Influx Of Phenylalanine Research Articles

Presumptive brain influx of large neutral amino acids and the effect of phenylalanine supplementation in patients with Tyrosinemia type 1.

IntroductionHereditary Tyrosinemia type 1 (HT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Current treatment consists of 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and a tyrosine and phenylalanine restricted diet. Recently, neuropsychological deficits have been seen in HT1 patients. These deficits are possibly associated with low blood phenylalanine concentrations and/or high blood tyrosine concentrations. Therefore, the aim of the present study was threefold. Firstly, we aimed to calculate how the plasma amino acid profile in HT1 patients may influence the presumptive brain influx of all large neutral amino acids (LNAA). Secondly, we aimed to investigate the effect of phenylalanine supplementation on presumptive brain phenylalanine and tyrosine influx. Thirdly, we aimed to theoretically determine minimal target plasma phenylalanine concentrations in HT1 patient to ensure adequate presumptive brain phenylalanine influx.MethodsData of plasma LNAA concentrations were obtained. In total, 239 samples of 9 HT1 children, treated with NTBC, diet, and partly with phenylalanine supplementation were collected together with 596 samples of independent control children. Presumptive brain influx of all LNAA was calculated, using Michaelis-Menten parameters (Km) and Vmax-values obtained from earlier articles.ResultsIn HT1 patients, plasma concentrations and presumptive brain influx of tyrosine were higher. However, plasma and especially brain influx of phenylalanine were lower in HT1 patients. Phenylalanine supplementation did not only tend to increase plasma phenylalanine concentrations, but also presumptive brain phenylalanine influx, despite increased plasma tyrosine concentrations. However, to ensure sufficient brain phenylalanine influx in HT1 patients, minimal plasma phenylalanine concentrations may need to be higher than considered thus far.ConclusionThis study clearly suggests a role for disturbed brain LNAA biochemistry, which is not well reflected by plasma LNAA concentrations. This could play a role in the pathophysiology of the neuropsychological impairments in HT1 patients and may have therapeutic implications.

A reassessment of the blood–brain barrier transport of large neutral amino acids during acute systemic inflammation in humans

We reassessed data from a previous study on the transcerebral net exchange of large neutral amino acids (LNAAs) using a novel mathematical model of blood-brain barrier (BBB) transport. The study included twelve healthy volunteers who received a 4-h intravenous lipopolysaccharide (LPS) infusion (total dose: 0·3ng/kg), a human experimental model of the systemic inflammatory response during the early stages of sepsis. Cerebral blood flow and arterial-to-jugular venous LNAA concentrations were measured prior to and after LPS, and the BBB transport and brain extracellular concentrations of LNAAs were calculated. The arterial concentration and unidirectional cerebral influx of phenylalanine increased after LPS. The BBB transport of tyrosine was unaffected, while its concentration in the brain extracellular fluid increased. These findings suggest that LPS infusion leads to an increased cerebral uptake of phenylalanine, which is then metabolized to tyrosine. This may reflect a neuroprotective mechanism that 'detoxifies' excess intracerebral phenylalanine in the clinical setting of sepsis.

Cerebral net exchange of large neutral amino acids after lipopolysaccharide infusion in healthy humans

IntroductionAlterations in circulating large neutral amino acids (LNAAs), leading to a decrease in the plasma ratio between branched-chain and aromatic amino acids (BCAA/AAA ratio), may be involved in sepsis-associated encephalopathy. We hypothesised that a decrease in the BCAA/AAA ratio occurs along with a net cerebral influx of the neurotoxic AAA phenylalanine in a human experimental model of systemic inflammation.MethodsThe BCAA/AAA ratio, the cerebral delivery, and net exchange of LNAAs and ammonia were measured before and 1 hour after a 4-hour intravenous infusion of Escherichia coli lipopolysaccharide (LPS) in 12 healthy young men.ResultsLPS induced systemic inflammation, reduced the BCAA/AAA ratio, increased the cerebral delivery and unidirectional influx of phenylalanine, and abolished the net cerebral influx of the BCAAs leucine and isoleucine. Furthermore, a net cerebral efflux of glutamine, which was independent of the cerebral net exchange of ammonia, was present after LPS infusion.ConclusionsSystemic inflammation may affect brain function by reducing the BCAA/AAA ratio, thereby changing the cerebral net exchange of LNAAs.

Double blind placebo control trial of large neutral amino acids in treatment of PKU: Effect on blood phenylalanine

Large neutral amino acids (LNAA) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In an open-label study using LNAA, a surprising decline of blood Phe concentration was found in patients with PKU in metabolic treatment centres in Russia, the Ukraine, and the United States. To validate the data obtained from this trial, a short-term double-blind placebo control study was done using LNAA in patients with PKU, with the participation of three additional metabolic centres--Milan, Padua and Rio de Janeiro. The results of the short trial showed significant lowering of blood Phe concentration by an average of 39% from baseline. The data from the double-blind placebo control are encouraging, establishing proof of principle of the role of orally administered LNAA in lowering blood Phe concentrations in patients with PKU. Long-term studies will be needed to validate the acceptability, efficacy and safety of such treatment.

Large neutral amino acids in the treatment of phenylketonuria (PKU)

Large neutral amino acids (LNAAs) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In earlier studies on mice with PKU (ENU(2)/ENU(2)), LNAAs were given and a surprising decline in blood Phe concentrations was observed. The formula used in the mouse experiment (PreKUnil) lacked lysine. Therefore, a new formulation of LNAAs (NeoPhe) was developed, introducing changes in the concentration of some amino acids and adding lysine, so that such a mixture could be used in humans. The new formula was found to be effective in reducing blood Phe concentration in mice by about 50% of the elevated levels. Patients with PKU were given LNAAs and blood Phe concentrations were determined in an open-label study. Three centers--in Russia, the Ukraine and the USA--took part in the study. NeoPhe was given at 0.5 g/kg per day in three divided doses to eight subjects with PKU and at 1.0 g/kg per day to three patients, for one week. The NeoPhe resulted in decrease of elevated blood Phe by 50% in both groups. The preliminary data from this study are encouraging and a double blind placebo-controlled trial will be required to show long-term efficacy and tolerance of LNAAs in the treatment of PKU.

Effect of enzymatic digestion on phenylalanine transport across the small intestine

1. 1. Phenylalanine accumulation in mucosal strips isolated from rat small intestine was significantly inhibited ( P<0.01) after preincubation with trypsin, chymotrypsin, phospholipase D and neuraminidase. 2. 2. Unidirectional phenylalanine influx across the small intestine was significantly reduced ( P<0.01) when the mucosal strips were preincubated with the above mentioned enzymes. 3. 3. Intestinal cell water and volume were not significantly changed ( P>0.6) when the intestinal tissues were preincubated with these enzymes.

Electrical properties of a Na +-dependent phenylalanine transport in lizard ( Lacerta galloti) duodenum

1. 1. The unidirectional transepithelial fluxes of l-phenylalanine across lizard duodenum were determined in flux chambers. 2. 2. Phenylalanine was preferentially transferred from the mucosal to the serosal fluid. 3. 3. This transport was accompanied by an accumulation of substrate from the mucosal medium into the tissue to a similar level and against a concentration gradient. 4. 4. There was no net movement of phenylalanine when the sodium was substituted by choline. 5. 5. The influx of l-phenylalanine into the epithalial cells of lizard duodenum was examined by incubating slices of intestine in radioactively-labelled solutions of the substrate for 2 min. 6. 6. The steady-state uptake was assessed after similar incubations lasting 45 min. 7. 7. Phenylalanine influx obeys the Michaelis-Menten equation with a K m of 5.1 and is dependent on the presence of sodium ions in the incubation medium. 8. 8. Phenylalanine has been used to induce changes in short-circuit current ( ΔI sc) across intestine. ΔI sc was a hyperbolic function of amino acid concentration characterized by the parameters J m (maximum change in ΔI sc and K m (concentration needed to attain an ΔI sc equal to half the J m). 9. 9. ΔI sc determined K m constants showed good agreement with values obtained from direct measurements of phenylalanine uptake into tissue.

Cerebral blood flow and blood-brain influx of some neutral amino acids in control and hypothyroid 16-day-old rats.

Rats were made hypothyroid by a daily subcutaneous injection of propylthiouracil beginning the first day after birth. CBF, brain plasma volume, blood-brain extraction, and influx of some neutral amino acids were studied in 16-day-old animals. In hypothyroid rats, the brain plasma volume was decreased by approximately 30%. CBF was decreased by greater than 50%. This decrease was the highest in cerebellum. Blood-brain extraction of small neutral amino acids (alanine, serine, cysteine) was greatly enhanced. This greater extraction compensated for the decreased supply of alanine brought about by its decreased plasma concentration and the lower CBF. In contrast, the extraction of the large amino acids tested (leucine, phenylalanine) was hardly increased, and the influx of phenylalanine was slightly decreased. These results suggest an alteration in the maturation of the brain capillary bed and capillary transport for neutral amino acids in hypothyroidism. The differential effect of hypothyroidism on some small and large amino acids is an additional argument for the existence of two systems of transport for neutral amino acids at the luminal membrane of brain capillary endothelial cells of immature rats.

Transport specificity for neutral and basic amino acids at maternal and fetal interfaces of the guinea-pig placenta.

1. The unidirectional influx of amino acids into the guinea-pig syncytiotrophoblast was measured using a single circulation paired-tracer dilution technique which allows separate characterization of both fetal and maternal interfaces. An in situ preparation perfused through the fetal circulation was used to examine the fetal side, while an isolated preparation perfused through both the fetal and maternal circulations was used to study both interfaces simultaneously.2. On the fetal side the maximal uptake (U(max)) determined at tracer concentrations was high for the short-chain neutral amino acid alanine (76%) and the long-chain neutrals, leucine (75%), phenylalanine (90%) and tyrosine (82%) and for the basic amino acid lysine (65%). In contrast, U(max) was negligible for alpha-methylaminoisobutyric acid and taurine, a beta-amino acid.3. The uptake of alanine and phenylalanine on the fetal side was inhibited by both short-chain (alanine, serine, cysteine) and long-chain (phenylalanine, methionine, leucine) neutral amino acids. d-alanine had no effect on l-alanine uptake whereas d-phenylalanine significantly inhibited that of l-phenylalanine. Diaminobutyric acid, lysine and arginine were effective inhibitors of alanine uptake but had no effect on phenylalanine uptake.4. On the maternal side uptake of alanine, phenylalanine and lysine was measured. Over a wide range of concentrations self-inhibition of alanine influx was similar to the cross-inhibition observed with phenylalanine. In contrast, the influx of phenylalanine, which was strongly self-inhibited, was only partially cross-inhibited by alanine.5. Influx of alanine and phenylalanine was measured at various perfusate concentrations and was found to be saturable on both maternal and fetal sides. The data were fitted to a single hyperbola and, on the maternal side, the K(m) for alanine (10.3+/-2.7 mm, mean+/-s.e., n = 3) was three-fold higher than the value measured for phenylalanine (3.1+/-0.8 mm). On the fetal side the K(m) values for alanine (8.4+/-1.4 mm, n = 4) and phenylalanine (11.9+/-1.9 mm, n = 3) were similar.6. The uptake of alanine, phenylalanine and lysine appeared to be highly sodium-dependent accounting for 40-70% of the total influx. However, the inhibited fractions were found to be different on the two sides of the placenta.7. The results of uptake, cross-inhibition and Na(+)-dependency experiments suggest the presence of an alanine-serine-cysteine (ASC) type system and a leucine (L) type system with markedly overlapping specificities at both the fetal and maternal interfaces. Separate kinetic characterization of a two carrier system was not possible under the conditions of these experiments. However, kinetic parameters for the over-all transport of alanine and phenylalanine were measured.

Structural and functional correlations in the hypertrophic mucosa of intestinal remnants following resection in rats.

Microdissection techniques were applied to determine the absolute increase in villus size and absorptive surface in the mucosa of rat intestinal remnants 3 weeks after 60% proximal resection. Adjacent tissues were used to evaluate the operational kinetic parameters describing the influx of L-phenylalanine and alpha-methyl-D-glucoside into intestinal rings. The villi of ileal remnants were enlarged significantly in all dimensions; the mean surface area of the villus was 2.5 times, and mucosal:serosal surface ratio 1.9 times larger than normal. The influx of phenylalanine and alpha-methyl-glucoside into the mucosa of ileal remnants was reduced, but the change could not be ascribed to an attenuation of one specific kinetic parameter. Using the microdissection data to transform the Vmax-values to a mucosal area basis, we could demonstrate that the Vmax for both substrates was smaller than normal. The results may derive from changes in the maturity of the cell population in intestinal remnants.

Amino acid supply to individual cerebral structures in awake and anesthetized rats.

The movement of phenylalanine from plasma into various cerebral structures was examined in detail in normal alert rats as well as in rats anesthetized with nitrous oxide, halothane, and sodium pentobarbital. Radioactive phenylalanine was infused intravenously in such a way as to rapidly establish and maintain a trace concentration in arterial blood. The rat was decapitated after 1.5 min, the brain removed, frozen, and thin sections cut for quantitative autoradiography. The plasma concentrations of phenylalanine as well as other neutral amino acids that compete with phenylalanine for entry into brain were measured. Phenylalanine influx varied considerably between structures, ranging from 5 nmol.min-1.g-1 in the globus pallidus to 12 nmol.min-1.g-1 in the inferior colliculus. Anesthetics reduced phenylalanine influx, but this effect was primarily due to alterations in the circulating neutral amino acid pattern. The anesthetics had little, if any, direct effect on the neutral amino acid transport process itself.

A kinetic study of the interactions between amino acids and monosaccharides at the intestinal brush-border membrane.

1. The influx of amino acids into guinea-pig intestinal rings in vitro is inhibited by monosaccharides, and that of monosaccharides by amino acids. Two hypotheses have been proposed to account for these heterologous interactions. According to the first, the cis hypothesis, there is an allosteric interaction between substrates binding to separate but related sites at the outer face of the brush-border membrane matrix. In contrast, the trans hypothesis envisages the interaction to result from a partial dissipation of the electrochemical sodium gradient due to the cotransport of each substrate with sodium ions. 2. In an attempt to distinguish between the merits of the two hypotheses, we examined the kinetics of the inhibition of phenylalanine influx by two sugars of widely different affinities, galactose and beta-methylglucoside. Since beta-methylglucoside carries more sodium into the cell than galactose, the trans hypothesis would predict it to be the stronger inhibitor, but in fact the opposite result is found. 3. Equations were developed to describe the inhibitions in accordance with the cis hypothesis. The satisfactory agreement between experimental observations and theoretical predictions provides support for the applicability of the model. Further implications of the polyfunctional carrier model are discussed.

Effect of papain treatment on phenylalanine transport in rabbit ileum

The unidirectional influx of phenylalanine across the mucosal brush border of rabbit ileum is reduced by pretreatment with papain. Phenylalanine influx is reduced to 10–15% of the control value by 60–90 minutes of preincubation with papain. Pretreatment with protease from Streptomyces griseus has no effect on phenylalanine influx. Kinetic analysis of the effect of papain indicates that the maximum velocity is reduced with little change in the apparent Michaelis-constant for phenylalanine. The data suggest that papain attacks a membrane protein required for transport. This protein is unaffected by protease which indicates the susceptible region of the protein is shielded by the membrane or inaccessible to protease.

Preliminary studies on the characteristics of phenylalanine and β-methyl-glucoside transport in the tench intestine in vitro

1. 1. The unidirectional transepithelial fluxes of L-phenylalanine, β-methyl-D-glucoside and sodium ions across emusculated sheets of tench mid-intestine were determined in flux chambers. 2. 2. No net sodium flux was detectable, but phenylalanine was preferentially transferred from the mucosal to the serosal fluid. 3. 3. There was also a net movement of β-methyl-glucoside towards the serosal medium, but it was much smaller than that of phenylalanine. 4. 4. This transport was accompanied by an accumulation of each substrate from the mucosal medium into the tissue to a similar level and against a concentration gradient. 5. 5. The poor transfer of the monosaccharide into the serosal medium could therefore be attributed to a low permeability of the baso-lateral membrane of the enterocyte for this substance. 6. 6. The influx of L-phenylalanine and of β-methyl- d-glucoside into the epithelial cells of tench midintestine was examined by incubating slices of emusculated intestine in radioactively-labelled solutions of the substrate for 2 min. 7. 7. The steady-state uptake was assessed after similar incubations lasting 45 min. 8. 8. Phenylalanine influx obeys the Michaelis-Menten equation with a K m of 2.9 mM and is dependent on the presence of sodium ions in the incubation medium. 9. 9. β-Methyl-glucoside influx reveals the same characteristics with a K m of 2.0 mM but a considerably lower V max; in addition, it is inhibited by galactose. 10. 10. The influx of both substrates is reduced by harmaline, which also inhibits the uptake of radioactive sodium by this preparation. 11. 11. The steady-state uptake of β-methyl-glucoside is also inhibited by ouabain and by 2.4-dinitrophenol. 12. 12. These results suggest that the mechanisms for sodium-dependent influx of monosaccharides and neutral amino-acids in the tench intestine are similar to those found in mammalian tissues. 13. 13. The principal difference appears to involve the release of monosaccharides across the baso-lateral membrane of the enterocyte.

Effect of metabolic inhibitors on phenylalanine influx in cerebral cortex slices from mature and immature rats.

Earlier investigations have shown that the transport of aromatic amino acids into cerebral cortex slices of rats changes during ontogenetic development. In order to illuminate the possible influence of the developmental alterations of energy metabolism in the brain on the changes of the amino acid transport the influx of phenylalanine into cerebral cortex slices of adult and 7-day-old rats was measured in the presence of metabolic inhibitors. Also the levels of lactate, ATP, ADP, AMP and creatine phosphate in the slices were measured. The used inhibitors cyanide, dinitrophenol and iodoacetate, which disturb certain steps of energy metabolism, had very similar effects in both age groups on phenylalanine influx and on the levels of energy-rich phosphates. The correlation between the level of ATP and the influx of phenylalanine was positive in both age groups, but the slopes of the regression lines were different. If ATP supplies energy for amino acid transport, its utilization, rather than its availability is a limiting factor in the slices from 7-day-old rats. Developmental changes in energy metabolism seem to have a minimal role in the changes of amino acid transport during the maturation of the brain. Changes in the properties of cell membrane carriers and in the transmembrane ionic gradients may be more important.

Effect of aromatic acids on the influx of aromatic amino acids in rat brain slices.

The influx of [3H]phenylalanine, [3H]tyrosine and [3H]tryptophan into brain cells was studied using brain slices from adult rats. Each aromatic amino acid inhibited the influx of the others into the brain cells. Tryptophan inhibited non-competitively the influx of phenylalanine, and phenylalanine similarly that of tyrosine and tryptophan. On the other hand, tyrosine inhibited competitively the influx of phenylalanine, and similarly tryptophan that of tyrosine, and tyrosine that of tryptophan. Among the aromatic organic acids tested, only phenylpyruvate and homogentisate had any inhibitory effect on the influx of the aromatic amino acids. These effects were generally competitive, non-competitive inhibition being obtained only in the inhibition of phenylalanine influx by homogentisate. The existence of only one common transport system for aromatic amino acids appear to be unlikely.

Kinetic analysis of phenylalanine-induced inhibition in the saturable influx of tyrosine, tryptophan, leucine and histidine into brain cortex slices from adult and 7-day-old rats.

Abstract—An attempt was made to isolate the saturable uptake from the unidirectional influx of amino acids into tissue slices and to estimate the transport constants and maximal velocities of saturable transport. The method was applied to studies on the inhibition of phenylalanine in the saturable influx of tyrosine, tryptophan, histidine and leucine into brain cortex slices from adult and 7‐day‐old rats. In both age groups phenylalanine inhibited the influx of the other amino acids, and vice versa. The apparent transport constants of the other amino acids increased in the presence of phenylalanine more noticeably in the slices from 7‐day‐old rats than in those from adult rats, whereas the concomitant influx of phenylalanine was inhibited less in the slices from 7‐day‐old rats. In immature animals in vivo competition between amino acids may play a more marked role in the supply of amino acids from plasma to brain, as the transport systems in brain slices from 7‐day‐old rats become saturated with extracellular amino acids more readily than do the transport systems in brain slices from adult rats.

Inhibition of amino acid transport in rabbit intestine by p-chloromercuriphenyl sulfonic acid.

Influx of phenylalanine across the brush border of rabbit intestine is markedly reduced by treatment with 5 mM p-chloromercuriphenyl sulfonate (PCMBS). The effect is rapidly and completely reversed by dithiothreitol. Phenylalanine influx into PCMBS-treated tissue can be competitively inhibited by other neutral amino acids and follows saturation kinetics. PCMBS causes an increase in the apparent Michaelis constant from the value observed in control tissue but does not alter the maximal influx significantly. Treatment of the tissue with PCMBS leads to a significant reduction in the Na-sensitivity of the transport, and a number of results indicate that the major effect of the reagent is to cause a marked reduction in the affinity of the transport system for Na. The transport system can be partially protected against reaction with PCMBS by phenylalanine and tryptophan but not by methionine or norleucine. The results suggest that PCMBS reacts with a sulfhydryl group in the region of the transport site and may alter conformational changes associated with the binding of substrates.

Kinetics of influx of phenylalanine, tyrosine, tryptophan, histidine and leucine into slices of brain cortex from adult and 7-day-old rats

The influx of phenylalanine, tyrosine, tryptophan, histidine and leucine into slices of brain cortex from 7-day-old and adult rats was analyzed kinetically. The initial velocity of the influx was measured using [ 3H]amino acids with various amino acid concentrations in the incubation medium. The influx was analyzed on the assumption that it was mediated by two mechanisms: a saturable transport conforming to Michaelis-Menten kinetics, and non-saturable physical diffusion. The two components in the influx were separated with the aid of a digital computer, and diffusion constants (K d) were determined. The maximal velocity of active transport (V m) and the transport constant (K t) were calculated from a logarithmically transformed Michaelis-Menten equation. There were no systematic differences in K d values between slices from adult and 7-day-old rats, but the V m and K t constants of all amino acids were about twice as great for adult rats as for 7-day-old rats.

Characteristics of the Amino Acid Transport System in the Mucosal Border of Rabbit Ileum

The specificity of the neutral amino acid transport system in the brush border was examined by studying the ability of amino acid analogues to inhibit the unidirectional influx of phenylalanine from mucosal solution into the cells. Effects were evaluated in terms of the affinity of various substrates for the amino acid site in the transport system. The affinity of amino acids for the site was proportional to the number of carbon atoms in the side chain. Electron-withdrawing substituents in the ring of phenylalanine increased affinity and electron-releasing groups decreased affinity. Removal of the alpha-amino group from phenylalanine decreased affinity by a factor of approximately 50 and removal of the carboxyl group decreased affinity 12-fold. Effects on affinity of variations in the side chain of the amino acid can be comparable in magnitude to that of the carboxyl group. The effect of sodium ion on the transport system appears to be similar for all compounds tested.