Phe Transport Research Articles

Phenylalanine Losses in Neutralization Dialysis: Modeling and Experiment.

A non-steady state mathematical model of an amino acid (phenylalanine (Phe)) and mineral salt (NaCl) solution separation by neutralization dialysis (ND) carried out in a batch mode is proposed. The model takes into account the characteristics of membranes (thickness, ion-exchange capacity, and conductivity) and solutions (concentration, composition). As compared to previously developed models, the new one considers the local equilibrium of Phe protolysis reactions in solutions and membranes and the transport of all the phenylalanine forms (zwitterionic, positively and negatively charged) through membranes. A series of experiments on ND demineralization of the NaCl and Phe mixed solution was carried out. In order to minimize Phe losses, the solution pH in the desalination compartment was controlled by changing the concentrations of the solutions in the acid and alkali compartments of the ND cell. The validity of the model was verified by comparison of simulated and experimental time dependencies of solution electrical conductivity and pH, as well as the concentration of Na+, Cl- ions, and Phe species in the desalination compartment. Based on the simulation results, the role of Phe transport mechanisms in the losses of this amino acid during ND was discussed. In the experiments carried out, the demineralization rate reached 90%, accompanied by minimal Phe losses of about 16%. Modeling predicts a steep increase in Phe losses when the demineralization rate is higher than 95%. Nevertheless, simulations show that it is possible to achieve a highly demineralized solution (by 99.9%) with Phe losses amounting to 42%.

Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria.

Phenylketonuria (PKU), a disease resulting in the disability to degrade phenylalanine (Phe) is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity. As an potential alternative to a protein-restricted diet, oral intake of engineered probiotics degrading Phe inside the body is a promising treatment, currently at clinical stage II (Isabella, et al., 2018). However, limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic’s activity. Here, we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL) as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917 (EcN) which overcomes the transportation problem. The metabolic engineering strategy was also combined with strengthening of Phe transportation, transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia. Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the PahF263S PKU mouse model reduced blood Phe concentration by 44.4% compared to the control EcN, independent of dietary protein intake. TYS8500 shows great potential in future applications for PKU therapy.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-2137-3. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

Predicting seasonal fate of phenanthrene in aquatic environment with a Markov chain.

Phenanthrene (Phe) with carcinogenicity is ubiquitous in the environment, especially in aquatic environment; its toxicity is greater. To help determine toxicity risk and remediation strategies, this study predicted seasonal fate of Phe in aquatic environment. Candidate mechanisms including biodegradation, sorption, desorption, photodegradation, hydrolysis and volatility were studied; the results for experiments under simulated conditions for normal, wet and dry seasons in the Yinma River Basin indicated that biodegradation in sediment, sorption, desorption, and volatility were important pathways for elimination of Phe from aquatic environment and showed seasonal variations. A microcosm which was used to mimic sediment/water system was set up to illustrate seasonal distribution and transport of Phe. A Markov chain was applied to predict seasonal fate of Phe in air/water/sediment environment, the predicted results were perfectly agreed with results of microcosm experiments. Predicted results with a Markov chain suggested that volatility and biodegradation in sediment were main elimination pathways, and contributions of elimination pathways showed seasonal variations; Phe was eliminated from water and sediment to negligible levels over around 250h in August and over 1000h in May; in November, Phe was eliminated from water to a negligible level while about 31% of Phe amount still remained in sediment over 1000h.

Transport of persistent organic pollutants by microplastics in estuarine conditions

Microplastics represent an increasing source of anthropogenic contamination in aquatic environments, where they may also act as scavengers and transporters of persistent organic pollutants. As estuaries are amongst the most productive aquatic systems, it is important to understand sorption behaviour and transport of persistent organic pollutants (POPs) by microplastics along estuarine gradients. The effects of salinity sorption equilibrium kinetics on the distribution coefficients (Kd) of phenanthrene (Phe) and 4,4′-DDT, onto polyvinyl chloride (PVC) and onto polyethylene (PE) were therefore investigated. A salinity gradient representing freshwater, estuarine and marine conditions, with salinities corresponding to 0 (MilliQ water, 690 μS/cm), 8.8, 17.5, 26.3 and 35 was used. Salinity had no significant effect on the time required to reach equilibrium onto PVC or PE and neither did it affect desorption rates of contaminants from plastics. Although salinity had no effect on sorption capacity of Phe onto plastics, a slight decrease in sorption capacity was observed for DDT with salinity. Salinity had little effect on sorption behaviour and POP/plastic combination was shown to be a more important factor. Transport of Phe and DDT from riverine to brackish and marine waters by plastic is therefore likely to be much more dependent on the aqueous POP concentration than on salinity. The physical characteristics of the polymer and local environmental conditions (e.g. plastic density, particle residence time in estuaries) will affect the physical transport of contaminated plastics. A transport model of POPs by microplastics under estuarine conditions is proposed. Transport of Phe and DDT by PVC and PE from fresh and brackish water toward fully marine conditions was the most likely net direction for contaminant transport and followed the order: Phe-PE >> DDT-PVC = DDT-PE >> Phe-PVC.

Advances in the nutritional and pharmacological management of phenylketonuria.

The purpose is to discuss advances in the nutritional and pharmacological management of phenylketonuria (PKU). Glycomacropeptide (GMP), a whey protein produced during cheese production, is a low-phenylalanine (phe) intact protein that represents a new dietary alternative to synthetic amino acids for people with PKU. Skeletal fragility is a long-term complication of PKU that based on murine research, appears to result from both genetic and nutritional factors. Skeletal fragility in murine PKU is attenuated with the GMP diet, compared with an amino acid diet, allowing greater radial bone growth. Pharmacologic therapy with tetrahydrobiopterin, acting as a molecular chaperone for phenylalanine hydroxylase, increases tolerance to dietary phe in some individuals. Large neutral amino acids inhibit phe transport across the intestinal mucosa and blood-brain barrier, and are most effective for individuals unable to comply with the low-phe diet. Although a low-phe synthetic amino acid diet remains the mainstay of PKU management, new nutritional and pharmacological treatment options offer alternative approaches to maintain lifelong low phe concentrations. GMP medical foods provide an alternative to amino acid formula that may improve bone health, and tetrahydrobiopterin permits some individuals with PKU to increase tolerance to dietary phe. Further research is needed to characterize the long-term efficacy of these new approaches for PKU management.

Interaction of phenanthrene and potassium uptake by wheat roots: a mechanistic model

BackgroundPolycyclic aromatic hydrocarbons (PAHs) are potentially carcinogenic, mutagenic and toxic to both human and non-human organisms. Dietary intake of PAHs is a dominant route of exposure for the general population where food crops are a major source of dietary PAHs. Over 20% of main food crops contain PAHs that exceed the control limits in China. However, the mechanisms on PAH accumulation in crops are not well understood.ResultsHere we report the physiological mechanism of potassium (K+)-stimulated uptake of phenanthrene (PHE, a model PAH) in wheat. PHE uptake is stimulated by the external K+. The addition of blockers (tetraethlyammonium and barium) for K+ channels does not suppress the process, suggesting that K+ channels are not involved. The introduction of PHE and K+ elicits a much greater depolarization in root cell membrane potential than that of either PHE or K+. K+ activates the plasma membrane proton (H+)-ATPase in a K+-dependent manner. The pattern is quite similar to that in PHE uptake in the presence of K+. The external medium pH treated with PHE and K+ is higher than that with K+, and lower than that with PHE, indicating that H+ pump involves in the interaction between PHE and K+ uptake.ConclusionsTherefore, it is concluded that a K+ influx/H+ efflux reaction is coupled with the transport of PHE into wheat root cells. Our results provide a novel insight into the PHE uptake by crop roots.

Non‐physiological amino acid (NPAA) therapy targeting brain phenylalanine reduction: pilot studies in PAHENU2 mice

Transport of large neutral amino acids (LNAA) across the blood brain barrier (BBB) is facilitated by the L-type amino acid transporter, LAT1. Peripheral accumulation of one LNAA (e.g., phenylalanine (phe) in PKU) is predicted to increase uptake of the offending amino acid to the detriment of others, resulting in disruption of brain amino acid homeostasis. We hypothesized that selected non-physiological amino acids (NPAAs) such as DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), 2-aminoisobutyrate (AIB), and N-methyl-aminoisobutyrate (MAIB), acting as competitive inhibitors of various brain amino acid transporters, could reduce brain phe in Pah (enu2) mice, a relevant murine model of PKU. Oral feeding of 5 % NL, 5 % AIB, 0.5 % NB and 3 % MAIB reduced brain phe by 56 % (p < 0.01), -1 % (p = NS), 27 % (p < 0.05) and 14 % (p < 0.01), respectively, compared to untreated subjects. Significant effects on other LNAAs (tyrosine, methionine, branched chain amino acids) were also observed, however, with MAIB displaying the mildest effects. Of interest, MAIB represents an inhibitor of the system A (alanine) transporter that primarily traffics small amino acids and not LNAAs. Our studies represent the first in vivo use of these NPAAs in Pah (enu2) mice, and provide proof-of-principle for their further preclinical development, with the long-term objective of identifying NPAA combinations and concentrations that selectively restrict brain phe transport while minimally impacting other LNAAs and downstream intermediates.

Dietary Glycomacropeptide Supports Growth and Reduces the Concentrations of Phenylalanine in Plasma and Brain in a Murine Model of Phenylketonuria ,

Phenylketonuria (PKU) is a genetic disorder caused by deficiency of phenylalanine hydroxylase (PAH) that requires life-long adherence to a low-phenylalanine (Phe) diet. Glycomacropeptide (GMP) is uniquely suited to the nutritional management of PKU, because pure GMP contains no Phe. Our aim was to assess how ingestion of diets containing GMP support growth and affect the concentrations of amino acids in plasma and brains of mice with a deficiency of PAH, the Pahenu2 mouse (PKU mouse). Experiments were conducted in 4- to 6-wk-old wild-type (WT) (C57Bl/6) and PKU mice fed diets containing 20% protein from casein, amino acids, or GMP supplemented with limiting indispensable amino acids (IAA). PKU mice fed the GMP diet showed gains in body weight, feed efficiency, and a protein efficiency ratio that did not differ from the amino acid diet. The concentrations of isoleucine and threonine in plasma showed a significant 2- to 3-fold increase for WT and PKU mice fed GMP compared with casein or amino acid diets, respectively. PKU mice fed the GMP diet had decreased concentrations of Phe in plasma (11% decrease) and in 5 regions of the brain (20% decrease) compared with the amino acid diet. The concentration of Phe in the brain was inversely correlated with the concentrations of isoleucine, threonine, and valine in plasma (R2 = 0.74; P < 0.0001), suggesting competitive inhibition of Phe transport into the brain. In summary, PKU mice fed GMP showed comparable growth and reduced concentrations of Phe in plasma and the brain compared with an amino acid diet. These data support the use of GMP supplemented with IAA as an alternative source of dietary protein for individuals with PKU.

Inter-individual variation in brain phenylalanine concentration in patients with PKU is not caused by genetic variation in the 4F2hc/LAT1 complex

It remains a question why some patients with phenylketonuria (PKU) have high IQ and low brain phenylalanine (Phe) concentrations in spite of high blood Phe levels. One possible explanation for the low brain Phe concentrations in these patients would be a reduced transport of Phe across the blood–brain barrier. The 4F2hc/LAT1 complex has been suggested to be the most important molecular component responsible for this transport. To test the hypothesis that structural variant(s) in the genes encoding 4F2hc and LAT1 might result in a complex with reduced affinity for Phe, we have screened the two genes for sequence variants in a group of 13 PKU patients with a low ratio of brain to blood Phe concentrations. Several common sequence variants were identified, but none of these is predicted to affect the resulting protein product. Our data suggest that individual vulnerability to Phe in patients with PKU is not due to structural variants in the 4F2hc/LAT1 complex.

Brain Imaging and Proton Magnetic Resonance Spectroscopy in Patients With Phenylketonuria

Magnetic resonance imaging studies in patients with phenylketonuria (PKU) revealed white matter alterations that correlated to most recent blood phenylalanine (Phe) concentrations as well as to brain Phe concentrations measured by magnetic resonance spectroscopy. The clinical significance of these changes is unknown. Magnetic resonance imaging data thus have no impact on therapeutic recommendations for adolescents and adults with PKU. Kinetic investigations of patients by magnetic resonance spectroscopy showed differences in brain Phe concentrations despite similar blood Phe levels. These were influenced by interindividual variations of blood-brain barrier Phe transport constants and by variations of the individual brain Phe consumption rate. Blood-brain barrier Phe transport characteristics as well as brain Phe consumption rates thus seem to be causative factors for the individual outcome in PKU.

Phenylalanine can be detected in brain tissue of healthy subjects by 1H magnetic resonance spectroscopy

Transport of phenylalanine (Phe) and the other large neutral amino acids across the blood-brain barrier plays a crucial role in the pathogenesis of phenylketonuria (PKU). Thus, investigation of Phe transport kinetics by means of proton magnetic resonance spectroscopy (1H MRS) became an important research area in the mid 1990s. As 1H MRS measurements of brain phenylalanine are restricted to tissue concentrations above 100-150 micromol/kg wet weight, this approach was possible only in PKU patients, and comparison with healthy controls was not achieved. Using standardized single-dose oral Phe loading in three healthy subjects, it was shown that Phe values increase steeply, peak at about 1 h post load, and decrease thereafter. In a single case study, repetitive Phe loading was then performed to achieve a plateau of high blood Phe concentrations for several hours. It was demonstrated that detection and monitoring of brain Phe concentrations is feasible by means of 1H MRS. This approach constitutes a prerequisite for describing carrier kinetics in health.

Facilitated transport and separation of aromatic amino acids through activated composite membranes

Activated composite membranes (ACM) containing bis-(2-ethyl-hexyl) phosphoric acid (DEHPA) have been found to facilitate the transport and separation of aromatic amino acids. The ACM, prepared according to a previously established procedure, were used to experimentally determine their behavior for the transport of phenylalanine (Phe), that was used as target amino acid. A general characterization of the Phe transport through DEHPA-ACM membranes including: the existence of facilitated counter transport, reproducibility and the effect of buffering the feed solution has been performed prior to a systematic study of the influence of some chemical transport parameters. In this concern, the initial Phe concentration, the nature and concentration of the stripping agent and the pH of the feed solution have been optimized (the feed phase was 0.5 mM of Phe in a 2 M NaCl solution at pH 3 and a 2 M HCl solution was used as stripping phase). Also, the influence of the DEHPA concentration in the casting solution used to prepare the membrane is established. It is found that the membrane phase controls the rate transport up to a DEHPA concentration of 1200 mM. Finally, although the affinity of the aromatic amino acids phenylalanine, tryptophan (Trp) and tyrosine (Tyr) for DEHPA is in the order Phe>Trp>Tyr, the selectivity of the DEHPA-ACM was found to be Trp>Phe>Tyr.

In vivo proton magnetic resonance spectroscopy in phenylketonuria.

In vivo nuclear magnetic resonance spectroscopy permits the non-invasive examination of metabolic characteristics of the human brain in a clinical environment. Methods to detect elevated phenylalanine (Phe) in patients with phenylketonuria (PKU) using difference spectroscopy and to estimate absolute brain Phe concentrations, [Phe]brain, have been developed. In patients with classical PKU, [Phe]brain typically varied between 0.14 and 0.78 mmol/l depending upon actual blood Phe concentrations, [Phe]blood, between 0.47 and 2.30 mmol/l. Dynamic investigations can be used to extract information about Phe transport at the human blood-brain barrier, which may be described by a symmetric Michaelis-Menten model. Carrier saturation and competitive inhibition of the influx of other large neutral amino acids can be expected at blood levels usually found in PKU patients. In single cases of untreated, normal intelligent patients, abnormally low [Phe]brain < or = 0.15 mmol/l were observed despite high stationary Phe levels ([Phe]blood = 1.15 +/- 0.10 mmol/l). Significant variations in phenylalanine transport parameters in untreated, normal intelligent patients indicated that blood-brain barrier transport or intracerebral phenylalanine consumption are causative factors for the individual vulnerability to phenylketonuria.

Modeling of Facilitated Transport of Phenylalanine by Emulsion Liquid Membranes with Di(2-ethylhexyl)phosphoric Acid as a Carrier

ABSTRACT A mathematical model is developed in this paper to simulate the facilitated transport of phenylalanine (Phe) in emulsion liquid membrane (ELM) systems with di(2-ethylhexyl)phosphoric acid as a carrier. The model takes into account the mass transfer in both the external aqueous phase and the organic membrane phase interfacial reaction as well as membrane breakage during agitation. The model is tested by comparing theoretical predications with experimental results using Phe extraction by ELM processes. It is found that the model is valid for simulating the facilitated transport of Phe with ELM under various experimental conditions.

Kinetics of phenylalanine transport at the human blood–brain barrier investigated in vivo

In vivo proton magnetic resonance spectroscopy was used to investigate intracerebral phenylalanine (Phe) concentrations in nine patients with classical phenylketonuria (PKU). The study included serial examinations ( n=31; plasma Phe levels: 0.47–2.24 mmol/l) of patients either receiving a Phe-restricted diet (200 mg Phe per day; four patients) or a diet rich in Phe (1000 mg Phe per day; three patients). No spectrum showed metabolic abnormalities besides elevated Phe. Difference spectroscopy yielded intracerebral Phe concentrations between 0.20 and 0.76 mmol/l. Regional variations between parieto-occipital periventricular brain, frontal brain, and cerebellum were not statistically significant. Data could be fitted assuming saturable Phe transport into the brain, based on a symmetric Michaelis–Menten model (characterized by an apparent Michaelis transport constant, K t,app, and a maximum transport velocity, T max) and constant Phe consumption in the brain cells (described by a velocity V max). Non-linear least-squares fitting of the combined data from all patients yielded K t,app=0.16±0.11 mmol/l and ( T max / V max)=9.0±4.1. Carrier saturation and competitive inhibition of the influx of other large neutral amino acids can be expected at plasma Phe levels usually found in PKU patients.

In-vivo NMR spectroscopy in patients with phenylketonuria: changes of cerebral phenylalanine levels under dietary treatment.

Localized proton magnetic resonance spectroscopy at short echo times was used to measure phenylalanine (Phe) in parieto-occipital periventricular brain. Six treated adult patients with phenylketonuria were investigated repeatedly following reinstitution of a Phe-restricted diet. Difference spectroscopy clearly enabled the identification of elevated cerebral Phe levels by subtracting spectra obtained from healthy volunteers. Estimates of absolute brain concentrations always yielded values well below the serum levels with ratios [Phe]brain/[Phe]serum ranging from 0.27 to 0.63. A plot of [Phe]brain versus [Phe]serum could be fitted to a straight line (R = 0.90) if [Phe]serum was below 1.3 mM. Measurements at higher serum levels could only be performed in one patient and yielded brain Phe concentrations of 0.63 +/- 0.10 mM suggesting a saturation of the carrier systems. The feasibility to quantify Phe transport across the blood-brain barrier in humans non-invasively employing in-vivo proton spectroscopy can effectively improve the prognostic significance of serum data.

Peptide transporter function and prolidase activities in Caco-2 cells: a lack of coordinated expression.

Peptide transport and prolidase activities were measured to determine whether the expression of these two components of protein nutrition are coordinately regulated; i.e., whether an increase in the peptide transporter function will necessarily lead to a higher prolidase activity, or vice versa. The results indicated that peptide transporter function and prolidase activity respond differently to cell differentiation and feeding schedules. The results also indicated that peptide transport and prolidase activities were different in two Caco-2 cell "clones", with S-K cells transported peptides at higher rates but had lower total prolidase activities, when compared to ATCC cells. These results suggest that the expression of the peptide transporter function and prolidase activity is not coordinated. In addition, both the transporter and the prolidase activities affected the overall transport of Phe when given as the dipeptide Phe-Pro, supporting the notion that intestinal absorption of peptides is an essential component of amino acid absorption. In conclusion, the evidence suggests that the peptide transporter function and prolidase activity are not coordinately expressed by the human intestinal Caco-2 cells.

Transport of a large neutral amino acid in a human intestinal epithelial cell line (Caco-2): uptake and efflux of phenylalanine

The processes of l-phenylalanine (Phe) uptake and efflux from the apical (AP) and basolateral (BL) sides of an intestinal epithelial cell line (Caco-2) were investigated to further characterize the mechanism of transcellular transport of this amino acid. The results indicated that the initial uptake rates of Phe were saturable with a K m of 2.7 nM for AP uptake and 0.18 mM for BL uptake. Unlike the uptake, the initial efflux rates were shown to be proportional to the intracellular concentrations of Phe. Based on these kinetic studies and determination of other characteristics (e.g., Na + dependency) of the uptake and efflux processes, it was concluded that AP uptake, BL uptake and BL efflux were distinctly different. This suggests that either different carriers or a different combination of carriers are responsible for the transmembrane transport of this amino acid. When the results of kinetic studies of Phe uptake and efflux were used to determine the rate-limiting step in the AP-to-BL transcellular transport of this amino acid, it was concluded that the BL efflux is the rate-limiting step in the transcellular transport of Phe in the Caco-2 cell monolayers.

Intestinal glycyl-L-phenylalanine and L-phenylalanine transport in a euryhaline teleost

The transport mechanisms for the dipeptide glycyl-L-phenylalanine (Gly-Phe) and L-phenylalanine (Phe) were characterized in fish intestinal brush-border membrane vesicles (BBMV). Gly-Phe was rapidly hydrolyzed only intravesicularly with almost total hydrolysis occurring even at 10 s. Dipeptide uptake was not stimulated by an inward gradient of Na, K, or H. Phe uptake was stimulated by an inward gradient of either Na or K but displayed an overshoot phenomenon only in the presence of an Na gradient. Kinetic analysis of the effect of substrate concentration on transport rate revealed that transport of both Gly-Phe and Phe occurred by a saturable process conforming to Michaelis-Menten kinetics. The Km for Gly-Phe was 9.8 +/- 3.5 mM, whereas that for Phe in the presence of Na or K, respectively, was 0.74 +/- 0.13 and 1.1 +/- 0.37 mM. Maximum uptake for Gly-Phe and for Phe in the presence of Na and K was 5.1, 0.9, and 0.4 nmol.mg and protein-1.5 s-1, respectively. Gly-Phe and Phe transport displayed different patterns of inhibition by dipeptides and amino acids. These results suggest that Gly-Phe and Phe are transported via different mechanisms, with Gly-Phe being hydrolyzed during a carrier-mediated, cation-independent process and Phe being transferred via a Na+ cotransport process similar to that described in mammals. During conditions of high luminal dipeptide concentrations, the Gly-Phe pathway may make a significant contribution to total Phe uptake.

Transport of a large neutral amino acid (phenylalanine) in a human intestinal epithelial cell line: Caco-2

We have characterized the transcellular transport of a large neutral amino acid (LNAA) in Caco-2 cell monolayers. The apical (AP) to basolateral (BL) transport of phenylalanine (Phe) was approximately 10-times faster than BL-to-AP transport. The evidence for the carrier-mediated AP-to-BL transport of Phe include: (a) temperature dependence and saturability, (b) Phe transport was not affected by a reverse gradient, (c) the activation energy for transport was 12.0 kcal/mol, and (d) an excess amount of unlabeled Phe caused a 75% reduction in transport rate and a delay (lag time) in the appearance of Phe in the BL side. The V m and K m for Phe transport were 572.4 pmol·mg protein −1·min −1 and 0.56 mM, respectively. Phe transport was decreased in the absence of glucose and in the presence of sodium azide or ouabain. The carrier interacted with LNAAs and with cationic amino acids but not with small neutral or anionic amino acids.