Reference Standardization for Quantification and Harmonization of Large-Scale Metabolomics.

Reference standardization was developed to address quantification and harmonization challenges for high-resolution metabolomics (HRM) data collected across different studies or analytical methods. Reference standardization relies on the concurrent analysis of calibrated pooled reference samples at predefined intervals and enables a single-step batch correction and quantification for high-throughput metabolomics. Here, we provide quantitative measures of approximately 200 metabolites for each of three pooled reference materials (220 metabolites for Qstd3, 211 metabolites for CHEAR, 204 metabolites for NIST1950) and show application of this approach for quantification supports harmonization of metabolomics data collected from 3677 human samples in 17 separate studies analyzed by two complementary HRM methods over a 17-month period. The results establish reference standardization as a method suitable for harmonizing large-scale metabolomics data and extending capabilities to quantify large numbers of known and unidentified metabolites detected by high-resolution mass spectrometry methods.

HPLC method for the assessment of tryptophan metabolism utilizing separate internal standard for each detector.

The development of a validated method, applicable for the measurement of tryptophan (TRP) and serotonin (5-HT), and that of the neuroprotective branch of the kynurenine pathway from several different biological matrices, including mouse brain, is described. Following the spectral analysis of the metabolites, they were quantified with reversed-phase high-performance liquid chromatography (HPLC), using separate internal standards (ISs) for UV (3-nitro-L-tyrosine) and fluorescent (the newly utilized 4-hydroxyquinazoline-2-carboxylic acid) detectors. With regard to validation parameters, selectivity, linearity, limit of detection, limit of quantification, precision and recovery were determined. Although the linearity ranges were different for the assessed matrices, the correlation coefficient was >0.999 in each case. Furthermore, good intra- and inter-day precision values were obtained with coefficient of variation <5%, and bias <6.5% (except the 5-HT level in brain samples), respectively. The recoveries varied between 82.5% and 116%. The currently developed methods yield opportunities for the assessment of concentration changes in the TRP metabolism from a wide range of biological matrices, therefore they may well be utilized in future clinical and preclinical studies, especially in view that so many metabolites with the application of ISs have not been detected from mouse brain with such a simple HPLC method before.

In vivo uptake and metabolism of alpha-[11C]methyl-L-tryptophan in human brain tumors.

Abnormal metabolism of tryptophan has been implicated in modulation of tumor cell proliferation and immunoresistance. alpha-[(11)C]Methyl-L-tryptophan (AMT) is a PET tracer to measure cerebral tryptophan metabolism in vivo. In the present study, we have measured tumor tryptophan uptake in 40 patients with primary brain tumors using AMT PET and standard uptake values (SUV). Tryptophan metabolism was further quantified in 23 patients using blood input data. Estimates of the volume of distribution (VD') and the metabolic rate constant (k(3)') were calculated and related to magnetic resonance imaging (MRI) and histology findings. All grade II to IV gliomas and glioneuronal tumors showed increased AMT SUV, including all recurrent/residual tumors. Gadolinium enhancement on MRI was associated with high VD' values, suggesting impaired blood-brain barrier, while k(3)' values were not related to contrast enhancement. Low-grade astrocytic gliomas showed increased tryptophan metabolism, as measured by k(3)'. In contrast, oligodendrogliomas showed high VD' values but lower k(3)' as compared with normal cortex. In astrocytic tumors, low grade was associated with high k(3)' and lower VD', while high-grade tumors showed the reverse pattern. The findings show high AMT uptake in primary and residual/recurrent gliomas and glioneuronal tumors. Increased AMT uptake can be due to increased metabolism of tryptophan and/or high volume of distribution, depending on tumor type and grade. High tryptophan metabolic rates in low-grade tumors may indicate activation of the kynurenine pathway, a mechanism regulating tumor cell growth. AMT PET might be a useful molecular imaging method to guide therapeutic approaches aimed at controlling tumor cell proliferation by acting on tryptophan metabolism.

From sugar beet molasses to Lyphan. Integrated quality management from the raw material to the drug.

L-tryptophan is produced at the AMINO GmbH (Frellstedt, FRG) via biocatalytical condensation of the amino acid L-serine with indole. As a biocatalyst, tryptophan synthetase is used which is produced in high activities by a natural mutant Escherichia coli strain. The enzyme mechanism and specificity and the individual process-parameters for the biotransformation procedure are explained as well as the purification process of educts and products. This includes a detailed description of the quality control of educts, intermediates and final product. The active ingredient L-tryptophan is subsequently used by AMINO's subsidiary company esparma GmbH to produce and distribute the pharmaceutical Lyphan. The quality management system and the production procedure for Lyphan are described and discussed.

Structural characterization of case-associated contaminants peak C and FF in L-tryptophan implicated in eosinophilia-myalgia syndrome.

We have characterized the structures of two case-associated contaminants of the Showa Denko L-tryptophan known to cause eosinophilia-myalgia syndrome (EMS). A combination of on-line accurate mass HPLC-electrospray ionization-mass spectrometry (LC-MS), HPLC-tandem mass spectrometry (LC-MS-MS) and HPLC-in source collision induced dissociation-MS-MS (LC-sCID-MS-MS) allowed the structure determination of both Peak C and FF. Peak C is identified as 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2-3b]-indole-2-carboxyl ic acid, whereas Peak FF is characterized as 2-(2-hydroxy-indoline)-tryptophan. Both contaminants contain indoline rings, and the significance of this finding is discussed.

Synthesis, formation, and occurrence of contaminants in biotechnologically manufactured L-tryptophan.

The pattern of contaminants in pharmaceutical and feed grade L-tryptophan (Trp) was investigated in a market survey of 22 lots of 6 different manufacturers. To date, 5 case associated contaminants in Showa Denko tryptophan (SD-Trp) known to cause the autoimmune disease eosinophilia-myalgia syndrome (EMS) have been structurally elucidated: 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid (PIC), an indoline compound, is one of the most abundant degradation compounds of unbound Trp during oxidative treatment. 2-(3-indolylmethyl)-L-tryptophan (IMT) and 2-(2-hydroxyindoline)-tryptophan (HIT) are both 2-substituted Trp-derivatives. IMT was synthesized by the reaction of Trp and indole-3-methanol or indole-3-acetaldehyde, respectively. From this finding it is proposed that Trp-metabolites can decompose under formation of transitional, mesomerism-stabilized cations that react with excess Trp to yield 2-substituted Trp derivatives. The decomposition of Trp-metabolites could be induced by elevated or low pH-values that occur during the downstream processing of the Trp fermentation broth. IMT was detected in pharmaceutical-grade and feed-grade Trp in amounts of < 20-1,400 mg/kg. 1,1'-Ethylidenebis-(L-tryptophan) (EBT) is formed from acetaldehyde and Trp under acidic conditions and serves as a marker for EMS-suspicious Trp. 3-(Phenylamino)alanine (PAA) is the only not Trp derived case associated contaminant. Low amounts of PAA (20 mg/kg) could be detected in feed-grade Trp of one manufacturer. Non-EMS correlated 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids of Trp and formaldehyde, acetaldehyde and indole-3-acetaldehyde could be detected in the examined Trp raw materials (< 10-13,500 mg/kg). In order to guarantee the safety of Trp containing drugs the amount of EBT (< 10 mg/kg Trp) and the sum of UV220 nm detectable contaminants (< 400 mg/kg Trp) are limited by the European authorities.

Synthesis and formation of an EMS correlated contaminant in biotechnologically manufactured L-tryptophan.

Contaminants in biotechnologically manufactured L-Tryptophan (Trp) are suspected to be responsible for the outbreak of an unknown autoimmune disease in 1989. The contaminants, found in Trp-lots of a Japanese manufacturer, are classified in EMS-correlated and non EMS-correlated substances. Up to now six EMS-correlated substances are known. One of these compounds is 2-(3'-indolylmethyl)-indole (IMT). IMT was detected as a major contaminant in two investigated EMS-associated trp-samples. In a seven step chemical synthesis IMT was obtained for use as a reference substance. A model system to investigate the formation of IMT was created using Trp and 3-indolylmethanol (IM). IMT formation was observed at acidic and alkaline pH-values and the optimal molar ratio of Trp to IM is 100:1. In addition an IMT formation was observed from indole, formaldehyde and Trp as well as from Trp and 3-indolylacetaldehyde.

Persistent microvasculopathy in chronic eosinophilia-myalgia syndrome.

Acute eosinophilia-myalgia syndrome (EMS) due to contaminated L-tryptophan (LT) exposure is an inflammatory microangiopathy of the dermis, fascia, and muscle. Select individuals evolve from acute EMS to have persistence of myalgia, fatigue, cramps, and skin changes for years. Many develop memory dysfunction and confusion. The objective of this study is to delineate the pathology in individuals with chronic EMS. Seventeen patients with ongoing symptoms representing chronic EMS are studied by skin, fascia, and muscle biopsies four to five years after exposure to contaminated LT and initial onset of EMS. All have microvascular disease. Most have lymphocytic inflammatory infiltrates. Several have dermal sclerosis. The findings indicate that persistent microvascular disease is present in chronic EMS. The pathologic changes are similar to those of acute EMS but with notable differences. Tissue eosinophil infiltration is rare in the chronic state as compared to acute EMS. The persistence of endothelial pathology indicates continuing microvascular dysfunction.

Efficacy of a lysine-tryptophan blend for growth of chicks.

Two chick experiments were conducted to compare the growth-promoting efficacy as well as the toxicity of a new source of L-tryptophan and L-lysine, Tryptosine (16.1% tryptophan, 56.3% lysine). A corn-feather meal-soybean meal basal diet was made singly deficient in either lysine or tryptophan, and graded doses of lysine or tryptophan from either Tryptosine or feed-grade sources of lysine and tryptophan were supplemented. Linear (P < .01) weight gain responses occurred, and responses to lysine or tryptophan in Tryptosine were similar to those obtained with equal doses of lysine or tryptophan provided by feed-grade sources of L-lysine.HCI or L-tryptophan. The toxicity trial involved additions of 1, 2, or 4% lysine with .29, .58, or 1.16% tryptophan to a lysine- and tryptophan-adequate corn-soybean meal diet. Both amino acids were provided as either Tryptosine or as feed-grade sources of lysine and tryptophan. Weight gain and feed intake were reduced in a linear fashion (P < .01) as levels of the two excess amino acids increased. The decreases caused by Tryptosine were similar to those caused by equivalent levels of excess feed-grade lysine and tryptophan.

Optimum ratio to lysine of threonine, tryptophan, and sulfur amino acids for finishing swine.

Forty-eight crossbred (PIC line 26 x Camborough 15) pigs were used in two finishing trials to compare the ideal ratios of threonine (Thr), tryptophan (Trp), and sulfur amino acids (SAA) to lysine (Lys) determined for young pigs to a proposed ratio of these amino acids for finishing pigs. Trial 1 involved 20 barrows and 20 gilts that were self-fed in sex groups of two. Trial 2 was a Latin square design that used four barrows and four gilts that were individually fed in metabolism cages. Separate diets were used for the early (EF = 56 to 90 kg) and late (LF = 90 to 112 kg) finishing periods. Diets were formulated from a corn-soybean meal mixture and contained 11% CP and .55% digestible lysine for EF pigs and 10% CP and .50% digestible lysine for LF pigs. Negative-control diets in both the EF and LF periods were designed to be slightly deficient in lysine and to contain digestible Thr (65%), Trp (18%), and SAA (60%) at the ideal ratio to digestible Lys determined for 10- to 20-kg pigs. The experimental diet in both the EF and LF periods was formulated to contain digestible Thr (70%), Trp (20%), and SAA (65%) at the proposed ideal ratio to digestible Lys for finishing pigs. In Trial 1, increased ratios of Thr, Trp, and SAA improved gain:feed ratio, whole-body and carcass protein concentration, and whole-body and carcass protein accretion. In Trial 2, LF pigs responded to the increased ratios of Thr, Trp, and SAA with decreased urinary nitrogen excretion and increased N retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Sorghum amino acid-supplemented diets for the 50- to 100-kilogram pig.

Three experiments were conducted to evaluate lysine (Lys), threonine (Thr), methionine (Met), tryptophan (Trp), and nonessential N (+N) supplementation of all sorghum grain (S) diets on growth and carcass characteristics of 50- to 100-kg pigs. In Exp. 1, four replicate pens of four pigs each were fed: 1) sorghum-soybean meal positive control (S-SBM); 2) S + Lys + Thr + Met + Trp to equal the amino acid concentrations in S-SBM (LTMT); 3) Diet 2 + N (LTMT+N); and 4) S negative control. Compared with pigs fed S-SBM, pigs fed LTMT or LTMT+N had reduced (P < .05) ADG, ADFI, serum urea N (SUN), pancreas weight, LMA, and percentage of muscling (PM) but higher dressing percentage (DP) and similar 10th rib fat thickness (TRF). Apparent N digestibility was lower (P < .05) in pigs fed LTMT than in pigs fed LTMT+N or S-SBM. In Exp. 2, two replicate pens of four pigs each were fed: 1) S-SBM; 2) S + Lys + Thr (LT); 3) S + Lys + Thr + Met (LTM); 4) S + Lys + Thr + Trp (LTT); 5) LTMT; 6 to 9) as 2 to 5 +N; and 10) S negative control. Compared with pigs fed S-SBM, pigs fed S, LT, LT+N, and LTM+N had lower (P < .05) ADG. Daily gain of pigs fed LTM, LTT, LTMT, LTT+N, or LTMT+N was not different (P > .10) from pigs fed S-SBM.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of tryptophan requirement of the commercial layer by using a corn-soybean meal basal diet.

An experiment was conduced with Hy-Line W36 hens to evaluate the requirement for Trp in a corn-soybean meal diet. Seven experimental diets were fed with Trp levels of 0.12, 0.13, 0.14, 0.15, 0.16, 0.17 and 0.18%. Supplemental amino acids (AA) were added to all diets to ensure that Trp was the first-limiting AA. A positive control diet (0.20% Trp) with Met supplementation was fed that had previously been shown to support maximum performance. Egg production (EP), egg weight (EW), and egg content (EC) were significantly increased by the addition of Trp to the basal diet. Broken-line regression indicated the Trp requirement for EP and EC was 139.8 and 149.0 mg per hen/d, respectively, for EP and EC when hens had a daily EC of 45.4 g per hen/d.