provision of LPSF within primary health care may indicate poor communication between specialists and primary healthcare professionals or a lack of scientific evidence demonstrating their clinical effectiveness.

Nutritional composition of low protein and phenylalanine-restricted dishes prepared for phenylketonuric patients

Nutritional management is essential for Phenylketonuria (PKU) treatment, consisting in a semi-synthetic and low phenylalanine (Phe) diet, which includes strictly controlled amounts of low protein natural foods (essentially fruits and vegetables) supplemented with Phe-free protein substitutes and dietetic low-protein products. PKU diet has to be carefully planned, providing the best ingredient combinations, so that patients can achieve good metabolic control and an adequate nutritional status. Hereupon, it is mandatory to know the detailed composition of natural and/or cooked foodstuffs prepared specifically for these patients.We intended to evaluate sixteen dishes specifically prepared for PKU patients, regarding the nutritional composition, Phe and tyrosine (Tyr) contents, fatty acids profile, and vitamins E and B12 amounts.The nutritional composition of the cooked samples was 15.5–92.0 g/100 g, for moisture; 0.7–3.2 g/100 g, for protein; 0.1–25.0 g/100 g, for total fat; and 5.0–62.0 g/100 g, for total carbohydrates. Fatty acids profile and vitamin E amount reflected the type of fat used. All samples were poor in vitamin B12 (0.3–0.8 μg/100 g). Boiled rice presented the highest Phe content: 50.3 mg/g of protein. These data allow a more accurate calculation of the diet portions to be ingested by the patients according to their individual tolerance.

Scientific Opinion on the re‐evaluation of aspartame (E 951) as a food additive

The EFSA ANS Panel provides a scientific opinion on the safety of aspartame (E 951). Aspartame is a sweetener authorised as a food additive in the EU. In previous evaluations by JECFA and the SCF, an ADI of 40 mg/kg bw/day was established based on chronic toxicity in animals. Original reports, previous evaluations, additional literature and data made available following a public call were evaluated. Aspartame is rapidly and completely hydrolysed in the gastrointestinal tract to phenylalanine, aspartic acid and methanol. Chronic and developmental toxicities were relevant endpoints in the animal database. From chronic toxicity studies in animals, a NOAEL of 4000 mg/kg bw/day was identified. The possibility of developmental toxicity occurring at lower doses than 4000 mg/kg in animals could not be excluded. Based on MoA and weight-of-evidence analysis, the Panel concluded that developmental toxicity in animals was attributable to phenylalanine. Phenylalanine at high plasma levels is known to cause developmental toxicity in humans. The Panel concluded that human data on developmental toxicity were more appropriate for the risk assessment. Concentration-response modelling was used to determine the effects of aspartame administration on plasma phenylalanine using human data after phenylalanine administration to normal, PKU heterozygote or PKU homozygote individuals. In normal and PKU heterozygotes, aspartame intakes up to the ADI of 40 mg/kg bw/day, in addition to dietary phenylalanine, would not lead to peak plasma phenylalanine concentrations above the current clinical guideline for the prevention of adverse effects in fetuses. The Panel concluded that aspartame was not of safety concern at the current aspartame exposure estimates or at the ADI of 40 mg/kg bw/day. Therefore, there was no reason to revise the ADI of aspartame. Current exposures to aspartame - and its degradation product DKP - were below their respective ADIs. The ADI is not applicable to PKU patients.

Phenylketonuria: Protein content and amino acids profile of dishes for phenylketonuric patients. The relevance of phenylalanine

Phenylketonuria is an inborn error of metabolism, involving, in most cases, a deficient activity of phenylalanine hydroxylase. Neonatal diagnosis and a prompt special diet (low phenylalanine and natural-protein restricted diets) are essential to the treatment. The lack of data concerning phenylalanine contents of processed foodstuffs is an additional limitation for an already very restrictive diet.Our goals were to quantify protein (Kjeldahl method) and amino acid (18) content (HPLC/fluorescence) in 16 dishes specifically conceived for phenylketonuric patients, and compare the most relevant results with those of several international food composition databases.As might be expected, all the meals contained low protein levels (0.67–3.15g/100g) with the highest ones occurring in boiled rice and potatoes. These foods also contained the highest amounts of phenylalanine (158.51 and 62.65mg/100g, respectively). In contrast to the other amino acids, it was possible to predict phenylalanine content based on protein alone. Slight deviations were observed when comparing results with the different food composition databases.

INITIATION OF DIETARY INTERVENTION AFTER 20 YEARS OF UNTREATED PKU

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism causing accumulation of phenylalanine resulting in cognitive, behavioural and pyramidal dysfunction. The prevalence of PKU is 1 per 10,000 live...

La phénylcétonurie : nouveaux traitements

Low phenylalanine diet has been the key treatment of phenylketonuria for more than 50years, allowing efficient management of thousands of PKU patients to date. However, non-compliance exists, mainly after adolescence. A medication for PKU received approval in Europe in 2009 (sapropterine dihydrochloride or Kuvan®) and can benefit to patients responsive to this drug. Other treatment options are available in some countries (glycomacropeptides, large neutral amino acids) or are currently under investigation (phenylalanine ammonia lyase, chaperones molecules, gene therapy).

Body mass index in adult patients with diet‐treated phenylketonuria

There is an increasing number of adults with phenylketonuria (PKU) on a low phenylalanine diet. In the general population, an increasing body mass index (BMI) in the UK is a major problem with associated co-morbidities. The present study aimed to identify whether patients with diet-treated PKU have obesity rates comparable to those in the general population. Two hundred and thirty-six PKU subjects (49% male, 51% female), aged >16years, who were diagnosed by newborn screening and were receiving a low phenylalanine diet, were identified from seven metabolic centres in the UK. Retrospective data were collated on age, sex, BMI and mean phenylalanine concentration over the previous 12months. Mean (SD) phenylalanine concentration for all 236 subjects was 789 (311)μm; mean (SD) BMI was 26 (5.4)kg m(-2) [males 25 (4.3)kg m(-2) , females 27 (6.2)kg m(-2) ]; mean (SD) age was 26 (7) years; and 55% had a BMI >25kg m(-2) . The percentage of subjects with a BMI >25kg m(-2) and >30kg m(-2) , as well as increasing obesity with age, was similar to the UK population. A correlation was observed between increasing BMI and a higher phenylalanine concentration (r=0.243, P=0.001). The number of overweight and obese patients with diet-treated PKU in the UK is a concern. This could lead to other obesity-related complications increasing the complexity of diet and the cost of their care. There is a need to educate patients with respect to adopting a healthy, low phenylalanine diet and lifestyle to prevent further rises in BMI.

Dietary phenylalanine requirement of adult Labrador Retrievers using the direct oxidation approach

Historically, the determination of amino acid (AA) requirements in dogs have been done in growing dogs and have been invasive in nature, requiring long periods of confinement and exposure to diets deficient in AA, which result in a reduction in the health and well being of dogs during the study period. Therefore, we were interested in examining whether carbon oxidation methods, with short periods of exposure to diets deficient in a single AA, could be successfully applied to determine AA requirements in dogs. Four adult Labrador Retrievers consumed 7 levels of phenylalanine (Phe), and excess tyrosine (Tyr) in random order and in combination with kibble that was formulated to be deficient in Phe and adequate in dietary energy (3730 kcal/kg as fed) and fed at ~13.5 g kg−1 BW d−1. Dogs were fed test diets for two days prior to conducting the tracer studies. The Phe requirement was determined by applying a 2‐phase linear regression crossover model to tracer oxidation (F13CO2). The mean Phe requirement was 3.27 g/kg diet (upper 95% CI= 4.96 g/kg diet) on a dry matter basis. The current NRC estimate of the adequate intake for Phe is 4.50 g/kg diet and based on growth and nitrogen balance in growing dogs. This magnitude of difference is similar to that seen when estimates of AA requirements in humans using nitrogen balance vs. carbon oxidation methods are compared.

Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single-Turnover Experiments

Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH(4)) and O(2). A complete kinetic mechanism for PheH was determined by global analysis of single-turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH(4)-phenylalanine complex begins with the rapid binding of BH(4) (K(d) = 65 μM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (K(d) = 130 μM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O(2) rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s(-1). Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines k(cat). Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.