L-Trp Concentration Research Articles

L-Tryptophan Metabolism in Pregnant Mice Fed a High L-Tryptophan Diet and the Effect on Maternal, Placental, and Fetal Growth

Excess L-tryptophan (L-Trp) in the diet decreases fetal body weight. However, the relationship between L-Trp concentration and its effects on maternal, placental, and fetal growth are not well-understood. We investigated the effects of excess L-Trp intake on maternal, placental, and fetal growth. Female mice were fed a 20% casein diet (control diet) or control diet plus 2% or 5% L-Trp during gestation. Pup weights did not differ between the control (L-Trp intake: 0.04 g/kg body weight (BW)/day) and 2% L-Trp groups (L-Trp intake: 3.3 g/kg BW/day), but were significantly lower in the 5% L-Trp group (L-Trp intake: 7.0 g/kg BW/day) than in the control and 2% L-Trp groups. These results show that less than 3.3 g/kg BW/day L-Trp intake in pregnant mice during gestation does not affect fetal growth or L-Trp homeostasis in the placenta or fetus.

Preparation of L -Tryptophan Electrochemical Sensor Based on Graphene Oxide/Carbon Nanotubes Nanocomposite Modified Electrode

A single-layer graphene oxide(GO) was prepared by Hummers method,and then mixed with multiwalled carbon nanotubes(MWCNT) in ultrasound bath to form stable GO/MWCNT nanocomposites.A novel L-tryptophan(L-Trp) electrochemical sensor was fabricated based on GO/MWCNT modified glassy carbon electrode.The modified electrode was characterized by transmission electron microscope(TEM),cyclic voltammetry(CV) and alternating current impedance(EIS),and the electrochemical behaviors and kinetic properties of L-Trp on the modified electrode were also investigated.The experimental results showed that a sensitive oxidation peak of L-Trp appeared at GO/MWCNT modified electrode(Epa=0.956 V),and the oxidation reaction was an irreversible process containing two electrons and two protons transfer.This electrode process controlled by the adsorption step,and the standard rate constant was 9.613×10-6 cm/s.Trace levels of L-Trp can be determined by this oxidation peak.In phosphate buffer of pH 6.0,the oxidation peak currents were linearly dependent on the L-Trp concentrations in the range of 1.00×10-6-1.00×10-4 mol/L with accumulation time of 25 s at 0.600 V and scan rate of 100 mV/s.The correlation coefficient was 0.995 and the detection limit was 3.50×10-7 mol/L.The prepared electrochemical sensor had favorable stability and could be applied to the quick determination of L-Trp in human serum,and the recovery of standard addition was from 97.8% to 104.2%.

Determination of L-tryptophan and L-kynurenine in Human Serum by using LC-MS after Derivatization with (R)-DBD-PyNCS

Concentrations of l-tryptophan (l-Trp) and its metabolite, l-kynurenine (l-KYN), in sera of 19 normal subjects (age: 23.6 ± 3.5 y, male: 8, female: 11) were determined by high-performance liquid chromatography with mass-spectrometric detection, following their derivatization with (R)-(−)-4-(N, N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-PyNCS). A significant positive correlation between l-Trp and l-KYN concentrations was observed (r = 0.532, P < 0.05). Serum l-Trp concentration in male subjects (95.65 ± 4.27 μM) was significantly higher than that in female subjects (79.20 ± 3.34 μM; P < 0.05), while no significant differences in l-KYN concentration or the l-KYN:l-Trp ratio were observed between male and female subjects.

Electrochemical Sensors for L-Tryptophan Based on Molecularly Imprinted Polymers

A novel sensing element for direct detection of L-Tryptophan (L-Trp) in human serum was proposed by combination of electrochemical transduction and molecular imprinting technique. The sensing element was prepared by electropolymerization of o-phenylenediamine (o-PD) on the gold electrode in the presence of template L-Trp. The electrochemical property of the sensor was characterized by differential pulse voltammetry (DPV). The linear response range of the sensor for L-Trp concentration was 1×10-8 mol/L~21×10-8 mol/L with a detection limit of 0.3×10-8 mol/L. The results show that this approach has the potential to generate a universal inexpensive assay methodology of high sensitivity, specificity and simplicity to detect L-Trp.

The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase catalyzes the O2-dependent oxidation of l-tryptophan (l-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high l-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with l-tryptophan (l-Trp) can be accounted for by the sequential, ordered binding of O2 and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O2 binds first followed by the binding of l-Trp; at higher concentrations of l-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (Em0) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between Em0 (that increases in the presence of l-Trp) and the rate constant for O2 binding. This means that the initial formation of ferric superoxide (Fe3+–O2•–) from Fe2+-O2 becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (kcat and Em0) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.

Poly-glutamic acid modified carbon nanotube-doped carbon paste electrode for sensitive detection of L-tryptophan

A novel poly-glutamic acid (PGA) film modified carbon paste electrode (CPE) incorporating carbon nanotubes (CNTs) was first prepared for the determination of l-tryptophan ( l-Trp). Scanning electron microscopy and Fourier transform infrared spectroscopy were applied for characterization of the surface morphology of the modified electrodes and cyclic voltammetry was used to investigate the electrochemical properties of the proposed electrode towards the oxidation of l-Trp. Optimization of the experimental parameters was performed with regard to pH, ratio of CNTs, concentration of glutamic acid, electro-polymerization cycles, accumulation time and concentration of sodium dodecylbenzene sulfonate. The linearity between the oxidation peak current and the l-Trp concentration was obtained in the range of 5.0 × 10 − 8 to 1.0 × 10 − 4 M with a detection limit of 1.0 × 10 − 8 M ( S/ N = 3) and the sensitivity was calculated to be 1143.79 μA∙mM − 1 ∙ cm − 2 . In addition, the PGA modified CPE incorporating CNTs displayed high selectivity, good stability and reproducibility, making it suitable for the routine analysis of l-Trp in clinical use.

Adsorption behavior of L-tryptophan on ion exchange resin

A batch method was applied to investigate the adsorption behavior and mechanisms of L-tryptophan (L- trp) on ion exchange resins. HZ-001 and JK006 were proved to be ideal adsorbents due to their large loading capacity and favorable selective adsorption for L-trp. Langmuir, Freundlich, and Dubinin-Radushkevich equations were applied to simulate the experimental data to describe the adsorption process of L-trp onto HZ-001 and JK006. The maximum loading capacity (at pH 5.0, 30 o C), determined by the Langmuir and Dubinin-Radushkevich models, was close to each other (833 mg/g vs. 874 mg/g) for HZ-001, while discrepant (833 mg/g vs. 935 mg/g) for JK006. Three diffusion-con- trolled kinetic models were utilized to analyze the results in order to identify the adsorption mechanism. The adsorption kinetics of L-trp onto cation exchange resins was investigated under different experimental conditions, including initial solution pH, temperature, initial L-trp concentration, and adsorbent dosage. Moreover, the diffusion process of L-trp onto HZ-001 and JK006 was evaluated at different initial adsorbate concentrations. The thermodynamic parameters, obtained from the kinetic data, demonstrated that L-trp could be adsorbed spontaneously onto both resins.

Ligand migration in human indoleamine‐2,3 dioxygenase

Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-tryptophan (L-Trp) and other indoleamine derivatives. Its activity follows typical Michaelis-Menten behavior only for L-Trp concentrations up to 50 μM; a further increase in the concentration of L-Trp causes a decrease in the activity. This substrate inhibition of hIDO is a result of the binding of a second L-Trp molecule in an inhibitory substrate binding site of the enzyme. The molecular details of the reaction and the inhibition are not yet known. In the following, we summarize the present knowledge about this heme enzyme.

Fluorescence determination of d- and l-tryptophan concentrations in rat plasma following administration of tryptophan enantiomers using HPLC with pre-column derivatization

Similar to l-tryptophan ( l-Trp), d-Trp can be converted to unique metabolites in the mammalian body. In the present study, the difference in the plasma half-life ( t 1/2) between Trp enantiomers was investigated by following the alterations in the plasma concentration of d- or l-Trp after intraperitoneal ( i.p.) administration of each enantiomer to male Sprague–Dawley rats (100 mg/kg). The investigation was performed using reversed-phase high-performance liquid chromatography (HPLC) and pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-( N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole ( R(−)-DBD-PyNCS). The t 1/2 value of d-Trp was significantly smaller than that of l-Trp, suggesting that d-Trp was eliminated from the plasma more rapidly than l-Trp. In addition, a significant increase in the plasma concentration of l-Trp was observed following administration of d-Trp, whereas no d-Trp was detected after l-Trp administration. Furthermore, the increase in the plasma concentration of l-Trp was significantly suppressed by pretreatment with an inhibitor of d-amino acid oxidase (DAAO), 3-methylpyrazole-5-carboxylic acid, which suggests that DAAO was involved in the conversion of d-Trp to l-Trp in vivo.

Ligand and Substrate Migration in Human Indoleamine 2,3-Dioxygenase

Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-tryptophan (L-Trp) and other indoleamine derivatives. Using Fourier transform infrared and optical absorption spectroscopy, we have investigated the interplay between ferrous hIDO, the ligand analog CO, and the physiological substrate L-Trp. These data provide the long-sought evidence for two distinct L-Trp binding sites. Upon photodissociation from the heme iron at T > 200 K, CO escapes into the solvent. Concomitantly, L-Trp exits the active site and, depending on the L-Trp concentration, migrates to a secondary binding site or into the solvent. Although L-Trp is spectroscopically silent at this site, it is still noticeable due to its pronounced effect on the CO association kinetics, which are significantly slower than those of L-Trp-free hIDO. L-Trp returns to its initial site only after CO has rebound to the heme iron.

Ligand and Substrate Migration in Human Indoleamine 2,3-Dioxygenase

Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-Trp and other indoleamine derivatives. Using Fourier transform infrared and optical absorption spectroscopy, we have investigated the interplay between ferrous hIDO, the ligand analog CO, and the physiological substrate L-Trp. These data provide the long sought evidence for two distinct L-Trp binding sites. Upon photodissociation from the heme iron at T > 200 K, CO escapes into the solvent. Concomitantly, L-Trp exits the active site and, depending on the l-Trp concentration, migrates to a secondary binding site or into the solvent. Although L-Trp is spectroscopically silent at this site, it is still noticeable due to its pronounced effect on the CO association kinetics, which are significantly slower than those of L-Trp-free hIDO. L-Trp returns to its initial site only after CO has rebound to the heme iron.

Cooperative Binding of l-Trp to Human Tryptophan 2,3-Dioxygenase: Resonance Raman Spectroscopic Analysis

Tryptophan 2,3-dioxygenase (TDO) is a tetrameric enzyme that catalyses the oxidative cleavage of l-tryptophan (l-Trp) to N-formylkynurenine by the addition of O(2) across the 2,3-bond of the indole ring. This reaction is the first and rate-limiting step in the kynurenine pathway in mammals. In the present study, we measured the conformational changes in the haem pocket of recombinant human TDO (rhTDO) in ferric form that are induced by l-Trp binding using both resonance Raman and optical absorption spectroscopies. The deconvolution analysis of the haem Raman bands at various concentrations of l-Trp revealed that the wild-type enzyme exhibits homotropic cooperativity in l-Trp binding, which was confirmed by a change in the optical absorption spectra. Mutation analysis showed that the Y42F mutant abolished the cooperative binding, and that the H76A mutant considerably reduced the catalytic activity. These data and the inter-subunit contacts reported in the bacterial TDO structure suggest that the Y42 of rhTDO is responsible for the cooperative binding of l-Trp by participating in the active site of the adjacent subunit.

Indoleamine 2,3-dioxygenase is highly expressed in human adult T-cell leukemia/lymphoma and chemotherapy changes tryptophan catabolism in serum and reduced activity

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-cell lymphotropic virus type 1 (HTLV-1). Indoleamine 2,3-dioxygenase (IDO), the l-tryptophan ( l-TRP)-degrading enzyme, plays a key role in the powerful immunomodulatory effects of several different types of immune cells. In this study, we investigated the IDO expression in ATLL cells and the effect of chemotherapy on IDO-initiating l-TRP catabolism in patients with ATLL. Serum l-kynurenine ( l-KYN) concentrations, l-KYN/ l-TRP ratio, and the level of IDO mRNA expression in ATLL cells were significantly increased in ATLL patients compared to those in healthy and HTLV-positive carrier subjects. On the other hand, l-TRP level was significantly decreased in ATLL patients compared to that in healthy subjects. In the immunohistochemical staining, IDO was strongly expressed in cytoplasm of ATLL cells. Interestingly, serum l-KYN as well as soluble IL-2 receptor concentrations was significantly reduced, and l-TRP concentrations were significantly increased after chemotherapy. These data provide evidence that IDO is highly expressed in ATLL cells, and that IDO-initiating l-TRP catabolism changes with chemotherapy.

Transfer and distribution of L-tryptophan between W/O and O/W, and between W/O and bicontinuous microemulsions through the study of interphase electric properties

The influence of L-tryptophan (L-Trp) on the interphase electric properties of sodium dodecyl sulfate (SDS)/ n-pentanol ( n-C 5H 11OH)/water (H 2O) system was studied by the method of AC impedance, and the distribution of L-Trp between the microemulsion phases with different structures in SDS/ n-C 5H 11OH/H 2O system was determined by UV–vis spectroscopy. The results show that L-Trp transfers from the lower phase (O/W or bicontinuous) to the upper phase (W/O), and that a small amount of SDS transfers from the upper phase to the lower phase correspondingly with the increase of the total SDS content at a constant weight ratio of n-C 5H 11OH/H 2O = 50/50 and a total L-Trp concentration of 1.0 × 10 −4 mol / L . In addition, the concentrations of L-Trp in both the upper and lower phases increase, but the SDS content decreases slightly in the upper phase and increases appreciably in the lower phase with the increase of the total L-Trp concentration at a constant total content of SDS. The interphase capacitance and the interphase charge-transfer current of the system increase, but the interphase resistivity decreases.

Inhibition and kinetic changes of brain tryptophan-5-hydroxylase during insulin-dependent diabetes mellitus in the rat

In the present study we report results on the possible mechanism of inhibition of tryptophan-5-hydroxylase activity induced by insulin-dependent diabetes mellitus (IDDM). Kinetic experiments were done with different l-tryptophan (l-Trp) concentrations in the rat brain at different days of evolution of the disease. Additionally, different activation conditions of the enzyme were evaluated, to gain information on the mechanism of the activity changes. Diabetes state was induced in normal male rats, by the administration of 75?mg/kg body weight of streptozotocin (STZ). The results showed an increase of the Km value and a decrease in the Vmax in the diabetic's brain as compared to controls. Interestingly, in the diabetic group, the response capacity to phosphorylation is significantly reduced. These shifts in the activity of tryptophan-5-hydroxylase developed during IDDM may not be explained only by a decrease of l-Trp, but also by a possible change in the enzyme itself, reflected in a diminished affinity for the substrate and a decreased response to phosphorylating conditions.

Mutations in Arabidopsis thaliana genes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces.

Arabidopsis thaliana roots grow in a wavy pattern upon a slanted surface. A novel mutation in the anthranilate synthase alpha 1 (ASA1) gene, named trp5-2wvc1, and mutations in the tryptophan synthase alpha and beta 1 genes (trp3-1 and trp2-1, respectively) confer a compressed root wave phenotype on tilted agar surfaces. When trp5-2wvc1 seedlings are grown on media supplemented with anthranilate metabolites, their roots wave like wild type. Genetic and pharmacological experiments argue that the compressed root wave phenotypes of trp5-2wvc1, trp2-1 and trp3-1 seedlings are not due to reduced IAA biosynthetic potential, but rather to a deficiency in L-tryptophan (L-Trp), or in a L-Trp derivative. Although the roots of 7-day-old seedlings possess higher concentrations of free L-Trp than the shoot as a whole, trp5-2wvc1 mutants show no detectable alteration in L-Trp levels in either tissue type, suggesting that a very localized shortage of L-Trp, or of a L-Trp-derived compound, is responsible for the observed phenotype.

Phase-transition changes of poly( N-( S- sec-butylacrylamide- co-N-isopropylacrylamide) in response to amino acids and its chiral recognition

In order to obtain a thermo-responsive polymer which exhibits hydration change in response to foreign optically active compounds as external stimuli, binary copolymers composed of N-( S)- sec-butylacrylamide (( S)- sec-BAAm) and N-isopropylacrylamide (IPAAm) were synthesized by ordinary radical copolymerization. Turbidity of the polymer aqueous solutions containing various concentrations of tryptophan (Trp) isomer was measured to determine lower critical solution temperature (LCST) values. Poly(( S)- sec-BAAm- co-IPAAm) having 50 mol% of ( S)- sec-BAAm content was a water-soluble polymer which exhibited LCST at 23.1°C. The LCST of the copolymer aqueous solution in the presence of l-Trp was shifted remarkably to 34.5°C, while that in the presence of d-Trp was 28.7°C. On the other hand, a racemic ( R,S)- sec-BAAm-containing copolymer showed the same phase transition temperatures in the presence of d- and l-Trp. There was no influence of Trp concentration on LCST for poly( N-isopropylacrylamide) aqueous solution. It was clear that the optically active ( S)- sec-BAAm in the copolymer stereospecifically interacted with l-Trp, showing a relatively higher LCST value. The LCSTs of the copolymer increased in response to increasing concentration of l-Trp. It was found that the introduction of the optically active moieties into the copolymer chains resulted in the phase-transition behavior changes of the copolymers in response to the aromatic amino acids in aqueous solution. The results, therefore, revealed that the thermo-responsive copolymer with optically active moieties demonstrated chiral recognition to the enantiomers.

光学分割膜 感温性光学活性ポリマーによる不斉認識

This paper describes that the optically active moieties, ((S) -sec-butylacrylamide (BAAm)), in N-isopropylacrylamide (IPAAm) copolymer chains affected the phase transition behavior and resulted in lower critical solution temperature (LCST) changes responding to foreign tryptophan (Trp). Copolymerization of (S) -sec-BAAm and IPAAm were carried out in DMF at 70°C for 25 h using AIBN. The copolymer was water-soluble and showed the characteristic LCST near 27°C. LCSTs of PIPAAm chains are affected by hydrophobic or hydrophilic nature of comonomers. Since sec-BAAm is insoluble in water, the component exhibited hydrophobic property. Therefore, poly ((S) -sec-BAAm-co-IPAAm) showed lower LCST as compared with that of PIPAAm. The LCSTs of the copolymer in the presence of L-Trp weve higher than those in the absence of the amino acid. It was understood that the optically active moieties in the copolymer stereospecifically interact with L-Trp, relatively enhancing hydrophilic property of the polymer chains. The copolymer increased the LCSTs in response to increasing L-Trp concentration. Hydrogels consisting of these comonomers will exhibit reversible swelling/ deswelling behavior in response to L-amino acids such as L-Trp. The polymers with the optically active moieties demonstrate the chiral discrimination to L-amino acids and are also good candidates to perform chiral separation.

Early nutritional changes modify the kinetics and phosphorylation capacity of tryptophan-5-hydroxylase

Gestational malnutrition induces an acceleration of the serotonin biosynthetic pathway in the developing brain with an increase in brain L-tryptophan (L-Trp), tryptophan-5-hydroxylase (TrpOH) activity and serotonin content. In the present work we report results on the possible mechanism of TrpOH activation. Kinetic experiments were done with different L-Trp concentrations in the rat brain at different ages. Also various phosphorylating conditions of the enzyme were tested in order to compare its activation in developmentally malnourished and normal brains. The results showed lower Km values and no changes in the Vmax in the malnourished as compared to controls. Interestingly, in the malnourished group, TrpOH showed an increased activity under the phosphorylating conditions employed. We propose that in the activation of brain TrpOH by developmental malnutrition, not only is an elevation of L-Trp involved, but also a change in the enzyme itself reflected in a higher affinity for L-Trp and in a greater response to phosphorylation. This allows us to propose the possibility that early chronic malnutrition induces structural changes in the enzymatic molecule.

Influence of L-tryptophan and auxins applied to the rhizosphere on the vegetative growth of Zea mays L.

Glasshouse experiments were conducted to evaluate the influence of L-TRP in comparison with indole-3-acetamide (IAM), tryptophol (TOL) and indole-3-acetic acid (IAA) on the growth of Zea mays L. var. Early Sunglow. L-TRP (25 to 2.5×10−5 mg kg−1 soil), IAM (22 to 2.2×10−5 mg kg−1 soil), TOL (20 to 2.0×10−5 mg kg−1 soil), and IAA (22 to 2.2×10−5 mg kg−1 soil) were applied as a soil drench to established uniform seedlings. All treatments were applied in a completely randomized design with 10 replicates. IAM had no significant effect on the plant growth parameters. Shoot height, uppermost leaf collar base distance, internodal distance, and shoot dry and fresh weights were significantly improved upon the addition of TOL (2.0×10−2 mg kg−1 soil), however, the highest concentration (20 mg kg−1 soil) caused a 14.6% reduction in leaf width. L-TRP (2.5×10−3 mg kg-1 soil) also had a significant influence on shoot height, uppermost leaf collar base distance, internodal distance and fresh weight of shoot compared with the control. The highest concentration of L-TRP (25=mg kg−1 soil) had a negative effect on leaf width and dry weight of the shoot. The most pronounced response on the corn growth parameters was observed with the application of IAA at lower concentrations (2.2×10−5 to 2.2×10−2 mg kg−1 soil) specifically improving root growth. The highest concentration (22 mg kg−1 soil) of IAA had a significant negative effect on plant height, leaf width, stem diameter, shoot fresh and dry weight. These findings indicate that L-TRP applied at the appropriate concentrations can have positive effects on corn growth comparable to pure auxins (TOL and IAA).