Recovery Of Tryptophan Research Articles

4-Vinylpyridine copolymers for improved LC–MS tryptophan and kynurenine determination in human serum

Tryptophan (an essential amino acid) and its clinically important metabolite—kynurenine contribute to several fundamental biological processes and methods that allow their determination in biological samples are in demand. The novelty of the work was a demonstration of the utility of two polymers: 4-vinylpyridine crosslinked with trimethylolpropane trimethacrylate (poly(4VP-co-TRIM)) or 1,4-dimethacryloyloxybenzene (poly(4VP-co-14DMB))—in terms of human serum clean-up for simultaneous LC–MS determination of tryptophan and kynurenine. The goal was to achieve a reduction of the matrix effect, which is responsible for signal suppression, with minimal capture of analytes. The adsorption properties of the polymeric beads were studied by evaluating the adsorption kinetics and isotherms in model matrices. Therefore, the adsorption capacities of both molecules were not efficient, the tested 4-vinylpyridine-based copolymers have shown great promise (especially poly(4VP-co-TRIM)) as sorbents for serum clean-up. In the model human serum matrix, poly(4VP-co-TRIM) provided good recoveries of tryptophan and kynurenine (76% and 87%, respectively) and allowed for the reduction of the matrix effect. Performances of both copolymers were compared to those of commercially available sorbents (octadecylsilane, activated charcoal, and primary secondary amine).

Assessment of Solvent Extraction using Sonication to Recover Tryptophan from Kappaphycus alvarezii (Doty) Doty ex Silva: Experimental and Modelling

The application of Kappaphycus alvarezii in the food system has attracted researchers due to its bioactive compounds, including tryptophan. The present study was conducted to extract tryptophan from K. alvarezii with the aid of sonication. A reduced multilevel factorial design was conducted to evaluate the effect of solvent, ultrasound power, duty-pulse cycle, time and temperature on the recovery of tryptophan. Analysis of variance suggested that the type of solvent and combination setting of power and duty-pulse cycle significantly influenced the extraction (p < 0.05). In contrast, extraction time and temperature did not alter the extraction (p > 0.05). The optimum was using ethanol, ultrasound power 80 %, pulse duty-cycle 0.8 s–1, extraction time 10 min, and temperature 25 °C and tryptophan concentration was 56.41 ± 2.42 mg L–1. Subsequently, a COnducto-like Screening MOdel for Real Solvent (COSMO-RS) was performed to clarify the impact of solvent affinity and polarity on the tryptophan extraction from K. alvarezii.

Selective extraction and preconcentration of melatonin mediated by hydrophobic natural deep eutectic systems

BackgroundSample preparation is the key to achieve the goals of Green Chemistry and represents the highest source of the total negative impact of analytical methodologies on the environment. Therefore, assessing the greenness of analytical procedures and, if possible, reducing their impact on the environment and workers is necessary. In this sense, the alternative analytical techniques must be cost-effective, eco-friendly, and sustainable, to focus these issues and ensure efficient extraction, preconcentration, and analysis of the targeted compounds. ResultsThe selective extraction and preconcentration of melatonin (MEL) mediated by hydrophobic natural deep eutectic systems (HPB-NADES) from three native plant growth-promoting rhizobacteria (PGPR) cultures was studied and performed. The HPB-NADES based on menthol:thymol showed excellent selectivity for MEL, evaluated through the 3-indole acetic acid and tryptophan recovery and the matrix effect values. The extraction recovery of MEL was 97.9 to 100.3%, with an RSD of less than 3% and an interesting enrichment factor of 14.7–15.2. Finally, the LOD and LOQ for MEL were 23.4 and 71.4 ng/L, with a linear range of 71.4–5000 ng/L showing good linearity (F less than 1.16). The sustainability of the methodology showed scores of 0.71 and 0.66, and a chemical risk corresponding to the use of 0.812 g of chloroform per extracted and analyzed sample. ConclusionsThe present study introduced, for the first time, a green analytical method for selectively extracting and quantifying MEL from three PGPR cultures using HPB-NADES. The methodology exhibited favorable linearity, LOD, LOQ, and extraction recoveries with negligible matrix effects. The use of environmentally friendly solvents and sample preparation techniques ensures the sustainability of the approach. Additionally, the present work highlighted the potential of PGPR in improving plant adaptation to abiotic stress under low MEL concentrations.

Tryptophan recovery index as a new biomarker for fitness.

The maximal oxygen uptake (VO2max) and maximal power output (Pmax) are commonly used parameters to evaluate the endurance fitness status. A connection between exercise and the kynurenine pathway (KP), which describes the metabolism of unused tryptophan, has already been reported. However, a potential association of the KP with endurance fitness levels remains unknown. In this study, adolescent competitive athletes performed an exhaustive incremental exercise test. Blood samples were taken before, directly after, and 30 minutes after the end of exercise. Tryptophan (Trp), kynurenine (Kyn) and kynurenic acid (KA) serum levels were determined by high-performance liquid chromatography (HPLC). Forty-four male and 27 female athletes (median age: 16 years) were recruited. During exhaustive exercise tests, Trp initially declined and then increased 30 minutes after discontinuing exercise. Similar findings were observed for Kyn, whereas KA levels behaved inversely. After incremental exhaustive exercise the relative increase of Trp concentrations, termed the tryptophan-recovery-index (TRI), showed a highly significant positive correlation with VO2max and Pmax (r=0.468 and 0.491, p-values <0.001). There was a significant gender-difference with higher levels of all metabolites at all measured time points in male participants. In the present study, a highly significant correlation was found between the TRI and the maximal oxygen uptake in well-trained athletes. The implementation of TRI can therefore be suggested as a biomarker for physical fitness.

LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites in Healthy Plant Foods.

Food plants contain hundreds of bioactive phytochemicals arising from different secondary metabolic pathways. Among these, the metabolic route of the amino acid Tryptophan yields a large number of plant natural products with chemically and pharmacologically diverse properties. We propose the identifier “indolome” to collect all metabolites in the Tryptophan pathway. In addition, Tryptophan-rich plant sources can be used as substrates for the fermentation by yeast strains to produce pharmacologically active metabolites, such as Melatonin. To pursue this technological development, we have developed a UHPLC-MS/MS method to monitor 14 Tryptophan, Tryptamine, amino-benzoic, and pyridine metabolites. In addition, different extraction procedures to improve the recovery of Tryptophan and its derivatives from the vegetal matrix were tested. We investigated soybeans, pumpkin seeds, sesame seeds, and spirulina because of their botanical diversity and documented healthy effects. Four different extractions with different solvents and temperatures were tested, and water extraction at room temperature was chosen as the most suitable procedure to extract the whole Tryptophan metabolites pattern (called by us “indolome”) in terms of ease, high efficiency, short time, low cost, and sustainability. In all plant matrices, Tryptophan was the most abundant indole compound, while the pattern of its metabolites was different in the diverse plants extracts. Overall, 5-OH Tryptamine and Kynurenine were the most abundant compounds, despite being 100–1000-fold lower than Tryptophan. Melatonin was undetected in all extracts, but sesame showed the presence of a Melatonin isomer. The results of this study highlight the variability in the occurrence of indole compounds among diverse food plants. The knowledge of Tryptophan metabolism in plants represents a relevant issue for human health and nutrition.

Metal Ion‐Assisted Infrared Optical Sensor for Selective Determination of Tryptophan in Urine Samples

AbstractIn this study, a simple infrared chemical sensor was developed for the selective detection of tryptophan in biological fluids. This sensor was capable of trapping tryptophan molecules through the formation of relatively stable metal ion complexes on the surface of the sensing element. A proline‐modified sensing phase was immobilized on the surface of the internal reflection element. With the assistance of appropriate metal ions, tryptophan molecules were selectively attracted nearby the evanescent field such that analytical signals were generated. Factors that affected the chemical equilibria in this detection system were examined including the species and concentration of metal ion, the pH of the sample solution, and the concentration of the chelating agent. Among the examined metal ions, nickel provided the best selectivity toward the detection of tryptophan as a result of its extremely high formation constant with tryptophan. Under the optimal conditions, the detected signals were related linearly (R2 &gt; 0.99) to concentrations of tryptophan up to 600 μM. Based on three times the baseline variation of blank samples, the detection limit was ca. 5 μM. From a study of possible interfering agents—metal ions and organic species—in the sample solution, the recoveries of tryptophan were greater than 95%.

Acid Hydrolysis of Protein in a Microcapillary Tube for the Recovery of Tryptophan

The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 microl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 degrees C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.

Determination of tryptophan by high-performance liquid chromatography of alkaline hydrolysates with spectrophotometric detection

A procedure for quantitation of tryptophan in feedstuff is described. It consist of hydrolysis in sodium hydroxide at 100 °C for 4 h, neutralization of the resulting hydrolysate to pH 7, dilution with sodium borate buffer (pH 9), and analysis by reverse-phase high-performance liquid chromatography with spectrophotometric determination of tryptophan at 280 nm. The recovery of tryptophan from lysozyme, added to some feedstuff before hydrolysis, ranges from 98.6 to 100%. A reduction of the time of analysis has been achieved as compared to previous methods for analysis of tryptophan.

Tryptophan in the sediments of lakes from Southwestern China Plateau

Methods using alkaline hydrolysis and HPLC were first developed to measure tryptophan concentrations in sediments. Early diagenesis of tryptophan was discussed and compared with that of other amino acids. Optimum alkaline hydrolysis conditions (120 °C, 20 h, 4.2 N sodium hydroxide) were chosen to hydrolyze sediments with a tryptophan recovery of 93%. Results show that tryptophan was a minor component in the sediments of four Chinese lakes, its concentrations ranging from 0.15 to 2.66 μmol g −1, and accounted for 0.17–9.18% of total hydrolyzable amino acids (THAA). In terms of relative abundance, tryptophan increased with sediment depth, while the other aromatic species (tyrosine and phenylalanine) decreased, showing contrasting geochemical behaviors among aromatic species during early diagenesis. Based on the good positive correlation between tryptophan and serine, it is suggested that tryptophan may be also related to the selective diatom cell wall preservation.

Influence of acute tryptophan depletion on mood and immune measures in healthy males.

Depressive illness has been associated with variations of several aspects of immune functioning, as well as alterations of cytokine production in stimulated lymphocytes. In the present investigation we sought to determine whether pharmacologically-induced reductions of mood in healthy, male subjects would be associated with alterations in the levels of circulating IL-1 beta or IL-6 or to in vitro lymphocyte proliferation in response to T cell mitogens, PHA and Con A. Lowering tryptophan levels by means of a tryptophan-deficient amino acid mixture, which reduced plasma tryptophan and serotonin (5-HT) levels, produced a lowering of mood in a subset of male subjects (that had no personal or family history of depression) relative to subjects that received a balanced amino acid mixture. Correlational analyses revealed that the change of mood (particularly depression and anger) in subjects that received the tryptophan-free mixture was related to the extent of the tryptophan or 5-HT reductions. However, while fenfluramine administration resulted in recovery of tryptophan and 5-HT levels, this was not accompanied by recovery of mood. Furthermore, it was observed that the lowering of tryptophan levels and the reduced mood were not accompanied by variations of the cytokine levels or cell proliferation. Evidently, transient and modest alterations of 5-HT or mood induced by a tryptophan-free amino acid mixture were insufficient to promote variations of immune activity or circulating IL-1 beta or IL-6 levels. Even if depression were related to immune disturbances, the mood and 5-HT alterations associated with this type of manipulation may be too brief to promote immune changes comparable with those ordinarily associated with severe or chronic depressive illness.

Fenazaquin Acaricide Specific Binding Sites in NADH: Ubiquinone Oxidoreductase and Apparently the ATP Synthase Stalk

Fenazaquin [4-(4-tert-butylphenethoxy)quinazoline] is one of several acaricides and insecticides, including rotenone and pyridaben, that are reported to act by inhibiting NADH:ubiquinone oxidoreductase (Complex I) (EC 1.6.99.3) in the range 1–10 nM. [3H]Fenazaquin is used here at 0.8 nMas a radioligand with electron transport particles (ETP) of bovine heart mitochondria to develop a new and rapid filtration assay with 42% specific binding (displaced by 2 μMrotenone). The potency of rotenoids, quinazolines, pyridaben, acetogenins, and several other disrupters of electron transport as inhibitors of NADH:ubiquinone oxidoreductase activity generally correlates with their effectiveness in displacing [3H]fenazaquin from its binding site in ETP (r2= 0.96,n= 18). This correlation extends to the fenazaquin analog with (trifluoromethyl)-diazirinyl replacing thetert-butyl substituent assayed at 6 nM,indicating that it also acts at a high-affinity site in Complex I. This diazirinyl analog of fenazaquin with tritium at theO-methylene position (4.7 Ci/mmol), on incubation at 435 nMwith ETP then irradiation at 350 nm, results in photoactivatable, irreversible specific binding which is fully protectable with rotenone. The site of labeling was examined by sequential isolation of the only band of specifically labeled protein(s) (which appeared at 22–24 kDa on SDS–PAGE),in situtryptic digestion of the protein(s) in the band, fractionation of the generated peptides by microbore HPLC, and amino acid sequence analysis. The single labeled HPLC peak of peptides in the tryptic digest was derived from two proteins, both components of ATP synthase, identified as subunit d and the oligomycin sensitivity conferral protein. An unusually low recovery of tryptophan at residue 12 of subunit d of specifically labeled ETP versus control ETP leads to the tentative proposal that labeling occurs at that site. Fenazaquin (and presumably rotenone as well) therefore has at least two specific binding sites in ETP, the anticipated high–affinity site in NADH:ubiquinone oxidoreductase and apparently a low-affinity site of unknown function, photoaffinity labeled in the stalk region of ATP synthase.

Tryptophan determination in feedstuffs: A critical examination of data from two collaborative studies through the evaluation of tryptophan recovery

The tryptophan recovery from various procedures used by 23 laboratories having participated in two collaborative studies (French for the one, European for the other) on the tryptophan determination of several feedstuff samples was evaluated by taking the corresponding data obtained by our laboratory, applying an accurate method to the same samples, as reference. The percentages resulting from this normalization represented a set of values based on about 500 determinations. They emphasized that all of the procedures developed for tryptophan quantitation from alkaline hydrolysate and without the help of an internal standard led to a mean underestimation of about 15%, irrespective of the nature of alkali used for hydrolysis, the kind of analyzed sample, and the conditions used for assaying liberated tryptophan. A mean underestimation of the same extent can be seen in the literature data, originating from individual as well as collaborative studies and pertaining to the recovery of a known amount of tryptophan added to samples prior hydrolysis, which lends additional support to the validity of our procedure. The use of 5-methyltryptophan but not alpha-methyltryptophan as an internal standard results in the improvement of assay accuracy with tryptophan recoveries close to 100%.

Determination of tryptophan in feedstuffs: comparison of sodium hydroxide and barium hydroxide as hydrolysis agents

The tryptophan content of nine samples of food and feedstuffs was determined using two procedures differing mainly by the hydrolysing agent. For a given smaple hydrolyses were conducted at the same time and in the same autoclave at 125°C for 16 h after a purging of the apparatus at 100°C for 5 min. Ba(OH) 2 hydrolysis yielded the highest values for tryptophan. As for NaOH hydrolysates, the tryptophan recovery evaluated from data obtained with Ba(OH) 2 hydrolysates and taken as reference varied from 74·5 to 98·2% with a mean of 88·8%. These values are identical to those reported in literature for recovery of tryptophan added to samples prior to their autoclaving in Ba(OH) 2 on NaOH but without purging at 100°C. The tryptophan recovery corrected from losses of added 5-methyltryptophan varied from 95·3 to 106·8% with a mean of 102·3%. The efficiency of autoclave purging at 100°C for the quantitative recovery of tryptophan from Ba(OH) 2 hydrolyses would be due to a thorough deoxygenation of medium through boiling.

High-performance liquid chromatography of tryptophan and other amino acids in hydrochloric acid hydrolysates

Tryptamine [3-(2-aminoethyl)indole] as an additive to 6 M HCl largely prevented the decomposition of tryptophan in proteins, and also in gas-phase hydrolysis, using a Pico-Tag Work Station, at 145°C for 4 h. This procedure proved to be excellent for amino acid analysis using conventional high-performance liquid chromatography with derivatization with phenyl isothiocyanate. The recovery of tryptophan from model solutions and from proteins was 80–98%. The results proved to be as good as any other obtained by the analysis of special alkaline or organic acid hydrolysates, most of which are suitable exclusively for the determination of tryptophan. The most important advantage of tryptamine was that it did not affect the quantitative recovery of other amino acids of proteins, as shown by the composition of lysozyme hydrolysates obtained in the absence and presence of various amounts of tryptamine. The reproducibility of the measurements was 3.7% (relative standard deviation) or less.

HPLC determination of tryptophan in foodstuffs using barium hydroxide hydrolysis at 140�C

Hydrolysis of samples of food and feedstuffs with 2.7 N Ba(OH)2 at 140°C for 4 h was tested for the recovery of tryptophan. On the basis of 100% yield for the tryptophan content, corresponding to samples determined after 16h-hydrolysis at 125°C, the recovery averaged 98.7 ± 0.9% SD or 99.4 ± 2% depending on how the bulk or aliquot of hydrolysate was analysed (conventional or simplified procedure). Tryptophan can be assayed by high performance liquid chromatography and fluorescence detection within 8h, 4h-hydrolysis at 140°C and a simplified procedure.

The direct hydrolysis of proteins containing tryptophanon polyvinylidene difluoride membranes by mercaptoethanesulfonic acid in the vapor phase

A procedure for the amino acid analysis of polypeptides that contain tryptophan on polyvinylidene difluoride membranes is described. Lysozyme, carbonic anhydrase, phytochrome, and ovalbumin were tested. The protein, which was separated from others by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was blotted from the gel onto a polyvinylidene difluoride membrane and directly hydrolyzed by 3 n aptoethanesulfonic acid vapor in a vacuum at 176°C for 25 min. The hydrolysate was extracted with 0.1 n HCl and 30% methanol and used for amino acid analysis. The tested proteins were adequately hydrolyzed, and the recovery of tryptophan was very efficient.

Development of an acid hydrolysis method with high recoveries of tryptophan and cysteine for microquantities of protein

High recoveries of tryptophan and cysteine were achieved by 12.5 min of hydrolysis with mercaptoethanesulfonic acid vapor. Proteins (1–100 μg) were modified by vapor-phase S-pyridylethylation before hydrolysis. The modified proteins were hydrolyzed with the vapor of 2.5 m mercaptoethanesulfonic acid at 176°C. This method promoted efficient hydrolysis of the peptide bonds in proteins and resulted in high recoveries of both tryptophan and cysteine, of 90% or greater, in addition to the other amino acids.

Effect of the structure and density of chemically bonded C 18 phase on the recovery of tryptophan and its metabolites from human urine

A series of materials with chemically bonded C 18 phase for use as the packings in clean-up columns for solid-phase extraction were prepared. The effects of the monomeric and/or polymeric structure of the chemically bonded phase and of the porous structure of the silica gel support on the recovery of tryptophan and two of its metabolites used as test substances were considered. It appeared that the best recoveries of at least 60% of the three test substances were obtained on material of the “monomer” type containing chemically bonded C 18 phase characterized by a high coverage density of α RP ≈ 3.8 μmol/m 2. The use of a silica gel support with a larger pore size and volume permits not only the effective isolation of individual substances, e.g., from urine, but also their 5-fold concentration.

Quantitation of tryptophan and other plasma amino acids by automated pre-column o-phthaldialdehyde derivatization high-performance liquid chromatography: improved sample preparation

Pre-column derivatization with o-phthaldialdehyde is a rapid and sensitive method for the quantitation of amino acids in biological fluids. This method uses acetonitrile as a deproteinizing reagent which gives improved recovery of tryptophan compared with 5-sulfosalicylic acid and permits the measurement of aspartic acid which coelutes with 5-sulfosalicylic acid. The method is automated to increase reproducibility and convenience. Mean coefficients of variation for peak areas relative to internal standard were 3.2 and 5.2% for amino acid standards and plasma samples, respectively. The presence of nitrilotriacetic acid stabilized the o-phthaldialdehyde reagent which is important in an automated system. The method is suitable for the analysis of large numbers of plasma samples where total tryptophan and aspartic acid are of interest.

Recovery of tryptophan in peptides and proteins by high-temperature and short-term acid hydrolysis in the presence of phenol

The addition of 3% ( w v ) phenol to 6 M HCl largely prevented the destruction of tryptophan during rapid hydrolysis of peptides and proteins at 166°C for 25 min or at 145°C for 4 h. This hydrolysis procedure was advantageous for amino acid microanalysis using conventional high-performance liquid chromatography with a precolumn derivatization technique. The recovery of tryptophan from proteins was at least 80%. The addition of phenol also improved the recovery of methionine and carboxymethylcysteine. The amount of tryptophan in proteins electroblotted onto a polyvinylidene difluoride membrane was determined by this method.