CHAGAS, A. C. de S.

COLNAGO, L. A.

OLIVEIRA, M. C. de S.

POSTIGO, M. P.

RCAAP - Repositório Científico de Acesso Aberto de Portugal

Metabolic Studies of Bovine Urine and Blood Plasma using 1H and 13C-SSPF High Resolution NMR after Treatment with Ivermectin

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MR2010Joint EUROMAR 2010and 17th ISMAR ConferenceBook of AbstractsFlorence, July 4-9,20107. PostersP199Metabolic Studies of Bovine Urine and Blood Plasma using 'n and 13C-SSPFHighResolution NMR after Treatment with IvermectinMatheus P. Postigoa,b, Ana Carolina de S. Chagas C, Márcia C. de S. Oliveira" and Luiz A. Colnago""Embrapa Agricultural Instrumentation, 1452 xv de Novembro St.,Zip Code 13560-970, São Carlos-SP, Brazil (m postigo@yahoo.com.brJ"Chemistry Institute in São Carlos, São Paulo 's University, 400 Trabalhador Sancarlense Ave.Zip Code 13560-970, São Carlos-SP, Brazil"Embrapa Cattle-Southeast, Km #234 Washington Luís Rd.,Zip Code 13560-970, São Carlos-SP, BrazilBovine Metabolomics represents a novel tool for inspecting meat and dairy products quality and helping to detcctanimal diseases or disorders. In our studies, 24 Dutch Holstein female calves received intravenous Ivermectin 4% (dosagcof 1 mL/50 Kg), from which urine and blood plasma were analysed before (blank), and 3and 6 hours after treatment. Samples were analysed by using IR NMR PRESATsequence, and l3C SSFP (Steady-State Free Precession) sequence. The l3C SSFPsequence leads to an average 3-fold increasing in the signal-to-noise ratio, whencompared to standard l3C sequences, avoiding the need for isotope labelling, and thusrepresenting a pioneer technique for metabolic analysis. Metabolites were identified byusing online databases, and chemometric studies were carried out to highlight thevariations in both urine and plasma samples (both IR and l3C-SSPF spectra). Severalchanges were observed in post-treatment samples, such as a considerable increasing in 1 o.:mconcentration of aminoacids and histamine (what c1early denotes an allergic reaction) R-NMR Unne S ectrumand a decreasing of citrate levels. The information from these studies are the very first step for establishing a chemicalfingerprint for ivermectin misuse and/or the non-observance of the drug withdrawal term and consequent contaminationof milk and meat.References:1. WishartO. S., et aI., Nucleic Acids Res., 35, 0521 - 6 (2007)2. Lindon J. C., et al., An. Rev. Ana/. Chem., 1,45 - 69 (2008)3. LundbergP., et aI., MDL - The Mag. Res. Metab. Database, ESMRMB,Basel, Switzerland(2005)Acknowledgrnents:Embrapa(CNPOIAandCPPSE),FAPESP,USP.P200Towards Understanding the Folding Mechanísm of a Four-Helix-Protein, ACBPthe Cooperative Formation of Specific Tertiary Contacts in the Unfolded StateSusanne W. Bruun, Vytautas Iesmantaviõius, Jens A. Danielsson and Flemming M. PoulsenDepartment of Bíology, University of Copenhagen, O/e Maalees Vej 5, DK-2200 Copenhagen, Denmark ffmp@.bio.ku.dkJInstudies of the ensembles of unfolded structures of a four helix bundle protein ACBP -acyl coenzyme A bindingprotein -we have detected the presence of potential precursors of nativctertiary structures. These observations were based on the perturbation ol'NMR chemical shifts of the protein backbone atoms by single sitcmutations. Some mutations change the chemical shifts of residucxremote from the site of mutation indicating the presence of an interactioubetween the mutated and the remote residues. This suggests that thcformation of helix segments and helix-helix interactions is cooperativo.We can begin to track down the folding mechanism ofthis protein usingonly experimental data by combining the information available for thcrate limiting structure formation during the folding process withmeasurements of the site specific hydrogen bond formation in the burstphase, and with the existence prior to the folding reaction of tertiarystructures in the ensemble of otherwise unfolded structures observed inthe present study. We envisage that the detection of long rangeinteractions in ensembles ofunfolded proteins is not only restricted to ACBP. For many proteins it has been noticed thatafter the unfolding transition residual structure prevails which disappears only upon increase of the denaturanlconcentration as observed either by CD and/or NMR or other techniques. It is the ensemble of transient residual structurein the unfolded state, which has been characterized in case of ACBP, however, it seems most likely that many othcrunfolded proteins could be candidates for a similar study. The method is therefore seen to have a potential to become animportant experimental tool for the advancement of our understanding ofthe protein folding problem..Rf236

Metabolic Studies of Bovine Urine and Blood Plasma using 1H and 13C-SSPF High Resolution NMR after Treatment with Ivermectin

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