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1. Antioxidants green tea extract and nordihydroguaiaretic acid confer species and strain-specific lifespan and health effects in Caenorhabditis nematodes.

Author

Banse, Stephen A., Sedore, Christine A., Johnson, Erik, Coleman-Hulbert, Anna L., Onken, Brian, Hall, David, Jackson, E. Grace, Huynh, Phu, Foulger, Anna C., Guo, Suzhen, Garrett, Theo, Xue, Jian, Inman, Delaney, Morshead, Mackenzie L., Plummer, W. Todd, Chen, Esteban, Bhaumik, Dipa, Chen, Michelle K., Harinath, Girish, and Chamoli, Manish

Subjects
TEA extracts, GREEN tea, CAENORHABDITIS, NEMATODES, CAENORHABDITIS elegans
Abstract

The Caenorhabditis Intervention Testing Program (CITP) is an NIH-funded research consortium of investigators who conduct analyses at three independent sites to identify chemical interventions that reproducibly promote health and lifespan in a robust manner. The founding principle of the CITP is that compounds with positive effects across a genetically diverse panel of Caenorhabditis species and strains are likely engaging conserved biochemical pathways to exert their effects. As such, interventions that are broadly efficacious might be considered prominent compounds for translation for pre-clinical research and human clinical applications. Here, we report results generated using a recently streamlined pipeline approach for the evaluation of the effects of chemical compounds on lifespan and health. We studied five compounds previously shown to extend C. elegans lifespan or thought to promote mammalian health: 17α-estradiol, acarbose, green tea extract, nordihydroguaiaretic acid, and rapamycin. We found that green tea extract and nordihydroguaiaretic acid extend Caenorhabditis lifespan in a species-specific manner. Additionally, these two antioxidants conferred assay-specific effects in some studies—for example, decreasing survival for certain genetic backgrounds in manual survival assays in contrast with extended lifespan as assayed using automated C. elegans Lifespan Machines. We also observed that GTE and NDGA impact on older adult mobility capacity is dependent on genetic background, and that GTE reduces oxidative stress resistance in some Caenorhabditis strains. Overall, our analysis of the five compounds supports the general idea that genetic background and assay type can influence lifespan and health effects of compounds, and underscores that lifespan and health can be uncoupled by chemical interventions. [ABSTRACT FROM AUTHOR]

Published
2024
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2. The Effect of Complex Instruction Team Product (CITP) Learning Model on Increase Student's Skills.

Author

Rumahlatu, Dominggus, Sangur, Kristin, and Liline, Sintje

Subjects
TEAM learning approach in education, HIGH school students, SCIENTISTS' attitudes, COGNITIVE learning, HIGH school seniors, BIOLOGY teachers
Abstract

This research aims at investigating the effect of Complex Instruction integrated with Team Product (CITP) learning model on students' creative thinking skills, critical thinking skills, metacognitive skills, scientific attitudes, cognitive learning results, and retention. This research used quasi-experimental design with pretestposttest nonequivalent control group design. This research was carried out in several Senior High Schools in Ambon, namely SMA 1 Ambon, SMA 4 Ambon, SMA 5 Ambon, SMA 13 Ambon, and SMA Kristen Ambon in the even semester of the academic year 2017/2018. The results of the research were analyzed using ANCOVA. A post hoc LSD test was also performed if there were differences in each variable. The results of the research show that the CITP learning model affects creative thinking skills, critical thinking skills, metacognitive skills, scientific attitudes, cognitive learning results, and retention of class X senior high school students on Ecosystem learning material. The syntax of the CITP learning model can improve students' creative thinking skills, critical thinking skills, metacognitive skills, scientific attitudes, cognitive learning results, and retention compared to the learning model commonly implemented by the biology teachers. [ABSTRACT FROM AUTHOR]

Published
2020
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3. Automated lifespan determination across Caenorhabditis strains and species reveals assay-specific effects of chemical interventions.

Author

Banse, Stephen A., Lucanic, Mark, Sedore, Christine A., Coleman-Hulbert, Anna L., Plummer, W. Todd, Chen, Esteban, Kish, Jason L., Hall, David, Onken, Brian, Presley, Michael P., Jones, E. Grace, Blue, Benjamin W., Garrett, Theo, Abbott, Mark, Xue, Jian, Guo, Suzhen, Johnson, Erik, Foulger, Anna C., Chamoli, Manish, and Falkowski, Ron

Subjects
CAENORHABDITIS, CHEMICAL testing, SPECIES, LIGHT elements
Abstract

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach toward aging-intervention screening. [ABSTRACT FROM AUTHOR]

Published
2019
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4. BLOOD LEVEL OF COLLAGEN BIOMARKERS AND SULFATED GLYCOSAMINOGLYCANS IN ACUTE TRANSMURAL MYOCARDIAL INFARCTION

Author

A. V. Govorin, E. V. Ratsina, N. V. Fetisova, and N. A. Sokolova

Subjects
transmural anterior mi, aneurysm, extracellular matrix, picp, citp, sgag, Diseases of the circulatory (Cardiovascular) system, RC666-701
Abstract

Aim. To study dynamics of blood levels of carboxyterminal propeptide of I type procollagen (PICP), carboxyterminal telopeptide of type I collagen (CITP) and sulfated glycosaminoglycans (sGAG) in the dynamics, in patients with transmural transmural myocardial infarction (MI) complicated with aneurysm.Material and methods. Totally, 46 patients included with acute transmural anterior MI, who underwent the assessment of PICP, CITP and sGAG in dynamics.Results. On the 1st to 3rd days of MI without aneurysm there was increase of PICP up to 61,4 ng/mL, on 10-12th and 18-22nd days its level did not change significantly, remaining high. In aneurysm MI the level of PICP increased just from the 10-12th days and in lower grade — comparing to patients without aneurysm. Level of CITP in patients without aneurysm was comparable to controls during the entire study. Level of sGAG in non-aneurysm patients increased from 10-12th days, and in MI with aneurysm — did not differ from healthy persons during the entire study.Conclusion. Increase of CITP and lower PICP and sGAG in MI patients with aneurysm witnesses’ probable predominance of degradation processes of connective tissue over its synthesis.

Published
2016
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5. Application of lactic acid bacteria for production of fermented beverages based on rice flour

Author

Michal MAGALA, Zlatica KOHAJDOVÁ, Jolana KAROVIČOVÁ, Mária GREIFOVÁ, and Jarmila HOJEROVÁ

Subjects
citp, hplc, fermentation, rice, sensory analysis, viscosity, Agriculture
Abstract

We investigated the suitability of rice flour for fermented beverage production using various strains of lactic acid bacteria. Fermentation led to a decrease in pH from 5.04-5.17 to 3.74-4.35. At the same time, total acidity increased (1.28-2.59 g/l) due to lactic acid (0.59-2.76 g/l) and acetic acid (0.11-0.30 g/l) production. Fermentation of rice beverages also caused a gradual decrease in glucose and fructose concentration. Lactic acid bacteria proliferated in the first phases of fermentation, and cell counts reached a maximum after 12 h. The highest growth rate (vLAB = 0.44 Log10 CFU/ml/h) was observed in a sample with the culture of Lactobacillus brevis CCM 1815. Viscosity of beverages decreased significantly after 24 h of fermentation. The highest values of sensory parameters were observed in a monoculture of Lactobacillus plantarum CCM 7039 and in a sample with a mixed culture of Lactobacillus plantarum CCM 7039 and Bifidobacterium longum CCM 4990.

Published
2015
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6. Application of Lactic Acid Bacteria for Production of Fermented Beverages Based on Rice Flour.

Author

MAGALA, MICHAL, KOHAJDOVÁ, ZLATICA, KAROVIČOVÁ, JOLANA, GREIFOVÁ, MÁRIA, and HOJEROVÁ, JARMILA

Subjects
LACTIC acid bacteria, FERMENTATION, VISCOSITY, RICE flour, FERMENTED beverages, ACETIC acid, GLUCOSE, FRUCTOSE
Abstract

We investigated the suitability of rice flour for fermented beverage production using various strains of lactic acid bacteria. Fermentation led to a decrease in pH from 5.04-5.17 to 3.74-4.35. At the same time, total acidity increased (1.28-2.59 g/l) due to lactic acid (0.59-2.76 g/l) and acetic acid (0.11-0.30 g/l) production. Fermentation of rice beverages also caused a gradual decrease in glucose and fructose concentration. Lactic acid bacteria proliferated in the first phases of fermentation, and cell counts reached a maximum after 12 h. The highest growth rate (vLAB = 0.44 Log10 CFU/ml/h) was observed in a sample with the culture of Lactobacillus brevis CCM 1815. Viscosity of beverages decreased significantly after 24 h of fermentation. The highest values of sensory parameters were observed in a monoculture of Lactobacillus plantarum CCM 7039 and in a sample with a mixed culture of Lactobacillus plantarum CCM 7039 and Bifidobacterium longum CCM 4990. [ABSTRACT FROM AUTHOR]

Published
2015
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7. A Saudi Child With Chronic Immune Thrombocytopenia and Vitiligo

Author

Hasanat Abbas, Maryam Attaf, Hidayah M Alotaibi, Arwa Al-Yamani, and Abdulqader Al-Hebshi

Subjects
vitiligo, Pediatrics, medicine.medical_specialty, tpo receptor agonist, Eltrombopag, Vitiligo, 030204 cardiovascular system & hematology, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, immune system diseases, hemic and lymphatic diseases, medicine, Thrombopoietin receptor, biology, business.industry, behcet, General Engineering, Genetic disorder, Hematology, citp, medicine.disease, tnf aip3 gene, Purpura, chemistry, biology.protein, Rituximab, medicine.symptom, Antibody, business, 030217 neurology & neurosurgery, medicine.drug, Medical literature
Abstract

Chronic immune thrombocytopenia (ITP) is less commonly found in the children presenting with ITP. Patients usually present with petechiae, purpura, or active bleeding in the form of epistaxis or hematuria. The main aim of treatment in chronic ITP is to prevent major bleeding and to increase the platelet count. High doses of corticosteroids, intravenous immunoglobulin, rituximab, and eltrombopag, a thrombopoietin receptor agonist (TPO-RA), are medications that can be used. In this report, we present a case of chronic ITP in a 12-year-old child. In addition to features of chronic ITP, he also has vitiligo around his eyes and limbs. During treatment, he was resistant to steroids and did not respond to rituximab or eltrombopag. To understand the cause of his presenting features, we did multiple diagnostic evaluations. The whole-exome sequencing raises the possibility of auto-inflammatory syndrome Behcet-like (AISBL), which is a rare genetic disorder and not frequently reported in the available medical literature. AISBL is caused by mutations in the TNFAIP3 gene. According to our best knowledge, this is the first Saudi child diagnosed with chronic ITP and vitiligo with the possibility of AISBL that needs further genetic work-up to confirm the diagnosis.

Published
2020

8. Comparison of cITP and 31 P NMR Methods for Determination of Polyphosphates in Meat Product and Sea Fruits

Author

A. Jastrzębska, P. Hrynczyszyn, and E. Szłyk

Subjects
polyphosphates, citp, 31p nmr, food samples, Agriculture
Abstract

Phosphates are widely used as additives in different kinds of food, including meat, poultry and seafood. These ions work synergistically with salt and allow meat processors to achieve equivalent water-holding capacity with reduced amounts of salt. Polyphosphates added to meat and seafood products hydrolysed quickly to phosphates and it is difficult to estimation of these compounds (Davis et al. 2004; Sheard & Tali 2004; Jastrzębska et al. 2008). In our work we compared capillary isotachophoresis (cITP) and nuclear magnetic resonance (31P NMR) methods for phosphate ions determination in food samples. Furthermore, the usefulness of the studied methods to monitoring of poly- and pyrophosphates hydrolysis in meat and sea fruits was discussed. The meat products and sea fruit samples purchased from a local market and acquired polyphosphates in composition were used for analyses. The food were minced, homogenised and extracted with redistilled water using an orbital shaker for 45 minutes. The extracts were separated using centrifuge at 9000 rpm for 30 min, followed by double filtration. All extracts were transferred into a 50 ml volumetric flask, made up to the mark and analysed with one-dimensional cITP and 31 P NMR methods. The obtained results for two proposed methods were discussed in respect of the accuracy, precision and compared by F-test and t--test. In concluded, cITP method was simpler, lower time consuming, uncomplicated and economical than 31 P NMR technique. Moreover, permitted on expiration date control by qualitative determination of polyphosphates.

Published
2009
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9. Application of capillary isotachophoretic method to the determination of nitrate and nitrite ions in meat products.

Author

Jastrzębska, A.

Subjects
*ISOTACHOPHORESIS, *ANIMAL products, *NITRITES, *NITRATES, *ELECTROLYTES, *IONS
Abstract

The application of capillary isotachophoretic method to the simultaneous determination of nitrates and nitrites in meat products was studied. Simple electrolyte system allowed separation of ions in standard solution with within-day and between-days coefficient of variation (CV) of relative step hight in the ranges 2.3-2.5% and 4.1-5.5%, respectively. The levels of nitrates and nitrites ranged from 3 to 65 mg/kg NaNO and from 36 to 111 mg/kg NaNO. The coefficients of variation for nitrites were 0.7-3.5% and for nitrates 2.3-5.9%, that indicated satisfactory precision of the proposed method. The recoveries for meat samples spiked with nitrites and nitrates varied between 95-99% and 91-102%, respectively. [ABSTRACT FROM AUTHOR]

Published
2010
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10. Automated Lifespan Determination Across Caenorhabditis Strains and Species Reveals Assay-Specific Effects of Chemical Interventions

Author

Daniel Edgar, Jian Xue, Erik Johnson, David Hall, Theo Garrett, Suzhen Guo, Ilija Melentijevic, Gordon J. Lithgow, Girish Harinath, Manish Chamoli, Michael P. Presley, Max Guo, Shobhna Patel, Patrick C. Phillips, Benjamin W. Blue, Jason L. Kish, Esteban Chen, Cody M. Jarrett, Anna C. Foulger, Brian Onken, Mark Abbott, Ron Falkowski, Monica Driscoll, Anna L. Coleman-Hulbert, Mark Lucanic, Phu Huynh, W. Todd Plummer, E. Grace Jones, Pankaj Kapahi, Stephen A. Banse, and Christine A Sedore

Subjects
0301 basic medicine, Aging, Longevity, Psychological intervention, Computational biology, Automation, 03 medical and health sciences, 0302 clinical medicine, Animals, CITP, Caenorhabditis elegans, 030304 developmental biology, Light exposure, Protocol (science), 0303 health sciences, Lifespan, biology, Geriatrics gerontology, Lasers, fungi, biology.organism_classification, Caenorhabditis, 030104 developmental biology, Lifespan machine, Thioflavin T, Ketoglutaric Acids, Original Article, Biological Assay, Geriatrics and Gerontology, Photic Stimulation, 030217 neurology & neurosurgery, Project design
Abstract

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach toward aging-intervention screening. Electronic supplementary material The online version of this article (10.1007/s11357-019-00108-9) contains supplementary material, which is available to authorized users.

Published
2019
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11. A hybrid approach for solving mixed binary integer programming problems

Author

Mat Shariff, Mastura Binti, Lucas, C, and Roman, D

Subjects
MBIP, VNS, LP relaxation, Metaheuristics, CITP
Abstract

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London Optimisation appears in many aspects of day to day life and more often, involves integer optimisation of very large scales. Although technology advancements have enabled many combinatorial optimisation problems to be solved exactly, this is only true for small and some of the medium instances. For large instances, they require high computational times and worse, fail to be solved due to the massive usage of the machine’s memory. In this research, we aim to develop a hybrid technique, focusing on solving the MBIP problem rather than finding the best solution for individual problem’s application. Therefore, we proposed a general framework of a hybrid technique that may need minor adjustment when applied to various optimisation problems, in particular to the mixed binary integer programming (MBIP) problems. The hybrid approach proposed in this research is the collaborative combination of the linear programming (LP) relaxation with variable neighbourhood search (VNS). We use LP relaxation solutions to generate initial solutions and use VNS to improve the solutions obtained. To illustrate the flexibility of the proposed method, we implement the proposed method on two similar MBIP problems; the constrained index tracking problem (CITP) and the gas supply chain problem. The proposed hybrid technique generates satisfactory solutions within significantly shorter amount of computational time. For the CITP problem, we compare the obtained solutions with the solutions provided by the CPLEX solver (with time and solution limit imposed) and a genetic algorithm (GA) approach. For most of the instances, our proposed hybrid technique gives better solutions with significant reduction of the computational time compared to the time taken by the CPLEX solver and the GA approach. For the gas supply chain problem, the proposed hybrid technique manage to replicate the solutions generated by the CPLEX solver (with time and solution limit imposed) within a shorter computational time. When we decrease the number of locations that were allowed to supply gas to a specific location, the proposed hybrid technique generated better solutions with lower total costs than the solutions given by the CPLEX solver. The proposed hybrid technique was successfully implemented for both problems by adjusting the optimal LP solutions of the decision variables that are used to guide the search process. Satisfactory solutions were obtained for both problems within a relatively shorter computational time. MARA

Published
2018

12. Role of collagen turnover biomarkers in the noninvasive assessment of myocardial fibrosis: an update.

Author

Ureche C, Nedelcu AE, Sascău RA, Stătescu C, Kanbay M, and Covic A

Subjects
Animals, Biomarkers metabolism, Collagen Type I genetics, Collagen Type III genetics, Fibrosis genetics, Fibrosis metabolism, Heart Diseases genetics, Heart Diseases pathology, Humans, Collagen Type I metabolism, Collagen Type III metabolism, Heart Diseases metabolism
Abstract

The pro-fibrotic milieu, as the result of the extracellular matrix remodeling, is a central feature in the pathophysiology of heart disease and contributes to its high morbidity and mortality. Fibrosis is a recognized risk factor for development of heart failure and arrythmias and is usually detected by cardiac magnetic resonance or endomyocardial biopsy. Collagen type I and type III are major components of the collagen network, and the assessment of their derived biomarkers could serve as estimate of the myocardial fibrotic content. This review summarizes data from numerous studies in which these biomarkers have proven their diagnostic and prognostic utility, setting the stage for further randomized clinical trials that might translate into early implementation of antifibrotic therapies.

Published
2020
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13. A Saudi Child With Chronic Immune Thrombocytopenia and Vitiligo.

Author

Alhebshi A, Abbas H, Alotaibi HM, Attaf M, and Al-Yamani A

Abstract

Chronic immune thrombocytopenia (ITP) is less commonly found in the children presenting with ITP. Patients usually present with petechiae, purpura, or active bleeding in the form of epistaxis or hematuria. The main aim of treatment in chronic ITP is to prevent major bleeding and to increase the platelet count. High doses of corticosteroids, intravenous immunoglobulin, rituximab, and eltrombopag, a thrombopoietin receptor agonist (TPO-RA), are medications that can be used. In this report, we present a case of chronic ITP in a 12-year-old child. In addition to features of chronic ITP, he also has vitiligo around his eyes and limbs. During treatment, he was resistant to steroids and did not respond to rituximab or eltrombopag. To understand the cause of his presenting features, we did multiple diagnostic evaluations. The whole-exome sequencing raises the possibility of auto-inflammatory syndrome Behcet-like (AISBL), which is a rare genetic disorder and not frequently reported in the available medical literature. AISBL is caused by mutations in the TNFAIP3 gene. According to our best knowledge, this is the first Saudi child diagnosed with chronic ITP and vitiligo with the possibility of AISBL that needs further genetic work-up to confirm the diagnosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Alhebshi et al.)

Published
2020
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14. The mechanism of the tyrosine transporter TyrP supports a proton motive tyrosine decarboxylation pathway in Lactobacillus brevis

Author

Wolken, WAM, Lucas, PM, Lonvaud-Funel, A, Lolkema, JS, Wolken, Wout A.M., Lucas, Patrick M., University of Groningen, Oenologie (UMRO), École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut National de la Recherche Agronomique (INRA)-Université Bordeaux Segalen - Bordeaux 2, Université de Bordeaux Ségalen [Bordeaux 2], Groningen Biomolecular Sciences and Biotechnology, Molecular Microbiology, and Faculty of Science and Engineering

Subjects
MECHANISM, EXPRESSION, Amino Acid Transport Systems, Operon, Decarboxylation, Levilactobacillus brevis, Tyramine, Biology, LACTIC-ACID BACTERIA, Microbiology, LACTOBACILLUS BREVIS, STREPTOCOCCACEAE, CITRATE, OXALOBACTER, Substrate Specificity, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, LACTOCOCCUS LACTIS, OPERON, CITP, Tyrosine, LACTOCOCCUS-LACTIS, EXCHANGE, Molecular Biology, 030304 developmental biology, 0303 health sciences, 030306 microbiology, Lactobacillus brevis, Lactococcus lactis, Proton-Motive Force, Transporter, biology.organism_classification, TYROSINE TRANSPORTER, Enzymes and Proteins, Tyrosine decarboxylase, Recombinant Proteins, CARRIER PROTEIN, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, chemistry, ESCHERICHIA-COLI, TYRP, BACTERIA, IOEB-9809
Abstract

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.

Published
2006
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15. Conserved residues R420 and Q428 in a cytoplasmic loop of the citrate/malate transporter CimH of Bacillus subtilis are accessible from the external face of the membrane

Author

Bastiaan P. Krom, Juke S. Lolkema, Groningen Biomolecular Sciences and Biotechnology, Molecular Microbiology, Faculty of Science and Engineering, and Preventive Dentistry

Subjects
Cytoplasm, Cell Membrane Permeability, Organic anion transporter 1, Glutamine, Malates, Organic Anion Transporters, PROTEIN, Bacillus subtilis, Biochemistry, Substrate Specificity, Conserved sequence, Cell membrane, Site-Directed, TOPOLOGY, Conserved Sequence, biology, Substrate Specificity/genetics, FAMILY, Transmembrane domain, medicine.anatomical_structure, Ethylmaleimide, ESCHERICHIA-COLI, Ethyl Methanesulfonate, Glutamine/genetics, KLEBSIELLA-PNEUMONIAE, Organic Anion Transporters/chemistry, SDG 6 - Clean Water and Sanitation, Bacillus subtilis/chemistry, Cytoplasm/chemistry, Plasmids, Cysteine/genetics, Ethyl Methanesulfonate/analogs & derivatives, INSERTION, Ethylmaleimide/chemistry, Cell Membrane/chemistry, Arginine, LACTIC-ACID BACTERIA, Citric Acid, Bacterial Proteins, Cell Membrane Permeability/genetics, Hydroxy Acids/chemistry, MALATE MLEP, Bacterial Proteins/chemistry, medicine, Cysteine, CITP, Arginine/genetics, Malates/chemistry, Lysine/genetics, Citric Acid/chemistry, Lysine, Cell Membrane, Substrate (chemistry), Carrier Proteins/chemistry, Citrate transport, biology.organism_classification, Mutagenesis, biology.protein, Mutagenesis, Site-Directed, Calcium, LEUCONOSTOC-MESENTEROIDES, Carrier Proteins, Hydroxy Acids
Abstract

CimH of Bacillus subtilis is a secondary transporter for citrate and malate that belongs to the 2-hydroxycarboxylate transporter (2HCT) family. Conserved residues R143, R420, and Q428.. located in putative cytoplasmic loops and R432, located at the cytoplasmic end of the C-terminal transmembrane segment XI were mutated to Cys to identify residues involved in binding of the substrates. R143C, R420C, and Q428C revealed kinetics similar to those of the wild-type transporter, while the activity of R432C was reduced by at least 2 orders of magnitude. Conservative replacement of R432 with Lys reduced the activity by I order of magnitude, by lowering the affinity for the substrate 10-fold. It is concluded that the arginine residue at position 432 in CimH interacts with one of the carboxylate groups of the substrates. Labeling of the R420C and Q428C mutants with thiol reagents inhibited citrate transport activity. Surprisingly, the cysteine residues in the cytoplasmic loops in both R420C and Q428C were accessible to the small, membrane-impermeable, negatively charged MTSES reagent from the external site of the membrane in a substrate protectable manner. The membrane impermeable reagents MTSET,(1) which is positively charged, and AMdiS, which is negatively charged like MTSES but more bulky, did not inhibit R420C and Q428C. It is suggested that the access pathway is optimized for small, negatively charged substrates. Either the cytoplasmic loop containing residues R420 and Q428 is partly protruding to the outside, possibly in a reentrant loop like structure, or alternatively, a water-filled substrate translocation pathway extents to the cytoplasm-membrane interface.

Published
2003
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16. Bacillus subtilis YxkJ is a secondary transporter of the 2-hydroxycarboxylate transporter family that transports L-malate and citrate

Author

Ronald Aardema, Juke S. Lolkema, Bastiaan P. Krom, Groningen Biomolecular Sciences and Biotechnology, Molecular Microbiology, and Preventive Dentistry

Subjects
Malates, Bacillus subtilis, medicine.disease_cause, LACTOCOCCUS, Substrate Specificity, Membrane Proteins/antagonists & inhibitors, Malates/metabolism, FERMENTATION, biology, Membrane transport protein, METABOLIC ENERGY, Proton-Motive Force, Carrier Proteins/antagonists & inhibitors, Cations, Monovalent, Transport protein, Biochemistry, ESCHERICHIA-COLI, HOMOLOGOUS PROTEINS, KLEBSIELLA-PNEUMONIAE, Protons, Active, Physiology and Metabolism, Biological Transport, Active, LACTIC-ACID BACTERIA, Microbiology, Malate dehydrogenase, Citric Acid, Monovalent, Bacterial Proteins, Citric Acid/metabolism, Cations, medicine, CITP, Membranes/metabolism, Malate transport, Molecular Biology, Escherichia coli, Membranes, Sodium, Membrane Proteins, Membrane Transport Proteins, Biological Transport, Citrate transport, biology.organism_classification, Molecular biology, Bacillus subtilis/genetics, Sodium/metabolism, Symporter, biology.protein, LEUCONOSTOC-MESENTEROIDES, Carrier Proteins, SYSTEM
Abstract

The genome of Bacillus subtilis contains two genes that code for membrane proteins that belong to the 2-hydroxycarboxylate transporter family. Here we report the functional characterization of one of the two, yxkJ , which codes for a transporter protein named CimHbs. The gene was cloned and expressed in Escherichia coli and complemented the citrate-negative phenotype of wild-type E. coli and the malate-negative phenotype of the E. coli strain JRG4008, which is defective in malate uptake. Subsequent uptake studies in whole cells expressing CimHbs clearly demonstrated the citrate and malate transport activity of the protein. Immunoblot analysis showed that CimHbs is a 48-kDa protein that is well expressed in E. coli . Studies with right-side-out membrane vesicles demonstrated that CimHbs is an electroneutral proton-solute symporter. No indications were found for the involvement of Na + ions in the transport process. Inhibition of the uptake catalyzed by CimHbs by divalent metal ions, together with the lack of effect on transport by the chelator EDTA, showed that CimHbs translocates the free citrate and malate anions. Among a large set of substrates tested, only malate, citramalate, and citrate competitively inhibited citrate transport catalyzed by CimHbs. The transporter is strictly stereoselective, recognizing only the S enantiomers of malate and citramalate. Remarkably, though citramalate binds to the transporter, it is not translocated.

Published
2001
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17. Stereoselectivity of the Membrane Potential-Generating Citrate and Malate Transporters of Lactic Acid Bacteria

Author

Michael Bandell, Juke S. Lolkema, Groningen Biomolecular Sciences and Biotechnology, and Molecular Microbiology

Subjects
EXPRESSION, Oxalobacter, Lactococcus, Lipid Bilayers, Carboxylic Acids, Malates, CARRIER, Organic Anion Transporters, FUNCTIONAL-PROPERTIES, LACTOCOCCUS, Biochemistry, Catalysis, OXALOBACTER, Membrane Potentials, chemistry.chemical_compound, Bacterial Proteins, NUCLEOTIDE-SEQUENCE, Lactic Acid, CITP, Carboxylate, SPECIFICITY, Binding Sites, Symporters, biology, Lactococcus lactis, Membrane Transport Proteins, Biological Transport, Stereoisomerism, Transporter, biology.organism_classification, GENE, Lactic acid, Kinetics, chemistry, Leuconostoc mesenteroides, KLEBSIELLA-PNEUMONIAE, Carrier Proteins, Leuconostoc, Bacteria
Abstract

The citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter of Lactococcus lactis (MleP) are homologous proteins that catalyze citrate-lactate and malate-lactate exchange, respectively. Both transporters transport a range of substrates that contain the 2-hydroxycarboxylate motif, HO-CR2-COO- [Bandell, M., et al. (1997) J. Biol. Chem. 272, 18140-18146]. In this study, we have analyzed binding and translocation properties of CitP and MleP for a wide variety of substrates and substrate analogues. Modification of the OH or the COO- groups of the 2-hydroxycarboxylate motif drastically reduced the affinity of the transporters for the substrates, indicating their relevance in substrate recognition. Both CitP and MleP were strictly stereoselective when the R group contained a second carboxylate group; the S-enantiomers were efficiently bound and translocated, while the transporters had no affinity for the R-enantiomers. The affinity of the S-enantiomers, and of citrate, was at least 1 order of magnitude higher than for lactate and other substrates with uncharged R groups, indicating a specific interaction between the second carboxylate group and the protein that is responsible for high-affinity binding. MleP was not stereoselective in binding when the R groups are hydrophobic and as large as a benzyl group. However, only the S-enantiomers were translocated by MleP. CitP had a strong preference for binding and translocating the R-enantiomers of substrates with large hydrophobic R groups. These differences between CitP and MleP explain why citrate is a substrate of CitP and not of MleP. The results are discussed in the context of a model for the interaction between sites on the protein and functional groups on the substrates in the binding pockets of the two proteins.

Published
1999
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18. Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403

Author

Juke S. Lolkema, Agata M. Pudlik, Groningen Biomolecular Sciences and Biotechnology, and Molecular Microbiology

Subjects
Oxaloacetic Acid, ATP citrate lyase, Carboxy-Lyases, PH, Physiology and Metabolism, Organic Anion Transporters, Biology, Microbiology, Gene Expression Regulation, Enzymologic, CLASSIFICATION, Substrate Specificity, ACID BACTERIA, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, BIOVAR DIACETYLACTIS, Citrate synthase, Citrates, MALIC ENZYME, CITP, Molecular Biology, 030304 developmental biology, 0303 health sciences, 030306 microbiology, Acetoin, Lactococcus lactis, Gene Expression Regulation, Bacterial, biology.organism_classification, TRANSPORTERS, Pyruvate carboxylase, Oxaloacetate decarboxylase, chemistry, Gluconeogenesis, Biochemistry, CORYNEBACTERIUM-GLUTAMICUM, SUBSP, biology.protein, Phosphoenolpyruvate carboxykinase, Gene Deletion, SYSTEM
Abstract

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706–714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of l -lactate, indicating exchange between oxaloacetate and l -lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.

Published
2011

19. Ultrasensitive CITP-MS based targeted proteomics technologies for protein identification and quantification

Author

Wang, Chenchen and Wang, Chenchen

Abstract

Mass Spectrometry (MS) based technologies have enabled efficient and comprehensive proteomic profiling for biomarker discovery. However, due to sample complexity and large concentration variation, the obtained data is usually biased to endogeneous high abundance proteins while the important disease-related information went missing. Targeted proteomics enables the delivery of precise and sensitive qualitative/quantitative data of interest to researchers by focusing analysis on a preselected population of cells or proteins. This project aims to develop targeted proteomic technologies through capillary isotachophoresis (CITP)-based technique which is capable of selectively enriching trace compounds for a further improved sensitivity in both discovery and validation studies. By employing tissue microdissection and a CITP-based multidimensional separation platform, homogeneous glioma cells were isolated from unwanted cells and analyzed in search of glioblastoma biomarker. Comparative proteomic profiling of pure tumor cells from different grades of infiltrative astrocytomas revealed disease specific protein expression variation among grades. Further validation using immunohistochemistry demonstrated consistent results. This targeted tissue analyzing platform provided a sensitive and confident methodology for biomarker discovery within minute amount of samples. With the demonstrated outstanding analyzing capacity on targeted biomarker discovery, we moved on to developing ultrasensitive targeted quantitation techniques. We demonstrated online coupling of transient-CITP/CZE (capillary zone electrophoresis) with selective reaction monitoring (SRM) MS for the first time via a sheathliquid interface for improved sensitivity and selectivity. Ultrasensitive targeted quantitation was achieved through the incorporation of the selective enrichment capability of CITP/CZE with SRM MS, giving a limit of quantitation (LOQ) of 50 pM with a total sample loading of 50 attomoles. In order t

Published
2014

20. Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403

Author

Pudlik, Agata M., Lolkema, Juke S., Pudlik, Agata M., and Lolkema, Juke S.

Abstract

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.

Published
2011

21. Sledování obsahu organických kyselin v alkoholických nápojích

Author

Benešová, Karolína, Běláková, Sylvie, Ostrihoňová, Katarína, Benešová, Karolína, Běláková, Sylvie, and Ostrihoňová, Katarína

Abstract

Cieľom diplomovej práce je stanovenie a optimalizácia stanovenia organických kyselín v pivách metódou kapilárnej izotachoforézy a vysokoúčinnej kvapalinovej chromatografie s UV detekciou. Pivo je zložitá matrica a preto vyžaduje úpravu pred analýzou HPLC. V diplomovej práci je diskutovaná optimalizácia metód (HPLC/UV a CITP) i optimalizácia úpravy vzorku, konkrétne extrakciou na pevnej fázy (SPE). V teoretickej časti je spracovaná história, charakteristika a technológia výroby piva. Ďalej sú popísané organické kyseliny a metódy stanovenia organických kyselín použité v tejto práci. Experimentálna časť obsahuje prípravy roztokov, vzoriek a kalibračných štandardov, parametre a postupy analýzy HPLC/UV, CITP a postup úpravy vzorku pomocou SPE. V diskusii a závere sú zhrnuté všetky výsledky a porovnané s aktuálnou literatúrou. Bolo analyzované 11 pív z obchodnej siete. Vo všetkých vzorkách bol stanovený obsah 6 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová, benzoová) metódou CITP a 5 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová) metódou HPLC/UV. Výsledky práce podávajú prehľad o obsahu organických kyselín vo vzorkách analyzovaných pív., The aim of the thesis is determination and optimization of organic acids in beers using the methods of capillary isotachophoresis (CITP) and high performance liquid chromatography with diode array detection (HPLC/UV). Beer is a complicated matrix therefore the samples need pretreatment using solid phase extraction (SPE). The diploma thesis discusses the optimization of the analytical methods (HPLC/UV and CITP) and optimization of SPE. In the theoretical part, history, characterization and technology of beer production are presented. Further, characteristics of organic acids and methods for organic acid determination are also discussed. The experimental part deals with the preparation of solutions, tested samples, calibration samples. Parameters and procedures of analytical methods (HPLC/UV and CITP) and pretreatment using the solid phase extraction are also described in the experimental part. All results are summarized and compared with the current literature in discussion and conclusion. Eleven beer samples from the retail stores were analyzed. In all samples, six organic acids (lactic, oxalic, succinic, acetic, citric, benzoic) were determined by CITP and five organic acids (lactic, oxalic, succinic, acetic, citric) by HPLC/UV were determined. Results of this study give an overview of the organic acid contents in beer samples.

22. Sledování obsahu organických kyselin v alkoholických nápojích

Author

Benešová, Karolína, Běláková, Sylvie, Ostrihoňová, Katarína, Benešová, Karolína, Běláková, Sylvie, and Ostrihoňová, Katarína

Abstract

Cieľom diplomovej práce je stanovenie a optimalizácia stanovenia organických kyselín v pivách metódou kapilárnej izotachoforézy a vysokoúčinnej kvapalinovej chromatografie s UV detekciou. Pivo je zložitá matrica a preto vyžaduje úpravu pred analýzou HPLC. V diplomovej práci je diskutovaná optimalizácia metód (HPLC/UV a CITP) i optimalizácia úpravy vzorku, konkrétne extrakciou na pevnej fázy (SPE). V teoretickej časti je spracovaná história, charakteristika a technológia výroby piva. Ďalej sú popísané organické kyseliny a metódy stanovenia organických kyselín použité v tejto práci. Experimentálna časť obsahuje prípravy roztokov, vzoriek a kalibračných štandardov, parametre a postupy analýzy HPLC/UV, CITP a postup úpravy vzorku pomocou SPE. V diskusii a závere sú zhrnuté všetky výsledky a porovnané s aktuálnou literatúrou. Bolo analyzované 11 pív z obchodnej siete. Vo všetkých vzorkách bol stanovený obsah 6 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová, benzoová) metódou CITP a 5 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová) metódou HPLC/UV. Výsledky práce podávajú prehľad o obsahu organických kyselín vo vzorkách analyzovaných pív., The aim of the thesis is determination and optimization of organic acids in beers using the methods of capillary isotachophoresis (CITP) and high performance liquid chromatography with diode array detection (HPLC/UV). Beer is a complicated matrix therefore the samples need pretreatment using solid phase extraction (SPE). The diploma thesis discusses the optimization of the analytical methods (HPLC/UV and CITP) and optimization of SPE. In the theoretical part, history, characterization and technology of beer production are presented. Further, characteristics of organic acids and methods for organic acid determination are also discussed. The experimental part deals with the preparation of solutions, tested samples, calibration samples. Parameters and procedures of analytical methods (HPLC/UV and CITP) and pretreatment using the solid phase extraction are also described in the experimental part. All results are summarized and compared with the current literature in discussion and conclusion. Eleven beer samples from the retail stores were analyzed. In all samples, six organic acids (lactic, oxalic, succinic, acetic, citric, benzoic) were determined by CITP and five organic acids (lactic, oxalic, succinic, acetic, citric) by HPLC/UV were determined. Results of this study give an overview of the organic acid contents in beer samples.

23. Sledování obsahu organických kyselin v alkoholických nápojích

Author

Benešová, Karolína, Běláková, Sylvie, Benešová, Karolína, and Běláková, Sylvie

Abstract

Cieľom diplomovej práce je stanovenie a optimalizácia stanovenia organických kyselín v pivách metódou kapilárnej izotachoforézy a vysokoúčinnej kvapalinovej chromatografie s UV detekciou. Pivo je zložitá matrica a preto vyžaduje úpravu pred analýzou HPLC. V diplomovej práci je diskutovaná optimalizácia metód (HPLC/UV a CITP) i optimalizácia úpravy vzorku, konkrétne extrakciou na pevnej fázy (SPE). V teoretickej časti je spracovaná história, charakteristika a technológia výroby piva. Ďalej sú popísané organické kyseliny a metódy stanovenia organických kyselín použité v tejto práci. Experimentálna časť obsahuje prípravy roztokov, vzoriek a kalibračných štandardov, parametre a postupy analýzy HPLC/UV, CITP a postup úpravy vzorku pomocou SPE. V diskusii a závere sú zhrnuté všetky výsledky a porovnané s aktuálnou literatúrou. Bolo analyzované 11 pív z obchodnej siete. Vo všetkých vzorkách bol stanovený obsah 6 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová, benzoová) metódou CITP a 5 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová) metódou HPLC/UV. Výsledky práce podávajú prehľad o obsahu organických kyselín vo vzorkách analyzovaných pív., The aim of the thesis is determination and optimization of organic acids in beers using the methods of capillary isotachophoresis (CITP) and high performance liquid chromatography with diode array detection (HPLC/UV). Beer is a complicated matrix therefore the samples need pretreatment using solid phase extraction (SPE). The diploma thesis discusses the optimization of the analytical methods (HPLC/UV and CITP) and optimization of SPE. In the theoretical part, history, characterization and technology of beer production are presented. Further, characteristics of organic acids and methods for organic acid determination are also discussed. The experimental part deals with the preparation of solutions, tested samples, calibration samples. Parameters and procedures of analytical methods (HPLC/UV and CITP) and pretreatment using the solid phase extraction are also described in the experimental part. All results are summarized and compared with the current literature in discussion and conclusion. Eleven beer samples from the retail stores were analyzed. In all samples, six organic acids (lactic, oxalic, succinic, acetic, citric, benzoic) were determined by CITP and five organic acids (lactic, oxalic, succinic, acetic, citric) by HPLC/UV were determined. Results of this study give an overview of the organic acid contents in beer samples.

24. Sledování obsahu organických kyselin v alkoholických nápojích

Author

Benešová, Karolína, Běláková, Sylvie, Benešová, Karolína, and Běláková, Sylvie

Abstract

Cieľom diplomovej práce je stanovenie a optimalizácia stanovenia organických kyselín v pivách metódou kapilárnej izotachoforézy a vysokoúčinnej kvapalinovej chromatografie s UV detekciou. Pivo je zložitá matrica a preto vyžaduje úpravu pred analýzou HPLC. V diplomovej práci je diskutovaná optimalizácia metód (HPLC/UV a CITP) i optimalizácia úpravy vzorku, konkrétne extrakciou na pevnej fázy (SPE). V teoretickej časti je spracovaná história, charakteristika a technológia výroby piva. Ďalej sú popísané organické kyseliny a metódy stanovenia organických kyselín použité v tejto práci. Experimentálna časť obsahuje prípravy roztokov, vzoriek a kalibračných štandardov, parametre a postupy analýzy HPLC/UV, CITP a postup úpravy vzorku pomocou SPE. V diskusii a závere sú zhrnuté všetky výsledky a porovnané s aktuálnou literatúrou. Bolo analyzované 11 pív z obchodnej siete. Vo všetkých vzorkách bol stanovený obsah 6 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová, benzoová) metódou CITP a 5 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová) metódou HPLC/UV. Výsledky práce podávajú prehľad o obsahu organických kyselín vo vzorkách analyzovaných pív., The aim of the thesis is determination and optimization of organic acids in beers using the methods of capillary isotachophoresis (CITP) and high performance liquid chromatography with diode array detection (HPLC/UV). Beer is a complicated matrix therefore the samples need pretreatment using solid phase extraction (SPE). The diploma thesis discusses the optimization of the analytical methods (HPLC/UV and CITP) and optimization of SPE. In the theoretical part, history, characterization and technology of beer production are presented. Further, characteristics of organic acids and methods for organic acid determination are also discussed. The experimental part deals with the preparation of solutions, tested samples, calibration samples. Parameters and procedures of analytical methods (HPLC/UV and CITP) and pretreatment using the solid phase extraction are also described in the experimental part. All results are summarized and compared with the current literature in discussion and conclusion. Eleven beer samples from the retail stores were analyzed. In all samples, six organic acids (lactic, oxalic, succinic, acetic, citric, benzoic) were determined by CITP and five organic acids (lactic, oxalic, succinic, acetic, citric) by HPLC/UV were determined. Results of this study give an overview of the organic acid contents in beer samples.

25. Sledování obsahu organických kyselin v alkoholických nápojích

Author

Benešová, Karolína, Běláková, Sylvie, Benešová, Karolína, and Běláková, Sylvie

Abstract

Cieľom diplomovej práce je stanovenie a optimalizácia stanovenia organických kyselín v pivách metódou kapilárnej izotachoforézy a vysokoúčinnej kvapalinovej chromatografie s UV detekciou. Pivo je zložitá matrica a preto vyžaduje úpravu pred analýzou HPLC. V diplomovej práci je diskutovaná optimalizácia metód (HPLC/UV a CITP) i optimalizácia úpravy vzorku, konkrétne extrakciou na pevnej fázy (SPE). V teoretickej časti je spracovaná história, charakteristika a technológia výroby piva. Ďalej sú popísané organické kyseliny a metódy stanovenia organických kyselín použité v tejto práci. Experimentálna časť obsahuje prípravy roztokov, vzoriek a kalibračných štandardov, parametre a postupy analýzy HPLC/UV, CITP a postup úpravy vzorku pomocou SPE. V diskusii a závere sú zhrnuté všetky výsledky a porovnané s aktuálnou literatúrou. Bolo analyzované 11 pív z obchodnej siete. Vo všetkých vzorkách bol stanovený obsah 6 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová, benzoová) metódou CITP a 5 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová) metódou HPLC/UV. Výsledky práce podávajú prehľad o obsahu organických kyselín vo vzorkách analyzovaných pív., The aim of the thesis is determination and optimization of organic acids in beers using the methods of capillary isotachophoresis (CITP) and high performance liquid chromatography with diode array detection (HPLC/UV). Beer is a complicated matrix therefore the samples need pretreatment using solid phase extraction (SPE). The diploma thesis discusses the optimization of the analytical methods (HPLC/UV and CITP) and optimization of SPE. In the theoretical part, history, characterization and technology of beer production are presented. Further, characteristics of organic acids and methods for organic acid determination are also discussed. The experimental part deals with the preparation of solutions, tested samples, calibration samples. Parameters and procedures of analytical methods (HPLC/UV and CITP) and pretreatment using the solid phase extraction are also described in the experimental part. All results are summarized and compared with the current literature in discussion and conclusion. Eleven beer samples from the retail stores were analyzed. In all samples, six organic acids (lactic, oxalic, succinic, acetic, citric, benzoic) were determined by CITP and five organic acids (lactic, oxalic, succinic, acetic, citric) by HPLC/UV were determined. Results of this study give an overview of the organic acid contents in beer samples.

26. Sledování obsahu organických kyselin v alkoholických nápojích

Author

Benešová, Karolína, Běláková, Sylvie, Ostrihoňová, Katarína, Benešová, Karolína, Běláková, Sylvie, and Ostrihoňová, Katarína

Abstract

Cieľom diplomovej práce je stanovenie a optimalizácia stanovenia organických kyselín v pivách metódou kapilárnej izotachoforézy a vysokoúčinnej kvapalinovej chromatografie s UV detekciou. Pivo je zložitá matrica a preto vyžaduje úpravu pred analýzou HPLC. V diplomovej práci je diskutovaná optimalizácia metód (HPLC/UV a CITP) i optimalizácia úpravy vzorku, konkrétne extrakciou na pevnej fázy (SPE). V teoretickej časti je spracovaná história, charakteristika a technológia výroby piva. Ďalej sú popísané organické kyseliny a metódy stanovenia organických kyselín použité v tejto práci. Experimentálna časť obsahuje prípravy roztokov, vzoriek a kalibračných štandardov, parametre a postupy analýzy HPLC/UV, CITP a postup úpravy vzorku pomocou SPE. V diskusii a závere sú zhrnuté všetky výsledky a porovnané s aktuálnou literatúrou. Bolo analyzované 11 pív z obchodnej siete. Vo všetkých vzorkách bol stanovený obsah 6 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová, benzoová) metódou CITP a 5 organických kyselín (mliečna, šťaveľová, jantárová, octová, citrónová) metódou HPLC/UV. Výsledky práce podávajú prehľad o obsahu organických kyselín vo vzorkách analyzovaných pív., The aim of the thesis is determination and optimization of organic acids in beers using the methods of capillary isotachophoresis (CITP) and high performance liquid chromatography with diode array detection (HPLC/UV). Beer is a complicated matrix therefore the samples need pretreatment using solid phase extraction (SPE). The diploma thesis discusses the optimization of the analytical methods (HPLC/UV and CITP) and optimization of SPE. In the theoretical part, history, characterization and technology of beer production are presented. Further, characteristics of organic acids and methods for organic acid determination are also discussed. The experimental part deals with the preparation of solutions, tested samples, calibration samples. Parameters and procedures of analytical methods (HPLC/UV and CITP) and pretreatment using the solid phase extraction are also described in the experimental part. All results are summarized and compared with the current literature in discussion and conclusion. Eleven beer samples from the retail stores were analyzed. In all samples, six organic acids (lactic, oxalic, succinic, acetic, citric, benzoic) were determined by CITP and five organic acids (lactic, oxalic, succinic, acetic, citric) by HPLC/UV were determined. Results of this study give an overview of the organic acid contents in beer samples.

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