Liquid chromatography-mass spectrometry untargeted metabolomics reveals increased levels of tryptophan indole metabolites in urine of metabolic syndrome patients.

Metabolic syndrome (MetS) is a multifactor condition predisposing for diabetes, cardiovascular diseases and other degenerative disorders. Although several diagnostic criteria have been established, none of them is specific and there is a call for better pathophysiological explanation of MetS and for the discovery of molecular biomarkers. Phenotype characterization at metabolome level might be useful for both purposes. To this end, our aim was to perform comparative untargeted metabolomics of urines from MetS patients and from the control group. The study participants included 52 diagnosticated and 50 healthy individuals from Leon city in central Mexico; 23 anthropometric and clinical parameters were measured and submitted to Principal Component Analysis (PCA). The obtained PCA model allowed us for selection of 11 MetS patients and 13 control subjects, correspondingly representative for each of the two groups (clearly separated in PCA). The first morning urines from these subjects were ambulatory collected and, after methanol extraction and acidification, were submitted to capillary liquid chromatography-high resolution mass spectrometry (LC-HRMS). The obtained data were analyzed on MetaboScape® platform (Bruker Daltonics). Specifically, t-test applied to LC-HRMS data revealed several ions presenting at least 3-fold higher intensities in MetS with respect to the control samples (p < 0.05). Data analysis and complementary experiments yielded the identification of the following metabolites: indole-3-acetic acid, indole-3-acetic acid-O-glucuronide, N-(indol-3-ylacetyl) glutamine, indole-3-carbaldehyde and hydroxyhexanoycarnitine. Additionally, indole-3-carboxylic acid was annotated with 2.13-fold higher abundance in MetS patients. To assess the contribution of individual metabolites in the difference between two groups of subjects, partial least square discriminant analysis was performed for LC-HRMS data and the obtained values of variable importance in projection (VIP), confirmed the association of six above mentioned compounds with MetS. Overall, this study provides direct evidence on the disturbed catabolism of tryptophan in metabolic syndrome.

Determination of copper and lead in tequila by conventional matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and partial least squares regression.

RATIONALE: Quantification of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is challenging yet attractive, due to micro-scale procedural simplicity, high throughput and lack of memory effects. Since these features are important while analyzing trace elements in quality control schemes, MALDI-TOFMS was used for the determination of copper (Cu) and lead (Pb) in tequila with quantification carried out by partial least squares regression (PLS2) and by univariate calibration (UC). METHODS: In the proposed procedure, Bi(III) was added as internal standard (IS), diethyldithiocarbamate complexes were formed (pH 7.4) and extracted into chloroform; after solvent evaporation and re-constitution in acetonitrile, the sample was co-crystallized with α-cyano-4-hydroxycinnamic acid on a steel target. From the acquired mass spectra, UC was performed using IS-normalized signals of the monoisotopic ions of analytes, and the m/z range 350-513 was used for PLS2. Accuracy was tested by recovery experiments and by inductively coupled plasma (ICP)-MS analysis. RESULTS: When compared with direct analyte signal measurements, application of IS yielded enhanced analytical performance using either UC or PLS2; the method quantification limits were: 11.1 µg L-1 , 23.4 µg L-1 for Cu and 89.8 µg L-1 , 97.1 µg L-1 for Pb, respectively. In tequila, MALDI-TOFMS and ICP-MS provided consistent results for Cu (165-2599 µg L-1 ); Pb was not detected in any sample by MALDI-TOFMS, yet recoveries obtained after standard addition were indicative of acceptable accuracy (400 µg L-1 Pb added; recoveries: 91.2-108% for UC and 98.8-120% for PLS2). CONCLUSIONS: New experimental evidence has been provided supporting the inclusion of trace metals quantification within a range of MALDI-TOFMS applications. Slightly better results were obtained for UC as compared with PLS2 yet both methods can be recommended for testing the compliance of Cu and Pb levels with Official Mexican Norm. Of note, while using PLS2, there is no need for signal integration nor for IS normalization.

Melatonin reduces lead levels in blood, brain and bone and increases lead excretion in rats subjected to subacute lead treatment.

Melatonin, a hormone known for its effects on free radical scavenging and antioxidant activity, can reduce lead toxicity in vivo and in vitro.We examined the effects of melatonin on lead bio-distribution. Rats were intraperitoneally injected with lead acetate (10, 15 or 20mg/kg/day) with or without melatonin (10mg/kg/day) daily for 10 days. In rats intoxicated with the highest lead doses, those treated with melatonin had lower lead levels in blood and higher levels in urine and feces than those treated with lead alone, suggesting that melatonin increases lead excretion. To explore the mechanism underlying this effect, we first assessed whether lead/melatonin complexes were formed directly. Electronic density functional (DFT) calculations showed that a lead/melatonin complex is energetically feasible; however, UV spectroscopy and NMR analysis showed no evidence of such complexes. Next, we examined the liver mRNA levels of metallothioneins (MT) 1 and 2. Melatonin cotreatment increased the MT2 mRNA expression in the liver of rats that received the highest doses of lead. The potential effects of MTs on the tissue distribution and excretion of lead are not well understood. This is the first report to suggest that melatonin directly affects lead levels in organisms exposed to subacute lead intoxication.

Natural decrease of dissolved arsenic in a small stream receiving drainages of abandoned silver mines in Guanajuato, Mexico.

Arsenic release from the abandoned mines and its fate in a local stream were studied. Physicochemical parameters, metals/metalloids and arsenic species were determined. One of the mine drainages was found as a point source of contamination with 309 µg L(-1) of dissolved arsenic; this concentration declined rapidly to 10.5 µg L(-1) about 2 km downstream. Data analysis confirmed that oxidation of As(III) released from the primary sulfide minerals was favored by the increase of pH and oxidation reduction potential; the results obtained in multivariate approach indicated that self-purification of water was due to association of As(V) with secondary solid phase containing Fe, Mn, Ca.

Selective derivatization of cytosine and methylcytosine moieties with 2-bromoacetophenone for submicrogram DNA methylation analysis by reversed phase HPLC with spectrofluorimetric detection.

In eukaryotes, actual DNA methylation patterns provide biologically important information, for which both, genome-wide and locus-specific methylation at cytosine residues have been extensively studied. The original contribution of this work relies on the selective derivatization of cytosine moieties with 2-bromoacetophenone for the determination of global DNA methylation by reversed phase high performance liquid chromatography with spectrofluorimetric detection. The important features of the proposed procedure are as follows: (1) no need for the elimination of RNA, (2) detection limits for cytidine, 2'-deoxycytidine, 5-methylcytidine, and 5-methyl-2'-deoxycytidine in the range of 14.4-22.7 fmol, (3) feasibility for the detection of 0.06% of methylation in a low amount of DNA (80 ng), (4) potential viability for the evaluation of RNA methylation, and (5) relative simplicity in terms of analytical instrumentation and personnel training. The results obtained in the analysis of salmon testes DNA and nucleic acids from plant, human blood, and earthworms demonstrate the utility of the proposed procedure in biological studies and, in particular, for evaluation of the potential effect of environmental factors on actual DNA methylation in different types of living organisms.