Quantification and Stereochemical Composition of R-(-) and S-(+)-Clenbuterol Enantiomers in Bovine Urine by Liquid Chromatography-Tandem Mass Spectrometry.

Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzylalcohol) is a ß2-adrenergic agonist. The consumption of meat contaminated with clenbuterol can lead to increased heart rate, blood pressure, anxiety, palpitations and skeletal muscle tremors. Several analytical methods have been developed to identify and quantify clenbuterol in different biological matrices. In this report, we have developed a specific and sensitive analytical method for quantifying clenbuterol and performed an in-depth enantiomeric analysis in bovine urine. The method was evaluated in accordance with international guidelines, and we used an isotopically labeled analog as an internal standard. The extraction efficiency for clenbuterol in bovine urine was > 98%, the limit of detection was 0.05 ng/mL and the limit of quantification was 0.10 ng/mL. Our assay showed high specificity, no carryover was observed and the assay was linear in the range 0.10-8.0 ng/mL. Fifteen bovine urine samples were analyzed (containing clenbuterol), and an enantiomeric analysis was performed. The clenbuterol concentration range was 0.10-10.56 ng/mL across these samples. The levorotatory enantiomer was detected at greater concentrations than the dextrorotatory enantiomer, the ratio being 1.7 ± 0.6 (n = 15), and a statistical difference was observed (P < 0.05) using the Wilcoxon test.

Effect of SNPs in HSP Family Genes, Variation in the mRNA and Intracellular Hsp Levels in COPD Secondary to Tobacco Smoking and Biomass-Burning Smoke.

Heat shock proteins (HSP) genes are a superfamily responsible for encoding highly conserved proteins that are important for antigen presentation, immune response regulation, and cellular housekeeping processes. These proteins can be increased by cellular stress related to pollution, for example, smoke from biomass burning and/or tobacco smoking. Single nucleotide polymorphisms (SNPs) in these genes could affect the levels of their proteins, as well as the susceptibility to developing lung diseases, such as chronic obstructive pulmonary disease (COPD), related to the exposure to environmental factors. Methods: The subjects included were organized into two comparison groups: 1,103 smokers (COPD patients, COPD-S = 360; smokers without COPD, SWOC = 743) and 442 never-smokers who were chronically exposed to biomass smoke (COPD patients, COPD-BS = 244; exposed without COPD, BBES = 198). Eight SNPs in three HSP genes were selected and genotyped: four in HSPA1A, two in HSPA1B, and two in HSPA1L. Sputum expectoration was induced to obtain pulmonary cells and relative quantification of mRNA expression. Subsequently, the intracellular protein levels of total Hsp27, phosphorylated Hsp27 (Hsp27p), Hsp60, and Hsp70 were measured in a sample of 148 individuals selected based on genotypes. Results: In the smokers' group, by a dominant model analysis, we found associations between rs1008438 (CA+AA; p = 0.006, OR = 1.52), rs6457452 (CT+TT; p = 0.000015, OR = 1.99), and rs2763979 (CT+TT; p = 0.007, OR = 1.60) and the risk to COPD. Among those exposed to biomass-burning smoke, only rs1008438 (CA+AA; p < 0.01, OR = 2.84) was associated. Additionally, rs1008438 was associated with disease severity in the COPD-S group (AA; p = 0.02, OR = 2.09). An increase in the relative expression level of HSPA1A was found (12-fold change) in the COPD-BS over the BBES group. Differences in Hsp27 and Hsp60 proteins levels were found (p < 0.05) in the comparison of COPD-S vs. SWOC. Among biomass-burning smoke-exposed subjects, differences in the levels of all proteins (p < 0.05) were detected. Conclusion: SNPs in HSP genes are associated with the risk of COPD and severe forms of the disease. Differences in the intracellular Hsp levels are altered depending on the exposition source.

SNPs in NRXN1 and CHRNA5 are associated to smoking and regulation of GABAergic and glutamatergic pathways.

AIM: To identify genetic variants associated with greater tobacco consumption in a Mexican population. PATIENTS & METHODS: Daily smokers were classified as light smokers (LS; n = 742), heavy smokers (HS; n = 601) and nonsmokers (NS; n = 606). In the first stage, a genotyping microarray that included 347 SNPs in CHRNA2-CHRNA7/CHRNA10, CHRNB2-CHRNB4 and NRXN1 genes and 37 ancestry-informative markers was used to analyze 707 samples (187 HS, 328 LS and 192 NS). In the second stage, 14 SNPs from stage 1 were validated in the remaining samples (HS, LS and NS; n = 414 in each group) using real-time PCR. To predict the role of the associated SNPs, an in silico analysis was performed. RESULTS: Two SNPs in NRXN1 and two in CHRNA5 were associated with cigarette consumption, while rs10865246/C (NRXN1) was associated with high nicotine addiction. The in silico analysis revealed that rs1882296/T had a high level of homology with Hsa-miR-6740-5p, which encodes a putative miRNA that targets glutamate receptor subunits (GRIA2, GRID2) and GABA receptor subunits (GABRG1, GABRA4, GABRB2), while rs1882296/C had a high level of homology with Hsa-miR-6866-5p, which encodes a different miRNA that targets GRID2 and GABRB2. CONCLUSION: In a Mexican Mestizo population, greater consumption of cigarettes was influenced by polymorphisms in the NRXN1 and CHRNA5 genes. We proposed new hypotheses regarding the putative roles of miRNAs that influence the GABAergic and glutamatergic pathways in smoking addiction.

Nicotine Addiction Development: From Epidemiology to Genetic Factors.

BACKGROUND: Nicotine addiction is a complex and multifactorial disease affecting the central nervous system and consists of a set of characteristic symptoms and signs. OBJECTIVE: The objective of this study was to provide an overview on smoking and the complexity of dependency, with special emphasis on the involvement of genetic factors, including neurexin and nicotinic cholinergic receptor genes. METHODS: The following two aspects are discussed in the present article: (i) epidemiology in Mexico; and (ii) a review of the published literature on genetic association studies using the National Center for Biotechnology Information (NCBI) database of the USA as a search tool. The search key words were: nicotine, smoking, dependence, genetic, tobacco, neurobiology and GWAS. The publication period of the reviewed articles was January 2005 to July 2015. RESULTS: There are numerous studies that provide evidence of the involvement of a genetic component that contributes to the risk of developing nicotine addiction, but the multifactorial nature of addiction requires coordinated research from multiple disciplines. CONCLUSION: Research is needed on the factors associated with genetic risk for nicotine addiction and their interaction with environmental factors.

Maternal age and family history are risk factors for ankylosing spondylitis.

OBJECTIVE: To investigate prevalence and gender distribution in parents of children with ankylosing spondylitis (AS). METHODS: Family history of AS (parents, uncles, and aunts), maternal age at delivery, and consecutive pregnancy number were assessed in the relatives of 40 Mexican Mestizo patients with definite AS (New York Criteria). RESULTS: We evaluated the family history of AS in 34 families of 40 AS patients; 12 with none, 4 with a paternal history (4 healthy fathers with a brother with AS) (odds ratio, OR, 1.37, p = 0.75), 15 with a maternal history of AS, (15 healthy mothers with a brother with AS) (OR 1.4, p = 0.55), and 3 with both lines (OR 0.84, p = 0.92). In these families AS was more frequent in males (29%) than in females (10%), OR 3.40 (95% confidence interval, CI: 1.43-8.29, p = 0.003). Juvenile onset was more common in the offspring of mothers with family history (72%) (OR 13.0, 95% CI: 1.68-147.48, p = 0.009). The number of first-born children with AS (18%) was similar to the later-born children (23%) (OR 1.37, 95% CI: 0.38-5.31, p = 0.78). The frequency of AS increased when the maternal age at delivery was < or = 30 years (OR 0.20, 95% CI: 0.04-0.75, p = 0.01). CONCLUSION: In Mexican Mestizo patients, there is no correlation between the risk for AS and the gender of the affected parent. However we found an association between juvenile onset and maternal family history with an increased incidence in patients with younger mothers.