HPLC Gradient Retention of Tryptophan and its Metabolites on Three Stationary Phases in Context of Lipophilicity Assessment.

This paper is a continuation of lipophilicity research on 14 compounds (tryptophan, kynurenine pathway products, auxin pathway products, serotonin pathway products, tryptamine, as well as two synthetic auxin analogs): indole-2-acetic acid sodium salt (IAA), serotonin, 5-hydroxy-L-tryptophan, tryptamine, L-tryptophan, L-kynurenine (KYN), kynurenic acid (KYA), 3-hydroxy-DL-kynurenine, naphtyl-1-acetamide, indole-3-propionic acid (IPA), naphthalene-1-acetic acid (NAA), indole-3-butyric acid (IBA), indole-3-pyruvic acid (IPV), as well as melatonin. They were chromatographed in high performance liquid chromatography gradient conditions on tree stationary phases (C18, CN, DIOL) using three modifiers on each phase (methanol, acetonitrile and acetone). The resulting retention data was correlated with computational lipophilicity indices. Six compounds were proven to be ionized in neutral pH physiological conditions (IAA, KYA, IPA, NAA, IBA and IPV) and they were rechromatographed with acidic mobile phase to enhance the resulting dataset. It can be concluded that the retention times are highly correlated with lipophilicity regardless of used modifier and column and the main differentiating trend can be only connected to presence of naphthalene or indole ring. The principal component analysis, additive linear modeling, as well as multiplicative trilinear parallel factor analysis (PARAFAC) modeling helped to understand the internal structure of the obtained results.

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.

Endogenous Fluorescence Dissimilarity Assessment of Four Potential Biomarkers of Early Liver Fibrosis by Preservation Media Effect.

Build-up of extracellular matrix in liver fibrosis results in changes on endogenous molecules expression that may be studied through the fluorescence characterization of ex vivo liver samples. To the best of our knowledge, no investigations have provided in-depth evidence and discussion on the changes of the endogenous fluorescence in ex vivo tissue due to the effects of the preservation media. In this work, we contrast and analyze the endogenous fluorescence from tryptophan, vitamin A, hydroxyproline and elastin cross-links potential biomarkers of the liver fibrosis, in in vivo measurements and liver samples preserved on formaldehyde, and two standard preservation media. As it is known, chemical changes in tissue, caused by formaldehyde fixation, alter the endogenous fluorescence spectra. We propose the use of phosphate-buffered saline (PBS), and Iscove's Modified Dulbecco's Medium (IMDM) to elude the fluorescence changes. PBS and IMDM showed to maintain the endogenous fluorescence characteristics similar to in vivo conditions. The results of this work point the way for a more reliable assessment of endogenous fluorescence in ex vivo hepatic studies.

Facile Preparation of Fe3O4/C Nanocomposite and Its Application for Cost-Effective and Sensitive Detection of Tryptophan.

In this study, we reported facile synthesis of Fe3O4/C composite and its application for the cost-effective and sensitive determination of tryptophan (Trp) in human serum samples. Fe3O4/C composites were prepared by a simple one-pot hydrothermal method followed by a mild calcination procedure, using FeCl3∙6H2O as Fe3O4 precursor, and glucose as reducing agent and carbon source simultaneously. The Fe3O4/C composite modified glassy carbon electrode (Fe3O4/C/GCE) was prepared by drop-casting method. The microstructure and morphology of Fe3O4/C composite was characterized by powder X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. Due to large specific surface area and synergistic effect from Fe3O4 nanoparticles and carbon coating, Fe3O4/C composite showed excellent electrocatalytic activity toward the oxidation of Trp. As a result, the proposed Fe3O4/C/GCE displayed superior analytical performances toward Trp determination, with two wide detection ranges (1.0-80 µM and 80-800 µM) and a low detection limit (0.26 µM, S/N = 3). Moreover, successful detection of Trp in human serum samples further validate the practicability of the proposed sensor.

Assessment of bioactive compounds and their in vitro bioaccessibility in whole-wheat flour pasta.

We studied the polyphenol profile and antioxidant properties of cooked whole-wheat pasta to evaluate its effective antioxidant capacity, including changes produced by its production and in vitro digestion. The polyphenol profile was studied by HPLC-ESI-MS/MS, while the antioxidant capacity was measured by TEAC and FRAP assays. Results show that the polyphenol profile and antioxidant capacity change along the elaboration of cooked pasta, being the cooking step important to increase the release of bound polyphenols, enhancing their antioxidant properties. On the other hand, the study of the bioaccessibility of polyphenols, using an experimental model that simulates human gastrointestinal digestion and subsequent absorption, showed that only a small fraction of the starting polyphenolic compounds, mainly free polyphenols, could be absorbed by the small intestine; thus, reducing their effective antioxidant capacity. To our knowledge, this is the first report showing the bioaccessibility of hydroxybenzoic acid glucoside, hydroxybenzoic acid diglucoside, tryptophan, 6-C-glucosyl-8-C-arabinosyl-apigenin and diferulic acids.

Toxicity assessment of 4-chlorophenol to aerobic granular sludge and its interaction with extracellular polymeric substances.

The main objective of this study was to evaluate the toxicity of 4-chlorophenol (4-CP) to aerobic granular sludge in the process of treating ammonia rich wastewater. In the short-term exposure of 4-CP of 5 and 10 mg/L, ammonia nitrogen removal efficiencies in the batch reactors decreased to 87.18±2.81 and 41.16±3.55%, which were remarkably lower than that of control experiment (99.83±0.54%). Correspondingly, the respirometric activities of heterotrophic and autotrophic bacteria of aerobic granular sludge were significantly inhibited in the presence of 4-CP. Moreover, the main components of extracellular polymeric substances (EPS) including polysaccharides and proteins increased from 18.74±0.29 and 22.57±0.34 mg/g SS to 27.79±0.51 and 24.69±0.38 mg/g SS, respectively, indicating that the presence of 4-CP played an important role on the EPS production. Three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy further showed that the intensities of EPS samples were obviously quenched with the increased of 4-CP concentrations. To be more detailed, synchronous fluorescence spectra indicated that the interaction between EPS and 4-CP was mainly caused by tryptophan residues. The mechanism of fluorescence quenching belongs to static quenching with a formation constant (KA) of 0.07×10(4) L/mol, implying the strong formation of EPS and 4-CP complex. The results could provide reliable and accurate information to determine the potential toxicity of 4-CP on the performance of aerobic granular sludge system.

Assessment of field fluorometers.

Two field fluorometers, devoted either to natural organic matter (NOM) or to tryptophan-like fluorescing substances, were tested for the characterization of a large set of water samples (n = 263) impacted to various degrees by untreated or poorly treated urban sewage. Both fluorometers yielded consistent results when testing discrete samples. A nonlinear correlation (coefficient of determination = 0.98) was found between the tryptophan concentration given by the tryptophan field fluorometer and the fluorescence intensity given by a bench-top fluorometer (excitation = 285 nm, emission = 335 nm), corresponding to tryptophan-like fluorescing substances. A linear correlation with a mediocre coefficient of determination (0.63) was found between the NOM concentration given by the NOM field fluorometer and the fluorescence intensity given by the bench-top fluorometer (excitation = 355 nm, emission = 405 nm). This could be related to the diversity of NOM present, as illustrated by the different shapes of synchronous fluorescence spectra collected for the same samples.

Visual chiral recognition of tryptophan enantiomers using unmodified gold nanoparticles as colorimetric probes.

A simple protocol to distinguish enantiomers is extremely intriguing and useful. In this study, we propose a low-cost, facile, sensitive method for visual chiral recognition of enantimers. It is based on the inherent chirality of gold nanoparticles (AuNPs), and the unmodified AuNPs are used as chiral selector for D- and L-Tryptophan (Trp). In the presence of D-Trp, an appreciable red-to-blue color change of AuNPs solution can be observed, whereas no color change is found in the presence of L-Trp. The method can be used to detect D-Trp in the range of 0.2-10 µM, and the limit of detection is 0.1 µM. The chiral assay described in this work is easily readout with the naked eye or using a UV-vis spectrometer. Furthermore, the AuNPs can selectively adsorb D-Trp, and simple centrifugation can allow the precipitation of D-Trp with AuNPs and leave a net excess of the other enantiomer in solution, thus resulting in enantioseparation. In this method, AuNPs do not need any labeling or modifying with chiral molecules. The method is more attractive because of its high sensitivity, low cost, ready availability and simple manipulation.

Assessment of natural fluorescence as a tracer of diffuse agricultural pollution from slurry spreading on intensely-farmed grasslands.

The value of natural fluorescence in tracing diffuse pollution, in liquid phase, following slurry application to land was assessed by field experiment using twelve one hectare lysimeters on a heavy clay soil in Devon, UK, during autumn 2007. A strong linear relationship was found between natural fluorescence intensity and slurry concentration. The ratio of indices of tryptophan-like and fulvic/humic-like fluorescence (TI:FI) varied between 2 and 5 for a range of slurries sampled from Devon farms and allowed slurry to be distinguished from uncontaminated drainage waters (TI:FI<1). Incidental losses of slurry, indicated by significantly enhanced TI:FI ratios, high TI and high ammonium levels, occurred via the drain flow pathway of the drained lysimeters during the first small event following slurry-spreading. The maximum estimated loss from a single lysimeter was 2-8kg or 0.004-0.016% of the applied slurry. In the second larger storm event, some five weeks later, significantly enhanced TI:FI ratios in the drain flows were not associated with high TI but with high nitrate levels and, compared to the earlier storm, an increase in the humification index. This implies the loss of slurry decomposition products during this event but further work is needed to validate this. There was no significant enhancement of TI:FI in the surface/throughflow pathways of the drained or undrained lysimeters in either of the events. The observed change over a period of weeks in the strength and nature of the fluorescence signal from spread slurry restricts quantification of slurry losses to those immediately after slurry spreading. Nonetheless, this study demonstrates the utility of fluorescence as an indicator of slurry in drainage waters and the importance of field drains in diffuse agricultural pollution.

Reliable and inexpensive colorimetric method for determining protein-bound tryptophan in maize kernels.

Biofortification programs in maize have led to the development of quality protein maize (QPM) with increased contents of the essential amino acids lysine and tryptophan, and increased nutritional value for protein deficient populations where maize is a staple food. Because multiple genetic systems control and modify the protein quality of QPM, tryptophan or lysine monitoring is required to maximize genetic gain in breeding programs. The objective of this work was to develop an accurate, reliable, and inexpensive method for tryptophan analysis in whole-grain maize flour to support QPM research efforts around the world. Tryptophan reacts with glyoxylic acid in the presence of sulfuric acid and ferric chloride, producing a colored compound that absorbs at 560 nm. A series of experiments varying the reagent concentrations, hydrolysis time, and length of the colorimetric reaction resulted in an optimized protocol which uses 0.1 M glyoxylic acid in 7 N sulfuric acid and 1.8 mM ferric chloride, and 30 min reaction time. This method produced stable and reproducible results for tryptophan concentration in whole-grain maize flour and was validated by comparison with data obtained using an acetic acid-based colorimetric procedure (r(2) = 0.80) and high pressure liquid chromatography (HPLC) (r(2) = 0.71). We describe adaptations that permit high throughput application of this tryptophan analysis method using a microplate platform.

Assessment of the performance of a fed-batch cultivation from the preculture quality using an electronic nose.

An electronic nose, a gas-phase multisensor system, was used to monitor precultivations of a recombinant tryptophan-producing Escherichia coli strain. The electronic nose signals showed a high correlation toward the main stages of the precultivations, namely, exponential growth, oxygen-limited growth, and glucose depletion. Principal component analysis (PCA) of the electronic nose signals was performed and shown to be useful for monitoring preculture progression. More importantly, PCA also allowed a qualitative assessment of the preculture performance during subsequent fed-batch cultivations. The electronic nose signals from the precultures showed, furthermore, a high correlation to the time of phosphate limitation and the tryptophan yield coefficient of the subsequent fed-batch cultivations, which allowed an accurate prediction of these process variables using partial least squares (PLS). The results demonstrate on data from 12 cultivations how the electronic nose can be a useful tool for the assessment of inoculum quality, thereby providing means of reducing batch-to-batch variation and increasing the productivity of bioprocesses.

Detection of water proximity to tryptophan residues in proteins by single photon radioluminescence.

We recently developed a single photon radioluminescence (SPR) technique to measure submicroscopic distances in biological samples [Bicknese et al., and Shahrokh et al., Biophys. J., 63 (1992) 1256-1279]. SPR arises from the excitation of a fluorophore by the energy deposited from a slowing beta decay electron. The purpose of this study was to detect 3H2O molecules near tryptophan residues in proteins by tryptophan SPR. To detect small SPR signals, a sample compartment with reflective ellipsoidal optics was constructed, and amplified signals from a cooled photomultiplier were resolved by pulse-height analysis. A Monte Carlo calculation was carried out to quantify the relationship between SPR signal and 3H2O-tryptophan proximity. Measurements of tryptophan SPR were made on aqueous tryptophan; dissolved melittin (containing a single tryptophan); native and denatured aldolase; dissolved aldolase, monellin, and human serum albumin; and the integral membrane proteins CHIP28 (containing a putative aqueous pore) and MIP26 using 3H2O or the aqueous-phase probe 3H-3-O-methylglucose (OMG). After subtraction of a Bremsstrahlung background signal, the SPR signal from aqueous tryptophan (cps.microCi-1 mumol-1 +/- SE) was 8.6 +/- 0.2 with 3H2O and 7.8 +/- 0.3 with 3HOMG (n = 8). With 3H2O as donor, the SPR signal (cps.microCi-1 mumol-1) was 9.0 +/- 0.3 for monomeric melittin in low salt (trytophan exposed) and 4.6 +/- 0.8 (n = 9) for tetrameric melittin in high salt (tryptophans buried away from aqueous solution). The ratio of SPR signal obtained for aldolase under denaturing conditions of 8 M urea (fluorophores exposed) versus non-denaturing buffer (fluorophores buried) was 1.53 +/- 0.07 (n = 6). Ratios of SPR signals normalized to fluorescence intensities for monellin, aldolase, and human serum albumin, relative to that for d-tryptophan, were 1.42, 1.09, and 1.04, indicating that the cross-section for excitation of fluorophores in proteins is greater than that for tryptophan in solution. For the CHIP28 and MIP26 proteins in membranes, the ratio of SPR signal obtained with 3H2O versus 3HOMG was 1.35 +/- 0.13 (CHIP28, n = 5) and 0.99 +/- 0.02 (MIP26). These data are consistent with the existence of an aqueous channel through CHIP28 that excludes small solutes. We conclude that tryptophan radioluminescence in proteins is measurable and provides unique information about the presence of local aqueous compartments.

Eosinophilia-myalgia syndrome: lessons for public health researchers.

OBJECTIVE: To review epidemiological investigations into the epidemic of eosinophilia-myalgia syndrome which occurred predominantly in the United States of America in mid to late 1989, and examine the implications for similar urgent public health research in Australia. DATA SOURCES: Published data from epidemiological research, and relevant regulatory statements. OUTCOME: Intensive epidemiological investigations established that eosinophilia-myalgia syndrome was strongly associated with ingestion of L-tryptophan produced by a single manufacturer. It is likely that an identified contaminant has a role in pathogenesis, although the mechanism of action remains unclear. CONCLUSIONS: Rapid epidemiological investigation led to early containment measures and prevention of further public exposure to the causative agent. The episode highlighted the importance of preparedness amongst public health organisations to promptly initiate investigations of disease outbreaks, and demonstrated the benefits of a nation-wide ability to coordinate these studies. There may be encumbrances to urgent public health research in the form of inadequate mechanisms of data linkage. It is recommended that attention be given to such barriers now rather than in a time of crisis.