Plasma Phe Research Articles

Protein intake and physical activity are associated with body composition in individuals with phenylalanine hydroxylase deficiency.

Determine whether body composition as it relates to dietary protein in patients with phenylalanine hydroxylase (PAH) deficiency is associated with genotype, dietary factors, and lifestyle choices. We examined associations between protein intake (intact and medical foods: MF) and body composition in PAH-deficient patients along with, physical activity, and genotype. Protein intakes (total, intact, and MF) were analysed from three-day food records with Nutrition Data System for Research (NDSR) in 59 children and 27 adults (N=86, median age=16.0years). The severity of PAH deficiency was classified using the genotype assigned value method (AV sum). Physical activity was assessed using a study-developed question (light vs. intense activity). Body composition was measured by DXA, including android:gynoid ratio (A:G), fat-free mass index (FFMI), fat mass index (FMI), and FMI:FFMI ratio. High intact protein intake was associated with high FFMI (rs=0.75, p=0.008) and low FMI:FFMI (rs=-0.59, p=0.04) in adults. Only in children, MF protein (rs=0.38, p=0.04) was directly proportional to FFMI. Median intact protein intakes of adults (25.1 vs. 9.9g/d, p<0.001) and children (11 vs. 6g/d, p<0.001) were higher than prescribed. Only in adults, the actual median MF protein intake was lower than prescribed (53 vs. 60g/d, p=0.03). In adults and children, light activity was associated with higher fat mass indices compared to intense activity (adults: FMI:FFMI: β=1.1, p=0.001, children: FMI:FFMI: β=1.1, p=0.007; FMI β=2.1, p=0.01; A:G β=1.1, p=0.04). All associations remained significant after covariate adjustment. Genotype was not associated with body composition. Although fat-free mass in adults was positively associated with intact protein intake, it should be consumed as prescribed per individual tolerance to maintain plasma Phe concentrations within treatment range. In children, total protein maximized with MF should be encouraged to promote lean mass. Nutrition counselling could be complemented with physical activity recommendations for optimal clinical outcomes.

The Indicator Amino Acid Oxidation Method with the Use of L-[1-13C]Leucine Suggests a Higher than Currently Recommended Protein Requirement in Children with Phenylketonuria

BackgroundPhenylketonuria is characterized by mutations in the Phe hydroxylase gene that leads to the accumulation of Phe in plasma and the brain. The standard of care for phenylketonuria is nutritional management with dietary restriction of Phe and the provision of sufficient protein and energy for growth and health maintenance. The protein requirement in children with phenylketonuria is empirically determined based upon phenylketonuria nutritional guidelines that are adjusted individually in response to biochemical markers and growth. ObjectiveWe determined dietary protein requirements in children with phenylketonuria with the use of the indicator amino acid oxidation (IAAO) technique, with L-[1-13C]Leu as the indicator amino acid. MethodsFour children (2 males; 2 females) aged 9–18 y with phenylketonuria [mild hyperphenylalanemia (mHPA); 6–10 mg/dL (360–600 μmol/L)] were recruited to participate in ≥7 separate test protein intakes (range: 0.2–3.2 g ⋅ kg−1 ⋅ d−1) with the IAAO protocol with the use of L-[1-13C]Leu followed by the collection of breath and urine samples over 8 h. The diets were isocaloric and provided energy at 1.7 times the resting energy expenditure. Protein was provided as a crystalline amino acid mixture based on an egg protein pattern, except Phe and Leu, which were maintained at a constant across intakes. Protein requirement was determined with the use of a 2-phase linear-regression crossover analysis of the rate of L-[1-13C]Leu tracer oxidation. ResultsThe mean protein requirement was determined to be 1.85 g ⋅ kg−1 ⋅ d−1 (R2 = 0.66; 95% CI: 1.37, 2.33). This result is substantially higher than the 2014 phenylketonuria recommendations (1.14–1.33 g ⋅ kg−1 ⋅ d−1; based on 120–140% above the current RDA for age). ConclusionsTo our knowledge, this is the first study to directly define a quantitative requirement for protein intake in children with mHPA and indicates that current protein recommendations in children with phenylketonuria may be insufficient. This trial was registered at clinicaltrials.gov as NCT01965691.

Low Serum Cholesterol Concentration in Adult Patients with Phenylketonuria- One Centre Experience

Introduction: Phenyloketonuria is a rare metabolic disorder caused by a deficiency of the phenylalanine (Phe) hydroxylase enzyme. A reduced intake of natural proteins helps optimise plasma Phe concentration. A relationship between high plasma Phe level and the inhibition of cholesterol synthesis was previously observed but the mechanisms are unclear. Low LDL-cholesterol concentrations were observed in children and adolescent PKU patients, but not in adults. This is the first paper to present lipid profile in adult patients with this condition. Methods: Lipid profile was analysed in adult patients with PKU. We examined associations between Phe and four outcomes: total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides. Confounding factors (predictors) were taken into account: body mass index (BMI), age and gender. The statistical analysis was performed using multiple linear regression. Results: Among 176 adult patients 91 were females (52%). The mean age was 32 ± 10.7 years. 82 patients (46%) were on strict PKU diet. Mean Phe was 1017 ± 440 μmol/L. Mean total cholesterol was 4.33 ± 0.94 mmol/L, LDL-cholesterol 2.48 ± 0.8 mmol/L, HDL-cholesterol 1.2 ± 0.34 mmol/L, triglycerides 1.6 ± 0.9 mmol/L. There was no correlation between Phe concentration and lipid profile in our cohort of adult patients with PKU. No cardiovascular events were documented in our cohort. Conclusions: In conclusion, the outcomes of the study demonstrate that mean total cholesterol, LDL-cholesterol and triglycerides concentrations were substantially lower as compared to healthy population, which may confer their reduced cardiovascular risk. Lipid profile remained low irrespectively of Phe concentration.

Demethylation of the promoter region of GPX3 in a newborn with classical phenylketonuria

ObjectivesPhenylketonuria (PKU) is characterized by a high phenylalanine (phe) in plasma and oxidative stress. However, the monitoring of oxidative stress in newborns with PKU using the activity levels of antioxidant enzymes is not optimal. We investigated the possibility of monitoring an increased reactive oxygen species (ROS) production using DNA methylation changes of an oxidative stress response element in the promoter region of an enzymatic antioxidant gene. Design and methodsUsing DNA extracted from blood leukocytes, the cytosine phosphodiester bond guanine positions of an overlapping CCAAT box/metal response element (CGATTGGCTG) of the glutathione peroxidase 3 promoter activated by oxidative stimuli and expressed in plasma were analysed for methylation changes in 20 newborns with hyperphenylalaninemia and 20 healthy controls. ResultsA demethylated allele was detected in a PKU patient at a phe level of 465μmol/L on day 2 after birth, but not in other patients (phe<465μmol/L, ≥day 2 after birth; phe>465μmol/L, ≥day 3 after birth) and healthy controls (phe<465μmol/L, ≥day 2 after birth). ConclusionsThe detection of the demethylated allele could be time and phe concentration dependent. The demethylated allele is suggested as an early epigenetic marker for an extracellular monitoring of an increased ROS production in newborns with PKU.

Lipid profile status and other related factors in patients with Hyperphenylalaninaemia.

BackgroundThe mainstay of treating patients with phenylketonuria (PKU) is based on a Phe-restricted diet, restrictive in natural protein combined with Phe-free L-amino acid supplements and low protein foods. This PKU diet seems to reduce atherogenesis and confer protection against cardiovascular diseases but the results from the few published studies have been inconclusive. The aim of our study was to evaluate the relationship between the lipid profile and several treatment-related risk factors in patients with hyperphenylalaninaemia (HPA) in order to optimize their monitoring.MethodsWe conducted a cross-sectional multicentre study. A total of 141 patients with HPA were classified according to age, phenotype, type of treatment and dietary adherence. Annual median blood phenylalanine (Phe) levels, Phe tolerance, anthropometric measurements, blood pressure (BP) and biochemical parameters [(triglycerides, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), apolipoprotein A (ApoA), apolipoprotein B (ApoB), vitamin B12, total homocysteine (tHcy), Methionine (Met), high sensitivity C-Reactive Protein (hsCRP)] were collected for each patient.ResultsPlasma TC levels were lower in patients with PKU than in the mild-HPA group (150 ± 31 vs. 164 ± 22 mg/dL), and there was a weak inverse correlation between plasma TC and Phe levels. HDL-C, LDL-C, ApoA and ApoB levels were lower in the PKU group than in mild-HPA. Patients with PKU had higher systolic BP than the mild-HPA group and there was found a quadratic correlation between median Phe levels and systolic BP (p = 6.42e-5) and a linear correlation between median Phe levels and diastolic BP (p = 5.65e-4). In overweight or obese PKU patients (24.11 %), biochemical parameters such as TC, triglycerides, LDL-C, tHcy, hsCRP and BP were higher. By contrast, HDL-C was lower in these patients.ConclusionOur data show a direct correlation between lipid profile parameters and good adherence to the diet in PKU patients. However, lipid profile in overweight or obese patients displayed an atherogenic profile, in addition to higher hsCRP concentrations and BP. Our study contributes to a better understanding of the relationship between phenotype and treatment in patients with HPA, which could be useful in improving follow-up strategies and clinical outcome.Trial registrationResearch Ethics Committee of Santiago-Lugo 2015/393. Registered 22 September 2015, retrospectively registered.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-016-0508-x) contains supplementary material, which is available to authorized users.

Glycomacropeptide for nutritional management of phenylketonuria: a randomized, controlled, crossover trial

Background: To prevent cognitive impairment, phenylketonuria requires lifelong management of blood phenylalanine (Phe) concentration with a low-Phe diet. The diet restricts intake of Phe from natural proteins in combination with traditional amino acid medical foods (AA-MFs) or glycomacropeptide medical foods (GMP-MFs) that contain primarily intact protein and a small amount of Phe.Objective: We investigated the efficacy and safety of a low-Phe diet combined with GMP-MFs or AA-MFs providing the same quantity of protein equivalents in free-living subjects with phenylketonuria.Design: This 2-stage, randomized crossover trial included 30 early-treated phenylketonuria subjects (aged 15–49 y), 20 with classical and 10 with variant phenylketonuria. Subjects consumed, in random order for 3 wk each, their usual low-Phe diet combined with AA-MFs or GMP-MFs. The treatments were separated by a 3-wk washout with AA-MFs. Fasting plasma amino acid profiles, blood Phe concentrations, food records, and neuropsychological tests were obtained.Results: The frequency of medical food intake was higher with GMP-MFs than with AA-MFs. Subjects rated GMP-MFs as more acceptable than AA-MFs and noted improved gastrointestinal symptoms and less hunger with GMP-MFs. ANCOVA indicated no significant mean ± SE increase in plasma Phe (62 ± 40 μmol/L, P = 0.136), despite a significant increase in Phe intake from GMP-MFs (88 ± 6 mg Phe/d, P = 0.026). AA-MFs decreased plasma Phe (−85 ± 40 μmol/L, P = 0.044) with stable Phe intake. Blood concentrations of Phe across time were not significantly different (AA-MFs = 444 ± 34 μmol/L, GMP-MFs = 497 ± 34 μmol/L), suggesting similar Phe control. Results of the Behavior Rating Inventory of Executive Function were not significantly different.Conclusions: GMP-MFs provide a safe and acceptable option for the nutritional management of phenylketonuria. The greater acceptability and fewer side effects noted with GMP-MFs than with AA-MFs may enhance dietary adherence for individuals with phenylketonuria. This trial was registered at www.clinicaltrials.gov as NCT01428258.

Validation of amino-acids measurement in dried blood spot by FIA-MS/MS for PKU management

ObjectivesPhenylketonuria (PKU) is a metabolic disorder leading to high concentrations of phenylalanine (Phe) and low concentrations of tyrosine (Tyr) in blood and brain that may be neurotoxic. This disease requires a regular monitoring of plasma Phe and Tyr as well as branched-chain amino-acids concentrations to adapt the Phe-restricted diet and other therapy that may be prescribed in PKU. We validated a Flow Injection Analysis tandem Mass Spectrometry (FIA-MS/MS) to replace the enzymatic method routinely used for neonatal screening in order to monitor in parallel to Phe, Tyr and branched-chain amino-acids not detected by the enzymatic method. Design and methodsWe ascertained the performances of the method: linearity, detection and quantification limits, contamination index, accuracy. We cross validated the FIA-MS/MS and enzymatic methods and we evaluated our own reference ranges to monitor Phe, Tyr, Leu, Val on 59 dried blood spots of normal controls. We also evaluated Tyr, Leu and Val concentrations in PKU patients to detect some potential abnormalities, not evaluated by the enzymatic method. ResultsWe developed a rapid method with excellent performances including precision and accuracy <15%. We noted an excellent correlation of Phe concentrations between FIA-MS/MS and enzymatic methods (p<0.0001) based on our database which are similar to references ranges published. We observed that 50% of PKU patients had lower concentrations of Tyr, Leu and/or Val that could not be detected by the enzymatic method. ConclusionBased on laboratory accreditation recommendations, we validated a robust, rapid and reliable FIA-MS/MS method to monitor plasma Phe concentrations but also Tyr, Leu and Val concentrations, suitable for PKU management. We evaluated our own reference ranges of concentration for a routine application of this method.

Phenylketonuria and Gut Microbiota: A Controlled Study Based on Next-Generation Sequencing.

Phenylketonuria (PKU) is an inborn error of metabolism associated with high blood levels of phenylalanine (Phe). A Phe-restricted diet supplemented with L-amino acids is the main treatment strategy for this disease; if started early, most neurological abnormalities can be prevented. The healthy human gut contains trillions of commensal bacteria, often referred to as the gut microbiota. The composition of the gut microbiota is known to be modulated by environmental factors, including diet. In this study, we compared the gut microbiota of 8 PKU patients on Phe-restricted dietary treatment with that of 10 healthy individuals. The microbiota were characterized by 16S rRNA sequencing using the Ion Torrent™ platform. The most dominant phyla detected in both groups were Bacteroidetes and Firmicutes. PKU patients showed reduced abundance of the Clostridiaceae, Erysipelotrichaceae, and Lachnospiraceae families, Clostridiales class, Coprococcus, Dorea, Lachnospira, Odoribacter, Ruminococcus and Veillonella genera, and enrichment of Prevotella, Akkermansia, and Peptostreptococcaceae. Microbial function prediction suggested significant differences in starch/glucose and amino acid metabolism between PKU patients and controls. Together, our results suggest the presence of distinct taxonomic groups within the gut microbiome of PKU patients, which may be modulated by their plasma Phe concentration. Whether our findings represent an effect of the disease itself, or a consequence of the modified diet is unclear.

Rapid quantitative method for the detection of phenylalanine and tyrosine in human plasma using pillar array columns and gradient elution.

This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field.

Impacts of adding ruminally protected phenylalanine to rations containing high levels of canola meal on performance of high producing Holstein cows

Even though studies supplementing Phe to dairy cattle are rare, it has been identified as limiting in corn silage based rations, after Lys and Met, as well as being important to the mammary gland for overall milk production. Since canola meal (CM) is low in Phe, plasma Phe concentrations decrease as more CM is included in dairy rations. A previous study fed 7.5g/cow/day of intestinally absorbable Phe, but results suggested that it was insufficient to support increased milk production since it was primarily used to support increased body condition score (BCS; Swanepoel et al., 2015a). Our objective was to determine if supplementing 15g/cow/day of intestinally absorbable Phe in a ruminally protected (RP) form (HCMP) to a ration containing 170g/kg CM (HCM) would support increased milk production after fulfilling its apparent 1st priority of restoring previously mobilized peptides to muscle protein synthesis, thereby regaining animal performance possibly lost with higher dietary CM inclusion levels (i.e., 130g/kg (LCM) to 165g/kg (HCM)) based upon Swanepoel et al. (2015a). Ruminally protected Met (2.0g/cow/day intestinally absorbable) was added as part of the treatment ration to HCM treatments to avoid a possible Met limitation. The experimental design was a 3×3 Latin square using 3 pens of ∼315 early lactation cows/pen with three 21day periods. Dry matter (DM) intake was not affected (avg: 27.5±0.5kg/day) by feeding RP Phe and there was no impact of treatment on milk and component yields, except a reduced lactose content (P=0.02) with Phe addition. Even though plasma Phe levels only differed numerically between treatments, its supplementation resulted in energy being diverted towards BCS gain as in Swanepoel et al. (2015a), but not at the expense of milk components, suggesting that higher Phe supplementation supplied enough Phe to replace mobilized muscle protein while maintaining milk production. The lack in change of plasma Phe concentrations could be due to extensive catabolization by the liver or hepatic conversion of Phe to Tyr, which is supported by the change in plasma Tyr concentrations. Interestingly, addition of Phe to the HCM ration increased whole tract neutral- and acid detergent fiber digestibility. Perhaps Phe released into the rumen when Phe was fed stimulated fibrolytic bacteria through a direct impact on microbes of free Phe, which has previously been shown to enhance growth and/or capabilities of cellulolytic bacteria. Total net energy output decreased with HCM feeding, but was restored to the level of the LCM ration for the HCMP treatment suggesting that further investigation to determine if an even higher Phe supplementation level may have additional benefits on milk production may have merit.

Clinical relevance of the discrepancy in phenylalanine concentrations analyzed using tandem mass spectrometry compared with ion-exchange chromatography in phenylketonuria

IntroductionMetabolic control of phenylketonuria (PKU) and compliance with the low-phenylalanine (phe) diet are frequently assessed by measuring blood phe concentrations in dried blood spots (DBS) collected by patients instead of plasma phe concentrations. ObjectiveOur objective was to investigate the difference in blood phe concentrations in DBS collected by subjects and analyzed using either a validated newborn screening tandem mass spectrometry (MS/MS) protocol or ion-exchange chromatography (IEC) compared to plasma phe concentrations obtained simultaneously and analyzed using IEC. DesignThree to four fasting blood samples were obtained from 29 subjects with PKU, ages 15–49years. Capillary blood was spotted on filter paper by each subject and the DBS analyzed using both MS/MS and IEC. Plasma was isolated from venous blood and analyzed using IEC. ResultsBlood phe concentrations in DBS analyzed using MS/MS are 28%±1% (n=110, p<0.0001) lower than plasma phe concentrations analyzed using IEC resulting in a blood phe concentration of 514±23μmol/L and a plasma phe concentration of 731±32μmol/L (mean±SEM). This discrepancy is larger when plasma phe is >600μmol/L. Due to the large variability across subjects of 13.2%, a calibration factor to adjust blood phe concentrations is not recommended. Analysis of DBS using IEC reduced the discrepancy to 15±2% lower phe concentrations compared to plasma analyzed using IEC (n=38, p=0.0001). This suggests that a major contributor to the discrepancy in phe concentrations is the analytical method. ConclusionUse of DBS analyzed using MS/MS to monitor blood phe concentrations in individuals with PKU yields significantly lower phe levels compared to plasma phe levels analyzed using IEC. Optimization of current testing methodologies for measuring phe in DBS, along with patient education regarding the appropriate technique for spotting blood on filter paper is needed to improve the accuracy of using DBS to measure phe concentrations in PKU management.

A Multiplatform Metabolomics Approach to Characterize Plasma Levels of Phenylalanine and Tyrosine in Phenylketonuria.

Different pathophysiological mechanisms have been described in phenylketonuria (PKU) but the indirect metabolic consequences of metabolic disorders caused by elevated Phe or low Tyr concentrations remain partially unknown. We used a multiplatform metabolomics approach to evaluate the metabolic signature associated with Phe and Tyr. We prospectively included 10 PKU adult patients and matched controls. We analysed the metabolome profile using GC-MS (urine), amino-acid analyzer (urine and plasma) and nuclear magnetic resonance spectroscopy (urine). We performed a multivariate analysis from the metabolome (after exclusion of Phe, Tyr and directly derived metabolites) to explain plasma Phe and Tyr concentrations, and the clinical status. Finally, we performed a univariate analysis of the most discriminant metabolites and we identified the associated metabolic pathways. We obtained a metabolic pattern from 118 metabolites and we built excellent multivariate models to explain Phe, Tyr concentrations and PKU diagnosis. Common metabolites of these models were identified: Gln, Arg, succinate and alpha aminobutyric acid. Univariate analysis showed an inverse correlation between Arg, alpha aminobutyric acid and Phe and a positive correlation between Arg, succinate, Gln and Tyr (p < 0.0003). Thus, we highlighted the following pathways: Arg and Pro, Ala, Asp and Glu metabolism. We obtain a specific metabolic signature related to Tyr and Phe concentrations. We confirmed the involvement of different pathophysiological mechanisms previously described in PKU such as protein synthesis, energetic metabolism and oxidative stress. The metabolomics approach is relevant to explore PKU pathogenesis.

Comprehensive elucidation of amino acid profile in human follicular fluid and plasma of in vitro fertilization patients

The assessment of oocyte quality in human in vitro fertilization (IVF) is in focus of intensive studies. Follicular fluid (FF) constitutes the major medium for the developing oocyte and therefore it is a perfect target to search for the biomarkers of female infertility. The objective of this study was to compare the amino acid (AA) profile of human FF and plasma (PL) (from 96 IVF patients) and to examine if the AA composition is related to oocyte quality, IVF results and women’s infertility. In both biological fluids, Gln, Gly and Ala appeared as dominant AAs. Most of AAs are more abundant in PL; the exceptions are Glu, Thr, Ala and Gly, which are higher in FF and Gln, Arg and Phe, the contents of which are similar in both biological fluids. Ser in FF and Met and Phe in PL were detected as potential biomarkers as their content varied depending on the IVF outcome. Significant differences were also detected between the groups of different infertility reasons. Our results suggest that intra-follicular AAs might reflect the condition of the preovulatory follicle and together with PL, AAs can be used to characterize the infertility etiology and oocyte quality related to IVF outcome.

Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the phenylalanine in human plasma

Phenylalanine (Phe) is the most reliable indicator for the diagnosis of phenylketonuria (PKU). The purpose of this study is to establish a reliable and quick method for the assignment of Phe in peripheral capillary blood from newborns and children by high performance liquid chromatography with ultraviolet detection (HPLC-UV). Phenylketonuria (PKU) is an inborn error of metabolism characterized by the inability of the body to use phenylalanine (Phe). A rapid and sensitive high performance liquid chromatographic (HPLC) method has been developed for determination of phenylalanine in plasma. The method uses a protein precipitation step with sulfosalicilic acid for sample preparation by separation on a Nova-pack C18 column using sodium acetate buffer and acetonitrile (94: 6 v/v) adjusted to pH 6.5 with glacial acetic acid. The eluted peaks detected by a UV detector was set at wavelength of 215 nm. The method was validated in the range of phenylalanine concentrations from 0.1 to 20µg/ml. The limits of detection (LOD) and quantitation (LOQ) of the method were 0.05 and 0.1µg/ml, respectively. The average drug recovery from plasma was 88.60 percent throughout the linear concentration range., with the average within-run and between-run accuracy values of 103.3 and 115.350, respectively. The method is quick, easy, very steady and precise for the screen, assignment, and evaluation of Phe in human plasma by HPLC, which is particularly a useful way for screening and diagnosis of PKU and monitoring of a diet therapy.

Effects of ruminally protected methionine and/or phenylalanine on performance of high producing Holstein cows fed rations with very high levels of canola meal

Canola meal is the second largest protein feed in the Northern latitudes and inclusion levels in dairy rations are expected to increase due to projected large increases in production of canola seed in Canada. However, a recent study (Swanepoel et al., 2014) showed that even though higher inclusions of canola meal (CM) had a positive effect on production when CM directly substituted for high protein corn based dried distillers grains (DDG), that there was an optimum point at 120–135g/kg of diet dry matter (DM) after which animal performance seemed to decline. Only the amino acids (AA), methionine (Met), phenylalanine (Phe) and leucine (Leu) could have limited production based upon plasma AA concentrations at the highest CM inclusion level. Our objective was to determine if either Met or Phe, or both, was limiting performance of early lactation dairy cows fed a ration containing 180g/kg of diet DM as CM, by supplementing a calculated target of 7.5g of intestinally absorbable Phe/cow/day and/or 8.0g of intestinally absorbable Met/cow/day in ruminally protected (RP) forms to four pens of ∼320 early lactation cows/pen in a 4×4 Latin square with 28 days experimental periods. Dry matter intake was not affected (avg.: 27.6±0.4kg/day) by feeding either of the RP AA, or the combination. Phenylalanine supplementation alone had no effect on milk production or composition, and body condition score (BCS) change compared to Control. Supplemental Met alone modestly increased (P<0.01) milk protein and fat content, while decreasing (P<0.01) milk lactose content and yield, but with no impact on BCS change compared to Control. Combination Met and Phe supplementation decreased milk and lactose yields, as well as lactose content (P<0.01), while increasing milk protein content and the BCS change (P<0.01). Urine volume (avg.: 16.7±0.31L/day) and flow of microbial protein (MCP) from the rumen (avg.: 2092±52.7g CP/day) were not affected by any treatment. Plasma Met levels increased (P<0.01) with both Met treatments and plasma tryptophan (Trp) levels decreased (P<0.01) with both Phe treatments. However, plasma Phe levels did not change with any treatment. Results are interpreted to suggest that delivery of Met with RP Met feeding was higher than animal requirements and caused an oversupply of Met. Addition of Phe to the Met supplementation changed the way energy was utilized by the cows, redirecting energy liberated by Met from milk components toward BCS gain. It remains unclear if Phe was limiting in the Control ration or if RP Phe was not fed at high enough levels to have a measurable response on production. However, it is clear that AA limitations, requirements and production responses are governed by much more than plasma AA levels. Results further suggest that AA are bioactive metabolites to the extent that they can change animal performance, even when they are not “limiting” per se, and that their supplementation to practical dairy cattle diets should be approached with extreme caution for this reason.

Effect of initiating enteral protein feeding on whole-body protein turnover in critically ill patients

Critically ill patients are susceptible to protein catabolism. Enteral feeding may ameliorate protein loss, but its effect is not well characterized in terms of protein kinetics. We established a method of quantifying the effect of enteral protein feeding on whole-body protein turnover and studied critically ill patients receiving early enteral nutrition. In a proof-of-concept study, we established, in healthy subjects (n = 6), a method of measuring the effect of continuous enteral protein feeding on whole-body protein turnover by using ¹³C-phenylalanine (¹³C-Phe) intrinsically labeled casein by a nasogastric feeding tube and an intravenous ²H₅-Phe tracer. The protocol was applied to study critically ill patients (n = 10) during the initial hypocaloric-hyponitrogenous dose of enteral nutrition. Patients were catabolic with a negative protein balance. The median splanchnic extraction fraction of hourly dietary Phe intake was 92% (range: 86-99%); that is, the availability of dietary Phe in arterial plasma was low. In patients with a stable parenteral amino acid supply (n = 7), the median net protein balance improved during enteral feeding from -8.6 to -5.8 μmol · kg body weight⁻¹ · h⁻¹ (P = 0.018). Whole-body protein turnover and the contribution of dietary protein can be quantified in critically ill patients by using intravenous and enteral stable-isotope Phe tracers. The whole-body protein balance improved during early hypocaloric-hyponitrogenous enteral protein feeding in these patients.

Metabolome-wide association study of phenylalanine in plasma of common marmosets.

Little systematic knowledge exists concerning the impacts of cumulative lifelong exposure, termed the exposome, on requirements for nutrients. Phenylalanine (Phe) is an essential dietary amino acid with an aromatic ring structure similar to endogenous metabolites, dietary compounds and environmental agents. Excess plasma Phe in genetic disease or nutritional deficiency of Phe has adverse health consequences. In principle, structurally similar chemicals interfering with Phe utilization could alter Phe requirement at an individual level. As a strategy to identify components of the exposome that could interfere with Phe utilization, we tested for metabolites correlating with Phe concentration in plasma of a non-human primate species, common marmosets (Callithrix jacchus). The results of tests for more than 5,000 chemical features detected by high-resolution metabolomics showed 17 positive correlations with Phe metabolites and other amino acids. Positive and negative correlations were also observed for 33 other chemicals, which included matches to endogenous metabolites and dietary, microbial and environmental chemicals in database searches. Chemical similarity analysis showed many of the matches had high structural similarity to Phe. Together, the results show that chemicals in marmoset plasma could impact Phe utilization. Such chemicals could contribute to early lifecycle developmental disorders when neurological development is vulnerable to Phe levels.

PO-0094 Anthropometric Characteristics And Bone Mineral Density In Patients With Phenylketonuria

Background Phenylketonuria (PKU) treatment requires a diet restricted from natural proteins and supplemented with phenylalanine (Phe)-free L-amino acid mixtures. Growth impairment and compromised bone mass have been described. This study aims to evaluate anthropometric characteristics and bone mineral density (BMD) in a cohort of PKU patients. Methods We conducted a retrospective longitudinal study collecting anthropometric characteristics (weight, height, body mass index (BMI) and BMD every 6 months from birth to 12 years of age in 34patients with diet restrictions. We compared the data results, expressed as z-scores, with the general population, as well as between patients with Phe 360 mmol/ml. Results Our PKU patients are shorter than the reference population; the sample mean was below z–score=0. Weight was comparable to that of the reference population and BMI had a tendency to be over the population mean. Growth impairment in PHA-deficiency is not related to plasma Phe concentration at birth but might be related to its levels throughout the follow up; patients with BMD was below the population mean in all cases (52% osteopenia). Conclusions PKU children in our study have a below than average height. Weight is consistent with the population average and BMI tends to be above it. Height seems more affected among those patients with better metabolic control. BMD is below the population mean. These data do not vary depending on the levels of Phe at diagnosis, but by the phenylalaninemia during growth. Further studies are needed to investigate the effect of diet restriction in PKU.

Tryptophan requirement of pregnant sows1

Amino acid requirements of sows may change from early to late gestation due to the accelerated growth of products of conception after d 70 of pregnancy. The objective of this study was to determine the Trp requirement, Phe kinetics, and energy expenditure in early (d 35 to 53) and late (d 92 to 111) pregnancy using the indicator amino acid oxidation method and indirect calorimetry. The same 6 second parity sows were fed 6 diets in a Latin square design in both early and late pregnancy. The diets based on corn, corn starch, and sugar provided 20 to 120% of the current recommended Trp intake (2.5 g/d) in early pregnancy and 60 to 180% in late pregnancy. Feed allowance was constant for each sow at 2.41 kg/d (SE 0.029). Expired air and blood were collected every 30 min for 5 1/2 h. After three 30-min periods to determine background (13)C enrichment in expired CO2 and plasma Phe, L[1-(13)C]Phe was given orally at a rate of 2 mg/(kg BW · h) with 8 1/2 hourly meals. Expired air and plasma were analyzed for (13)CO2 and (13)C-Phe enrichment, respectively. Requirements were determined as the breakpoint of 2-phase linear models. Sows grew from 167.7 kg (SE 3.93) at breeding to 211.9 kg (SE 5.18) post-farrowing and had litters of 14.5 piglets (SE 0.43) weighing 19.0 kg (SE 1.41) at birth. The Trp requirement was 1.7 g/d (SE 0.29, P = 0.001) in early pregnancy and 2.6 g/d (SE 0.37, P = 0.013) in late pregnancy, or 0.7 g/kg and 1.1 g/kg diet of total Trp, respectively, for a feed allowance of 2.4 kg/d. The Trp requirement in late pregnancy tended (P = 0.056) to be greater than in early pregnancy. Quantitative Phe kinetics were not affected by Trp intake except for a quadratic response of Phe oxidation and retention (P < 0.1) to Trp intake in early pregnancy. In late pregnancy, sows oxidized less Phe and retained more Phe (P = 0.001) than in early pregnancy, indicating that young, growing pregnant sows increase the efficiency of utilizing AA in late pregnancy to maintain protein synthesis in both maternal and fetal tissues. Oxidation and body protein breakdown contributed less to Phe flux in late than early pregnancy while protein synthesis contributed more (P < 0.01). Heat production and energy retention were not affected by Trp level or stage of gestation. To meet both energy and AA requirements in late gestation, a phase feeding program with 2 diets is recommended. The feed allowance in late pregnancy should be greater than in early pregnancy to account for the increased energy expenditure.

Modelo de atención de la fenilcetonuria (PKU) en Uruguay

The Newborn screening program (PN) in Uruguay began in 1990; in 2007 the PN on filter paper was mandatory for phenylketonuria (PKU) and currently has a near 100% coverage, which ensures the detection, validation, monitoring and treatment of all patients. Many public institutions are committed to the National Newborn Screening (SNPN) but sample processing is centralized at the PN Laboratory which belongs to the Management of Social Welfare Bank Laboratories for Social Security Institute in Montevideo (BPS). From 2008 phenylalanine (Phe) quantification is perfomed in tandem mass spectrometry (MS/MS). The amount of Phe tolerance is titrated in patients with levels between 150 and 360 uml /L; above 360 uml /L treatment is started by an interdisciplinary health professional team. Phe-free formula is purchased by the BPS and provided to the patients at no cost, regardless if it is a public or private health system. The biochemical goal is to maintain Phe levels below 360 umol /L with frequent analysis of plasma Phe with a simultaneous 3 day diet diary. Phe control is performed weekly for the first six months and then every 15 days until the age of five. From 2007 to 2011, the PN has found: 7 patients with PKU, 5 persistent hyperphenylalaninemias, 3 patients with transient hyperphenylalaninemias, one patient with tetrahydrobiopterin deficiency, 8 patients were diagnosed late but had an improved behavior and attention when they were treated.