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Analytical and Bioanalytical Chemistry (v.392, #6)
Cytochrome P450 by P. H. Roos; N. Jakubowski (pp. 1015-1017).
is biologist, lecturer in physiological chemistry, and head of the Molecular Toxicology group at Leibniz Research Centre of Working Environment and Human Factors (IfADo). His research focuses on effects and interactions of chemicals with cells with regard to adverse processes including chemically induced cancerogenesis, where cytochromes P450 play an important role. is a senior scientist in ISAS—Institute for Analytical Sciences. His research interest is related to the development of methods and instrumentation in plasma-based inorganic mass spectrometry and their application to the material and bio-related sciences.
Cytochrome P450 by P. H. Roos; N. Jakubowski (pp. 1015-1017).
is biologist, lecturer in physiological chemistry, and head of the Molecular Toxicology group at Leibniz Research Centre of Working Environment and Human Factors (IfADo). His research focuses on effects and interactions of chemicals with cells with regard to adverse processes including chemically induced cancerogenesis, where cytochromes P450 play an important role. is a senior scientist in ISAS—Institute for Analytical Sciences. His research interest is related to the development of methods and instrumentation in plasma-based inorganic mass spectrometry and their application to the material and bio-related sciences.
Substrate binding to cytochromes P450 by Emre M. Isin; F. Peter Guengerich (pp. 1019-1030).
P450s have attracted tremendous attention owing to not only their involvement in the metabolism of drug molecules and endogenous substrates but also the unusual nature of the reaction they catalyze, namely, the oxidation of unactivated C–H bonds. The binding of substrates to P450s, which is usually viewed as the first step in the catalytic cycle, has been studied extensively via a variety of biochemical and biophysical approaches. These studies were directed towards answering different questions related to P450s, including mechanism of oxidation, substrate properties, unusual substrate oxidation kinetics, function, and active-site features. Some of the substrate binding studies extending over a period of more than 40 years of dedicated work have been summarized in this review and categorized by the techniques employed in the binding studies.
Keywords: Substrate binding; Cytochrome P450; Cooperativity; Ligands; Drug development
Substrate binding to cytochromes P450 by Emre M. Isin; F. Peter Guengerich (pp. 1019-1030).
P450s have attracted tremendous attention owing to not only their involvement in the metabolism of drug molecules and endogenous substrates but also the unusual nature of the reaction they catalyze, namely, the oxidation of unactivated C–H bonds. The binding of substrates to P450s, which is usually viewed as the first step in the catalytic cycle, has been studied extensively via a variety of biochemical and biophysical approaches. These studies were directed towards answering different questions related to P450s, including mechanism of oxidation, substrate properties, unusual substrate oxidation kinetics, function, and active-site features. Some of the substrate binding studies extending over a period of more than 40 years of dedicated work have been summarized in this review and categorized by the techniques employed in the binding studies.
Keywords: Substrate binding; Cytochrome P450; Cooperativity; Ligands; Drug development
Fourier transform infrared spectroscopy as a tool to study structural properties of cytochromes P450 (CYPs) by Christiane Jung (pp. 1031-1058).
Cytochrome P450 proteins (CYPs) are a big class of heme proteins which are involved in various metabolic processes of living organisms. CYPs are the terminal catalytically active components of monooxygenase systems where the substrate binds and is hydroxylated. In order to be functionally competent, the protein structures of CYPs possess specific properties that must be explored in order to understand structure–function relationships and mechanistic aspects. Fourier transform infrared spectroscopy (FTIR) is one tool that is used to study these structural properties. The application of FTIR spectroscopy to the secondary structures of CYP proteins, protein unfolding, protein–protein interactions and the structure and dynamics of the CYP heme pocket is reviewed. A comparison with other thiolate heme proteins (nitric oxide synthase and chloroperoxidase) is also included. Figure The protein secondary structure, protein unfolding, redox-partner protein–protein interaction, structural changes induced by the reduction of the heme iron, and the structure and dynamics of the active site of cytochromes P450 (CYP) can be studied using Fourier transform infrared spectroscopy (FTIR). FTIR spectroscopy is a good approach for gaining a deeper insight into structure–function relationships in CYPs.
Keywords: Fourier transform infrared spectroscopy; Cytochrome P450; CYP; Nitric oxide synthase; Chloroperoxidase
Fourier transform infrared spectroscopy as a tool to study structural properties of cytochromes P450 (CYPs) by Christiane Jung (pp. 1031-1058).
Cytochrome P450 proteins (CYPs) are a big class of heme proteins which are involved in various metabolic processes of living organisms. CYPs are the terminal catalytically active components of monooxygenase systems where the substrate binds and is hydroxylated. In order to be functionally competent, the protein structures of CYPs possess specific properties that must be explored in order to understand structure–function relationships and mechanistic aspects. Fourier transform infrared spectroscopy (FTIR) is one tool that is used to study these structural properties. The application of FTIR spectroscopy to the secondary structures of CYP proteins, protein unfolding, protein–protein interactions and the structure and dynamics of the CYP heme pocket is reviewed. A comparison with other thiolate heme proteins (nitric oxide synthase and chloroperoxidase) is also included. Figure The protein secondary structure, protein unfolding, redox-partner protein–protein interaction, structural changes induced by the reduction of the heme iron, and the structure and dynamics of the active site of cytochromes P450 (CYP) can be studied using Fourier transform infrared spectroscopy (FTIR). FTIR spectroscopy is a good approach for gaining a deeper insight into structure–function relationships in CYPs.
Keywords: Fourier transform infrared spectroscopy; Cytochrome P450; CYP; Nitric oxide synthase; Chloroperoxidase
Engineering and assaying of cytochrome P450 biocatalysts by Kersten S. Rabe; Valérie J. Gandubert; Mark Spengler; Michael Erkelenz; Christof M. Niemeyer (pp. 1059-1073).
Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.
Keywords: Analytical methods; Cytochrome P450; Enzyme engineering; High-throughput screening; P450 substrates
Engineering and assaying of cytochrome P450 biocatalysts by Kersten S. Rabe; Valérie J. Gandubert; Mark Spengler; Michael Erkelenz; Christof M. Niemeyer (pp. 1059-1073).
Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.
Keywords: Analytical methods; Cytochrome P450; Enzyme engineering; High-throughput screening; P450 substrates
Intracellular transport and localization of microsomal cytochrome P450 by Etienne P. A. Neve; Magnus Ingelman-Sundberg (pp. 1075-1084).
The cytochrome P450 (P450) enzymes are mainly localized to the endoplasmic reticulum (ER), where they function within catalytic complexes metabolizing xenobiotics and some endogenous substrates. However, certain members of families 1–3 were also found in other subcellular compartments, such as mitochondria, plasma membrane, and lysosomes. The physiological function of these enzymes in non-ER locations is not known, although plasma-membrane-associated P450s have been described to be catalytically active and to participate in immune-mediated reactions with autoantibody formation that can trigger drug-induced hepatitis. Several retention/retrieval mechanisms are active in the ER retention of the P450s and inverse integration of the translated P450 into the ER membrane appears to be responsible for transport to the plasma membrane. Furthermore, hydrophilic motifs in the NH2-terminal part have been suggested to be important for mitochondrial import. Phosphorylation of P450s has been described to be important for increased rate of degradation as well as for targeting into mitochondria. It was also suggested that the mitochondria-targeted P450s from families 1–3 could be active in drug metabolism using an alternative electron transport chain. In this review we present an update of the field emphasizing studies concerning localization, posttranslational modification, such as phosphorylation, and intracellular transport of microsomal P450s.
Keywords: Microsomal P450s; Mitochondria; Phosphorylation; Plasma membrane; Protein targeting; Degradation
Intracellular transport and localization of microsomal cytochrome P450 by Etienne P. A. Neve; Magnus Ingelman-Sundberg (pp. 1075-1084).
The cytochrome P450 (P450) enzymes are mainly localized to the endoplasmic reticulum (ER), where they function within catalytic complexes metabolizing xenobiotics and some endogenous substrates. However, certain members of families 1–3 were also found in other subcellular compartments, such as mitochondria, plasma membrane, and lysosomes. The physiological function of these enzymes in non-ER locations is not known, although plasma-membrane-associated P450s have been described to be catalytically active and to participate in immune-mediated reactions with autoantibody formation that can trigger drug-induced hepatitis. Several retention/retrieval mechanisms are active in the ER retention of the P450s and inverse integration of the translated P450 into the ER membrane appears to be responsible for transport to the plasma membrane. Furthermore, hydrophilic motifs in the NH2-terminal part have been suggested to be important for mitochondrial import. Phosphorylation of P450s has been described to be important for increased rate of degradation as well as for targeting into mitochondria. It was also suggested that the mitochondria-targeted P450s from families 1–3 could be active in drug metabolism using an alternative electron transport chain. In this review we present an update of the field emphasizing studies concerning localization, posttranslational modification, such as phosphorylation, and intracellular transport of microsomal P450s.
Keywords: Microsomal P450s; Mitochondria; Phosphorylation; Plasma membrane; Protein targeting; Degradation
Phosphorylation of xenobiotic-metabolizing cytochromes P450 by B. Oesch-Bartlomowicz; F. Oesch (pp. 1085-1092).
The regulation of cytochromes P450 (CYPs) by induction mediated by xenobiotics is well known. Our team has discovered an additional important regulation of xenobiotic-metabolizing CYPs by phosphorylation. Individual CYPs are phosphorylated by different protein kinases, leading to CYP isoenzyme-selective changes in the metabolism of individual substrates and consequent profound changes in the control of mutagenic and cytotoxic metabolites. Some CYPs are phosphorylated by protein kinase C and some by the cyclic adenosine monophosphate (cAMP) dependent protein kinase A. We found that cAMP not only leads to drastic changes in the activity of individual CYPs, but also drastic changes in the nuclear localization of the CYP-related transcription factor Ah receptor (AHR). The consequences are very different from those of AHR nuclear translocation mediated by its classic ligands (such as dioxin and many polycyclic aromatic hydrocarbons) and may represent the long-sought physiological function of the AHR. The disturbance of this physiological function of AHR by extremely persistent high-affinity xenobiotic ligands such as dioxin may represent the most important contributing factor for their potent toxicity.
Keywords: Cytochromes P450; Phosphorylation; Protein kinase A; cAMP; Drug metabolism; Ah receptor nuclear translocation
Phosphorylation of xenobiotic-metabolizing cytochromes P450 by B. Oesch-Bartlomowicz; F. Oesch (pp. 1085-1092).
The regulation of cytochromes P450 (CYPs) by induction mediated by xenobiotics is well known. Our team has discovered an additional important regulation of xenobiotic-metabolizing CYPs by phosphorylation. Individual CYPs are phosphorylated by different protein kinases, leading to CYP isoenzyme-selective changes in the metabolism of individual substrates and consequent profound changes in the control of mutagenic and cytotoxic metabolites. Some CYPs are phosphorylated by protein kinase C and some by the cyclic adenosine monophosphate (cAMP) dependent protein kinase A. We found that cAMP not only leads to drastic changes in the activity of individual CYPs, but also drastic changes in the nuclear localization of the CYP-related transcription factor Ah receptor (AHR). The consequences are very different from those of AHR nuclear translocation mediated by its classic ligands (such as dioxin and many polycyclic aromatic hydrocarbons) and may represent the long-sought physiological function of the AHR. The disturbance of this physiological function of AHR by extremely persistent high-affinity xenobiotic ligands such as dioxin may represent the most important contributing factor for their potent toxicity.
Keywords: Cytochromes P450; Phosphorylation; Protein kinase A; cAMP; Drug metabolism; Ah receptor nuclear translocation
Functional pharmacogenetics/genomics of human cytochromes P450 involved in drug biotransformation by Ulrich M. Zanger; Miia Turpeinen; Kathrin Klein; Matthias Schwab (pp. 1093-1108).
We investigated the elimination routes for the 200 drugs that are sold most often by prescription count in the United States. The majority (78%) of the hepatically cleared drugs were found to be subject to oxidative metabolism via cytochromes P450 of the families 1, 2 and 3, with major contributions from CYP3A4/5 (37% of drugs) followed by CYP2C9 (17%), CYP2D6 (15%), CYP2C19 (10%), CYP1A2 (9%), CYP2C8 (6%), and CYP2B6 (4%). Clinically well-established polymorphic CYPs (i.e., CYP2C9, CYP2C19, and CYP2D6) were involved in the metabolism of approximately half of those drugs, including (in particular) NSAIDs metabolized mainly by CYP2C9, proton-pump inhibitors metabolized by CYP2C19, and beta blockers and several antipsychotics and antidepressants metabolized by CYP2D6. In this review, we provide an up-to-date summary of the functional polymorphisms and aspects of the functional genomics of the major human drug-metabolizing cytochrome P450s, as well as their clinical significance.
Keywords: Drug metabolism; Functional genomics; Genotype; Mutation; Pharmacogenetics; Phenotype; Single-nucleotide polymorphism
Functional pharmacogenetics/genomics of human cytochromes P450 involved in drug biotransformation by Ulrich M. Zanger; Miia Turpeinen; Kathrin Klein; Matthias Schwab (pp. 1093-1108).
We investigated the elimination routes for the 200 drugs that are sold most often by prescription count in the United States. The majority (78%) of the hepatically cleared drugs were found to be subject to oxidative metabolism via cytochromes P450 of the families 1, 2 and 3, with major contributions from CYP3A4/5 (37% of drugs) followed by CYP2C9 (17%), CYP2D6 (15%), CYP2C19 (10%), CYP1A2 (9%), CYP2C8 (6%), and CYP2B6 (4%). Clinically well-established polymorphic CYPs (i.e., CYP2C9, CYP2C19, and CYP2D6) were involved in the metabolism of approximately half of those drugs, including (in particular) NSAIDs metabolized mainly by CYP2C9, proton-pump inhibitors metabolized by CYP2C19, and beta blockers and several antipsychotics and antidepressants metabolized by CYP2D6. In this review, we provide an up-to-date summary of the functional polymorphisms and aspects of the functional genomics of the major human drug-metabolizing cytochrome P450s, as well as their clinical significance.
Keywords: Drug metabolism; Functional genomics; Genotype; Mutation; Pharmacogenetics; Phenotype; Single-nucleotide polymorphism
Focus on the intermediate state: immature mRNA of cytochromes P450—methods and insights by Christina U. Köhler; Peter H. Roos (pp. 1109-1122).
The scattered and limited data on hnRNAs (pre-mRNAs) of cytochromes P450 (CYP) are compiled and discussed for the first time. The methods for determination and quantification of hnRNAs are compared. In most cases, CYP hnRNA levels were determined as a parameter of transcriptional activity. It is known, however, that some CYPs, in particular CYP2E1, are in addition specifically and extensively regulated by post-transcriptional processes. Obviously, these processes also influence the processing of CYP hnRNAs so that their levels cannot be considered a mere result of transcription. The underlying mechanisms of post-transcriptional CYP hnRNA and mRNA regulation are not well understood. It is our aim therefore to bring together available data on CYP hnRNA and to discuss them in the light of recent advances in knowledge concerning pre-mRNA processing and interactions between RNA and low molecular weight interfering RNAs. By doing this, we hope to drive research in a direction which appears promising in providing some long-awaited answers with respect to mechanisms of post-transcriptional CYP regulation.
Keywords: hnRNA; mRNA; Cytochrome P450; Transcript level; Post-transcriptional regulation; Pre-mRNA
Focus on the intermediate state: immature mRNA of cytochromes P450—methods and insights by Christina U. Köhler; Peter H. Roos (pp. 1109-1122).
The scattered and limited data on hnRNAs (pre-mRNAs) of cytochromes P450 (CYP) are compiled and discussed for the first time. The methods for determination and quantification of hnRNAs are compared. In most cases, CYP hnRNA levels were determined as a parameter of transcriptional activity. It is known, however, that some CYPs, in particular CYP2E1, are in addition specifically and extensively regulated by post-transcriptional processes. Obviously, these processes also influence the processing of CYP hnRNAs so that their levels cannot be considered a mere result of transcription. The underlying mechanisms of post-transcriptional CYP hnRNA and mRNA regulation are not well understood. It is our aim therefore to bring together available data on CYP hnRNA and to discuss them in the light of recent advances in knowledge concerning pre-mRNA processing and interactions between RNA and low molecular weight interfering RNAs. By doing this, we hope to drive research in a direction which appears promising in providing some long-awaited answers with respect to mechanisms of post-transcriptional CYP regulation.
Keywords: hnRNA; mRNA; Cytochrome P450; Transcript level; Post-transcriptional regulation; Pre-mRNA
Quantitative analysis of highly homologous proteins: the challenge of assaying the “CYP-ome” by mass spectrometry by Elmar Langenfeld; Helmut E. Meyer; Katrin Marcus (pp. 1123-1134).
Cytochrome P450 enzymes comprise families of highly homologous proteins. These proteins play a pivotal role in oxidative drug metabolism and are important targets in drug discovery research. Proteomics today is a valuable tool for the analysis of proteins. In the past, qualitative analysis of the proteome was the main focus of research, but in the last few years interest in the mathematical modelling of protein networks has been growing and so has the demand on quantitative proteome analysis. As a thorough understanding of cytochrome P450 dependent metabolism is crucial for drug discovery, it is thus not astounding that cytochrome P450 enzymes are a target for quantitative proteomics research. In this article, we review the techniques available for quantitative proteome analysis and to what extent these techniques have been used for the quantification of cytochrome P450 enzymes and give a brief outlook of the techniques that have promising potential for the analysis of these proteins in the future.
Keywords: Proteomics; Quantification; Protein label; Mass spectrometry; Target compound analysis
Quantitative analysis of highly homologous proteins: the challenge of assaying the “CYP-ome” by mass spectrometry by Elmar Langenfeld; Helmut E. Meyer; Katrin Marcus (pp. 1123-1134).
Cytochrome P450 enzymes comprise families of highly homologous proteins. These proteins play a pivotal role in oxidative drug metabolism and are important targets in drug discovery research. Proteomics today is a valuable tool for the analysis of proteins. In the past, qualitative analysis of the proteome was the main focus of research, but in the last few years interest in the mathematical modelling of protein networks has been growing and so has the demand on quantitative proteome analysis. As a thorough understanding of cytochrome P450 dependent metabolism is crucial for drug discovery, it is thus not astounding that cytochrome P450 enzymes are a target for quantitative proteomics research. In this article, we review the techniques available for quantitative proteome analysis and to what extent these techniques have been used for the quantification of cytochrome P450 enzymes and give a brief outlook of the techniques that have promising potential for the analysis of these proteins in the future.
Keywords: Proteomics; Quantification; Protein label; Mass spectrometry; Target compound analysis
Detection of electrophoretically separated cytochromes P450 by element-labelled monoclonal antibodies via laser ablation inductively coupled plasma mass spectrometry by Peter H. Roos; Arunachalam Venkatachalam; Andreas Manz; Larissa Waentig; Christina U. Koehler; Norbert Jakubowski (pp. 1135-1147).
Numerous structurally and enzymatically similar cytochromes P450 (CYPs) are involved in the metabolism of xenobiotics and are present in different amounts and with different enzyme profiles in human tissues and cells. Analysis of their adaptively regulated and individually variable patterns is a peculiar analytical challenge. We developed a laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) based method for concomitant detection and semiquantitative determination of electrophoretically separated and blotted CYPs. The first results are given here for the two enzymes CYP1A1 and CYP2E1. Specific monoclonal antibodies directed against the enzymes were differentially labelled with europium via a covalently linked chelator and with iodine, respectively. Analysis of the modified antibodies shows that both europium and iodine are coupled to the heavy and the light chains of the antibodies. Also, the antibodies maintained their antigen-binding properties after labelling as demonstrated by LA-ICP-MS-analysed immunoblots. The method allowed us to detect specifically and concomitantly both CYP enzymes in complex biological samples, i.e. microsomes of rat liver and minipig duodenum, which are characterized by different levels and proportions of the two CYP enzymes. A strong CYP1A1 signal is found in liver microsomes of 3-methylcholanthrene-treated rats, while it is (nearly) absent in liver microsomes of rats treated with isonocotinic acid hydrazide (isoniazid). The constitutively expressed CYP2E1 is found in microsomes of both treatment groups. Duodenal microsomes of minipigs orally exposed to polycyclic aromatic hydrocarbons show a clear CYP1A1 signal. Low levels of CYP2E1 can also be detected in these microsomes. The LA-ICP-MS method allows concomitant determination of CYPs, thereby exhibiting sensitivity similar to that of conventional chemoluminescence detection via peroxidase-labelled secondary antibodies. The latter method allows readout of a single CYP protein in a 1D separation. Although the results presented here are only for labelling by use of the elements iodine and europium, the same strategy can be applied also for other lanthanide elements in combination with chelating compounds, so LA-ICP-MS of western blots offers a new capability to be applied for highly multiplexed CYP determinations via labelled antibodies.
Keywords: Laser ablation inductively coupled plasma mass spectrometry; Cytochrome P450; 1, 4, 7, 10-Tetraazacyclododecane- 1, 4, 7, 10-tetraacetic acid; Iodination; Europium; Electrophoresis
Detection of electrophoretically separated cytochromes P450 by element-labelled monoclonal antibodies via laser ablation inductively coupled plasma mass spectrometry by Peter H. Roos; Arunachalam Venkatachalam; Andreas Manz; Larissa Waentig; Christina U. Koehler; Norbert Jakubowski (pp. 1135-1147).
Numerous structurally and enzymatically similar cytochromes P450 (CYPs) are involved in the metabolism of xenobiotics and are present in different amounts and with different enzyme profiles in human tissues and cells. Analysis of their adaptively regulated and individually variable patterns is a peculiar analytical challenge. We developed a laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) based method for concomitant detection and semiquantitative determination of electrophoretically separated and blotted CYPs. The first results are given here for the two enzymes CYP1A1 and CYP2E1. Specific monoclonal antibodies directed against the enzymes were differentially labelled with europium via a covalently linked chelator and with iodine, respectively. Analysis of the modified antibodies shows that both europium and iodine are coupled to the heavy and the light chains of the antibodies. Also, the antibodies maintained their antigen-binding properties after labelling as demonstrated by LA-ICP-MS-analysed immunoblots. The method allowed us to detect specifically and concomitantly both CYP enzymes in complex biological samples, i.e. microsomes of rat liver and minipig duodenum, which are characterized by different levels and proportions of the two CYP enzymes. A strong CYP1A1 signal is found in liver microsomes of 3-methylcholanthrene-treated rats, while it is (nearly) absent in liver microsomes of rats treated with isonocotinic acid hydrazide (isoniazid). The constitutively expressed CYP2E1 is found in microsomes of both treatment groups. Duodenal microsomes of minipigs orally exposed to polycyclic aromatic hydrocarbons show a clear CYP1A1 signal. Low levels of CYP2E1 can also be detected in these microsomes. The LA-ICP-MS method allows concomitant determination of CYPs, thereby exhibiting sensitivity similar to that of conventional chemoluminescence detection via peroxidase-labelled secondary antibodies. The latter method allows readout of a single CYP protein in a 1D separation. Although the results presented here are only for labelling by use of the elements iodine and europium, the same strategy can be applied also for other lanthanide elements in combination with chelating compounds, so LA-ICP-MS of western blots offers a new capability to be applied for highly multiplexed CYP determinations via labelled antibodies.
Keywords: Laser ablation inductively coupled plasma mass spectrometry; Cytochrome P450; 1, 4, 7, 10-Tetraazacyclododecane- 1, 4, 7, 10-tetraacetic acid; Iodination; Europium; Electrophoresis
Analysis of CYP1A1 induction in single cells of urothelial cell populations by flow cytometry by Sabine Plöttner; Gisela H. Degen; Peter H. Roos; Wolfram Föllmann (pp. 1149-1158).
As carcinogenesis is a process starting at the single-cell level it is desirable to study carcinogen-mediated effects in individual cells. A primary step in chemically induced carcinogenesis is the formation of reactive DNA-binding metabolites by cytochromes P450 (CYP). We applied indirect immunofluorescence to stain CYP1A1 in urothelial cells for quantification by flow cytometry. Our studies were carried out with metabolically competent primary porcine urinary bladder epithelial cells (PUBECs) and the human urothelial cell line 5637 for which we have previously demonstrated CYP1A1 mRNA induction by the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) applying real-time RT-PCR. Flow cytometric analysis revealed that for PUBEC and 5637 cells the fraction of CYP1A1-induced cells increased with B[a]P concentration. Furthermore, in 5637 cells this effect was time-dependent, being more pronounced after 48 h than after 24 h. However, CYP1A1 induction could not be detected in all analyzed PUBEC and 5637 cells after treatment with up to 50 μM B[a]P. The reason for this remains unknown at the moment. Overall, B[a]P-treated cells could be divided into fractions of clearly CYP1A1-induced and clearly uninduced cells. Another fraction of “unclear” CYP1A1-induced cells and one of unclassifiable cells remained, as quantification of CYP1A1 induction by flow cytometry was hampered by B[a]P-related fluorescence. This is ascribed to phenolic B[a]P metabolites formed by CYP1A1 and which are known to fluoresce at wavelengths above 500 nm, whereas B[a]P does not. Overall, the method permits the detection of CYP1A1 protein level in large numbers of individual cells, thereby providing an adequate basis for statistical analyses. Flow cytometric detection of CYP1A1 induction in individual cells allows further insight into the metabolic competence of single cells and therefore could be a valuable tool for toxicological studies. Figure Flow cytometric analysis of immunostained PUBEC cells reveals that CYP1A1 is induced by B[a]P in a concentration-dependent manner
Keywords: Cytochrome P450 1A1 induction; Flow cytometry; Single-cell analysis; Benzo[a]pyrene
Analysis of CYP1A1 induction in single cells of urothelial cell populations by flow cytometry by Sabine Plöttner; Gisela H. Degen; Peter H. Roos; Wolfram Föllmann (pp. 1149-1158).
As carcinogenesis is a process starting at the single-cell level it is desirable to study carcinogen-mediated effects in individual cells. A primary step in chemically induced carcinogenesis is the formation of reactive DNA-binding metabolites by cytochromes P450 (CYP). We applied indirect immunofluorescence to stain CYP1A1 in urothelial cells for quantification by flow cytometry. Our studies were carried out with metabolically competent primary porcine urinary bladder epithelial cells (PUBECs) and the human urothelial cell line 5637 for which we have previously demonstrated CYP1A1 mRNA induction by the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) applying real-time RT-PCR. Flow cytometric analysis revealed that for PUBEC and 5637 cells the fraction of CYP1A1-induced cells increased with B[a]P concentration. Furthermore, in 5637 cells this effect was time-dependent, being more pronounced after 48 h than after 24 h. However, CYP1A1 induction could not be detected in all analyzed PUBEC and 5637 cells after treatment with up to 50 μM B[a]P. The reason for this remains unknown at the moment. Overall, B[a]P-treated cells could be divided into fractions of clearly CYP1A1-induced and clearly uninduced cells. Another fraction of “unclear” CYP1A1-induced cells and one of unclassifiable cells remained, as quantification of CYP1A1 induction by flow cytometry was hampered by B[a]P-related fluorescence. This is ascribed to phenolic B[a]P metabolites formed by CYP1A1 and which are known to fluoresce at wavelengths above 500 nm, whereas B[a]P does not. Overall, the method permits the detection of CYP1A1 protein level in large numbers of individual cells, thereby providing an adequate basis for statistical analyses. Flow cytometric detection of CYP1A1 induction in individual cells allows further insight into the metabolic competence of single cells and therefore could be a valuable tool for toxicological studies. Figure Flow cytometric analysis of immunostained PUBEC cells reveals that CYP1A1 is induced by B[a]P in a concentration-dependent manner
Keywords: Cytochrome P450 1A1 induction; Flow cytometry; Single-cell analysis; Benzo[a]pyrene
Concomitant detection of CYP1A1 enzymatic activity and CYP1A1 protein in individual cells of a human urothelial cell line using a bilayer microfluidic device by Claus A. Schumann; Angelika Dörrenhaus; Joachim Franzke; Peter Lampen; Petra S. Dittrich; Andreas Manz; Peter H. Roos (pp. 1159-1166).
To understand molecular networking at the cellular level, analyses of processes and effects at the single-cell level are most appropriate. Usual biochemical or molecular biological analyses are based on integrated signals of numerous cells which differ, however, in their expression and activity profiles. Here we show that it is possible to determine different types of properties of individual cells by means of a specifically designed microfluidic device. As part of investigations to characterize the human urothelial cell line 5637 as a potential model system for studies of toxic and carcinogenic effects on urothelial cells, we use this cell line to assign cytochrome P450 activity, and expression of the enzymes involved, to individual cells. It is shown that the cell population is very heterogeneous with respect to the extent and kinetics of CYP1A1-dependent ethoxyresorufin O-deethylase (EROD). This is also true for the cells’ CYP1A1 protein content. With some exceptions, the EROD activity largely coincides with the presence of CYP1A1 protein in the cells. The results obtained with the microfluidic device are promising and open up new perspectives with regard to multi-property determinations in individual cells and to studies focusing on the biochemical and molecular heterogeneity of cells. Figure Formation of fluorescent resorufin from ethoxyresorufin by cytochrome P450 activity in urothelial cells attached within the chamber of a microfluidic device
Keywords: Microfluidic device; Single cell analysis; Cytochrome P450; CYP1A1; EROD activity; PDMS
Concomitant detection of CYP1A1 enzymatic activity and CYP1A1 protein in individual cells of a human urothelial cell line using a bilayer microfluidic device by Claus A. Schumann; Angelika Dörrenhaus; Joachim Franzke; Peter Lampen; Petra S. Dittrich; Andreas Manz; Peter H. Roos (pp. 1159-1166).
To understand molecular networking at the cellular level, analyses of processes and effects at the single-cell level are most appropriate. Usual biochemical or molecular biological analyses are based on integrated signals of numerous cells which differ, however, in their expression and activity profiles. Here we show that it is possible to determine different types of properties of individual cells by means of a specifically designed microfluidic device. As part of investigations to characterize the human urothelial cell line 5637 as a potential model system for studies of toxic and carcinogenic effects on urothelial cells, we use this cell line to assign cytochrome P450 activity, and expression of the enzymes involved, to individual cells. It is shown that the cell population is very heterogeneous with respect to the extent and kinetics of CYP1A1-dependent ethoxyresorufin O-deethylase (EROD). This is also true for the cells’ CYP1A1 protein content. With some exceptions, the EROD activity largely coincides with the presence of CYP1A1 protein in the cells. The results obtained with the microfluidic device are promising and open up new perspectives with regard to multi-property determinations in individual cells and to studies focusing on the biochemical and molecular heterogeneity of cells. Figure Formation of fluorescent resorufin from ethoxyresorufin by cytochrome P450 activity in urothelial cells attached within the chamber of a microfluidic device
Keywords: Microfluidic device; Single cell analysis; Cytochrome P450; CYP1A1; EROD activity; PDMS
Determination of CYP activity in precision-cut liver slices: whether to use intact slices or slice homogenate by R. Glöckner; A. Lieder; A. Lupp (pp. 1167-1172).
Despite CYP induction in vitro in precision-cut liver slices (LS) is well documented, there are no standardised assays for determining CYP activity as a major end-point. In this paper, short-term assays with intact and homogenised LS from male and female rats were directly compared. We obtained similar results for 7-ethoxycoumarine O-deethylation (ECOD) with LS from both sexes: higher basal activities were measured in LS homogenate, whereas slightly stronger induction by BNF was found with intact LS. CYP3A-dependent basal and dexamethasone (Dex)-induced 2β-, 15β- and 6β-testosterone hydroxylation (TH) rates were higher in both intact and homogenised LS from male compared to female rats. CYP3A induction in vitro could likewise be detected in intact and homogenised LS preferentially by determining 2β- and 15β-TH, with higher induction factors observed in LS from females. 6β-TH seems to be less inducible in intact LS of males. In vivo pretreatment of liver donors with BNF and Dex did not substantially disturb the subsequent in vitro induction of ECOD and TH, respectively.
Keywords: Ethoxycoumarin O-deethylation; CYP induction; Intact liver slices; Liver slice homogenate; Testosterone hydroxylation
Determination of CYP activity in precision-cut liver slices: whether to use intact slices or slice homogenate by R. Glöckner; A. Lieder; A. Lupp (pp. 1167-1172).
Despite CYP induction in vitro in precision-cut liver slices (LS) is well documented, there are no standardised assays for determining CYP activity as a major end-point. In this paper, short-term assays with intact and homogenised LS from male and female rats were directly compared. We obtained similar results for 7-ethoxycoumarine O-deethylation (ECOD) with LS from both sexes: higher basal activities were measured in LS homogenate, whereas slightly stronger induction by BNF was found with intact LS. CYP3A-dependent basal and dexamethasone (Dex)-induced 2β-, 15β- and 6β-testosterone hydroxylation (TH) rates were higher in both intact and homogenised LS from male compared to female rats. CYP3A induction in vitro could likewise be detected in intact and homogenised LS preferentially by determining 2β- and 15β-TH, with higher induction factors observed in LS from females. 6β-TH seems to be less inducible in intact LS of males. In vivo pretreatment of liver donors with BNF and Dex did not substantially disturb the subsequent in vitro induction of ECOD and TH, respectively.
Keywords: Ethoxycoumarin O-deethylation; CYP induction; Intact liver slices; Liver slice homogenate; Testosterone hydroxylation
Precision-cut liver slices from rats of different ages: basal cytochrome P450-dependent monooxygenase activities and inducibility by Amelie Lupp; Reinhild Glöckner; Joachim Etzrodt; Dieter Müller (pp. 1173-1184).
The biotransformation capacity – of the cytochrome P450 (CYP) system for example – is lower but inducibility is more pronounced in neonates than in adults. On the other hand, both enzyme activities and inducibility decline with senescence. Precision-cut rat liver slices are widely used as an in vitro tool for the examination of drug toxicity, xenobiotic metabolism or enzyme induction. The aim of the present study was to assess whether age-related changes in CYP activities and induction observed in vivo are also mirrored in vitro in liver slices. For this purpose, different CYP model reactions were measured in precision-cut liver slices from one-day-old, 40-day-old and one-year-old rats after in vitro exposure to various inducers. Similar to the in vivo situation, basal CYP activities were distinctly lower and inducibility was much more pronounced in liver slices from neonatal than in those from adult animals. Also, enzyme activities were mostly somewhat lower in liver slices from aged rats compared to those from 40-day-old rats. However, CYP inducibility was less pronounced than with younger animals too. Thus, precision-cut rat liver slices are a suitable in vitro tool for investigating age-related changes in CYP activities and induction as well as developmental differences in drug metabolism and toxicity.
Keywords: Rat; Liver slices; Precision-cut; Age dependency; Cytochrome P450; Induction
Precision-cut liver slices from rats of different ages: basal cytochrome P450-dependent monooxygenase activities and inducibility by Amelie Lupp; Reinhild Glöckner; Joachim Etzrodt; Dieter Müller (pp. 1173-1184).
The biotransformation capacity – of the cytochrome P450 (CYP) system for example – is lower but inducibility is more pronounced in neonates than in adults. On the other hand, both enzyme activities and inducibility decline with senescence. Precision-cut rat liver slices are widely used as an in vitro tool for the examination of drug toxicity, xenobiotic metabolism or enzyme induction. The aim of the present study was to assess whether age-related changes in CYP activities and induction observed in vivo are also mirrored in vitro in liver slices. For this purpose, different CYP model reactions were measured in precision-cut liver slices from one-day-old, 40-day-old and one-year-old rats after in vitro exposure to various inducers. Similar to the in vivo situation, basal CYP activities were distinctly lower and inducibility was much more pronounced in liver slices from neonatal than in those from adult animals. Also, enzyme activities were mostly somewhat lower in liver slices from aged rats compared to those from 40-day-old rats. However, CYP inducibility was less pronounced than with younger animals too. Thus, precision-cut rat liver slices are a suitable in vitro tool for investigating age-related changes in CYP activities and induction as well as developmental differences in drug metabolism and toxicity.
Keywords: Rat; Liver slices; Precision-cut; Age dependency; Cytochrome P450; Induction
A new method for the detection of ATP using a quantum-dot-tagged aptamer by Zhang Chen; Guang Li; Lan Zhang; Junfeng Jiang; Zhao Li; Zhihui Peng; Le Deng (pp. 1185-1188).
Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3′-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3′-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3′-biotin-modified DNA and 3′-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3′-biotin-modified DNA and 3′-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3′-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer–ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection.
Keywords: Aptamer; Quantum dot; FRET; ATP detection
A new method for the detection of ATP using a quantum-dot-tagged aptamer by Zhang Chen; Guang Li; Lan Zhang; Junfeng Jiang; Zhao Li; Zhihui Peng; Le Deng (pp. 1185-1188).
Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3′-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3′-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3′-biotin-modified DNA and 3′-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3′-biotin-modified DNA and 3′-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3′-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer–ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection.
Keywords: Aptamer; Quantum dot; FRET; ATP detection
In vitro nonenzymatic glycation of guanosine 5′-triphosphate by dihydroxyacetone phosphate by Yuyuan Li; Menashi A. Cohenford; Udayan Dutta; Joel A. Dain (pp. 1189-1196).
Dihydroxyacetone phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosephosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triphosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.
Keywords: AGEs; Advanced glycation endproducts; Nonenzymatic glycation; GTP; Dihydroxyacetone phosphate; DHAP
In vitro nonenzymatic glycation of guanosine 5′-triphosphate by dihydroxyacetone phosphate by Yuyuan Li; Menashi A. Cohenford; Udayan Dutta; Joel A. Dain (pp. 1189-1196).
Dihydroxyacetone phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosephosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triphosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.
Keywords: AGEs; Advanced glycation endproducts; Nonenzymatic glycation; GTP; Dihydroxyacetone phosphate; DHAP
Synthesis and assessment of molecular recognizability by RP-HPLC of an N-alkyl-β-Ala-L-Phe-derived organic phase with self-assembling ability by M. Mizanur Rahman; Makoto Takafuji; Hirotaka Ihara (pp. 1197-1208).
An N-alky-β-Ala-L-Phe derivative, N'-octadecyl-N α -[(N-acryloyl)-β-alanyl]-L-phenylalanineamide (1), with a polymerizable head group has been synthesized and telomerized with the silane coupling agent 3-mercaptopropyltrimethoxysilane (MPS). SEM and DSC observations indicated that both 1 and its telomer (T-1) could self-assemble into fibrillar forms with highly ordered structures in organic media such as benzene through complementary hydrogen bonding between the amide moieties. T-1 was grafted onto porous silica gels through the terminal trimethoxysilyl group and then packed into a stainless steel column. RP-HPLC results for polycyclic aromatic hydrocarbons (PAHs) demonstrated that significantly higher molecular shape recognition could be achieved by silica-supported T-1 (Sil-T-1). In this paper, the mechanism of the selectivity enhancement in HPLC by Sil-T-1 is discussed on the basis of comparing with the corresponding L-Phe derivative N'-octadecyl-N α -(acryloyl)-L-phenylalanineamide (2) without β -Ala and the stationary phase (Sil-T-2) obtained from it. The HPLC column materials Sil-T-1 and Sil-T-2 were characterized by DSC, TGA, DRIFT-IR, and 13C and 29Si CP-MAS NMR spectroscopic measurements.
Keywords: Fibrillar aggregates; Intermolecular hydrogen bonding; Molecular recognition; Ordered carbonyl moieties; Self-assembling ability
Synthesis and assessment of molecular recognizability by RP-HPLC of an N-alkyl-β-Ala-L-Phe-derived organic phase with self-assembling ability by M. Mizanur Rahman; Makoto Takafuji; Hirotaka Ihara (pp. 1197-1208).
An N-alky-β-Ala-L-Phe derivative, N'-octadecyl-N α -[(N-acryloyl)-β-alanyl]-L-phenylalanineamide (1), with a polymerizable head group has been synthesized and telomerized with the silane coupling agent 3-mercaptopropyltrimethoxysilane (MPS). SEM and DSC observations indicated that both 1 and its telomer (T-1) could self-assemble into fibrillar forms with highly ordered structures in organic media such as benzene through complementary hydrogen bonding between the amide moieties. T-1 was grafted onto porous silica gels through the terminal trimethoxysilyl group and then packed into a stainless steel column. RP-HPLC results for polycyclic aromatic hydrocarbons (PAHs) demonstrated that significantly higher molecular shape recognition could be achieved by silica-supported T-1 (Sil-T-1). In this paper, the mechanism of the selectivity enhancement in HPLC by Sil-T-1 is discussed on the basis of comparing with the corresponding L-Phe derivative N'-octadecyl-N α -(acryloyl)-L-phenylalanineamide (2) without β -Ala and the stationary phase (Sil-T-2) obtained from it. The HPLC column materials Sil-T-1 and Sil-T-2 were characterized by DSC, TGA, DRIFT-IR, and 13C and 29Si CP-MAS NMR spectroscopic measurements.
Keywords: Fibrillar aggregates; Intermolecular hydrogen bonding; Molecular recognition; Ordered carbonyl moieties; Self-assembling ability
Separation of Amadori peptides from their unmodified analogs by ion-pairing RP-HPLC with heptafluorobutyric acid as ion-pair reagent by Andrej Frolov; Ralf Hoffmann (pp. 1209-1214).
Glycation is a common class of nonenzymatic posttranslational modifications relevant for several diseases and cell aging in general, such as D-glucose-derived modifications at the ɛ-amino groups of lysine residues in blood proteins, especially albumin, immunoglobulin, and hemoglobin, for diabetic patients. These Amadori compounds are identified on the peptide level after enzymatic digestion and chromatographic separation by mass spectrometry. Their syntheses usually rely on a global glycation approach. Both areas require the reliable separation of glycated peptides from their unmodified congeners present in different ratios, which is typically not achieved by standard eluent systems in ion-pairing RP-HPLC (IP-RPLC). Here, we compare aqueous acetonitrile and methanol gradients containing either trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as ion-pairing agents to separate such peptide pairs. TFA-containing eluents resulted in rather low resolutions, and the glycated and unglycated peptides often coeluted. HFBA increased the retention times of the unmodified peptide more than for the glycated peptide thereby improving the separation of all eight studied peptide pairs, even achieving baseline separations for some sequences. Thus the use of HFBA as ion-pair reagent provides a universally applicable eluent system in IP-RPLC to separate glycated peptides from their unmodified counterparts, even at the preparative scale required for synthetic peptides.
Keywords: Electrospray ionization (ESI); Glycation; Mass spectrometry (MS); Matrix-assisted laser desorption/ionization (MALDI); Solid-phase peptide synthesis (SPPS)
Separation of Amadori peptides from their unmodified analogs by ion-pairing RP-HPLC with heptafluorobutyric acid as ion-pair reagent by Andrej Frolov; Ralf Hoffmann (pp. 1209-1214).
Glycation is a common class of nonenzymatic posttranslational modifications relevant for several diseases and cell aging in general, such as D-glucose-derived modifications at the ɛ-amino groups of lysine residues in blood proteins, especially albumin, immunoglobulin, and hemoglobin, for diabetic patients. These Amadori compounds are identified on the peptide level after enzymatic digestion and chromatographic separation by mass spectrometry. Their syntheses usually rely on a global glycation approach. Both areas require the reliable separation of glycated peptides from their unmodified congeners present in different ratios, which is typically not achieved by standard eluent systems in ion-pairing RP-HPLC (IP-RPLC). Here, we compare aqueous acetonitrile and methanol gradients containing either trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as ion-pairing agents to separate such peptide pairs. TFA-containing eluents resulted in rather low resolutions, and the glycated and unglycated peptides often coeluted. HFBA increased the retention times of the unmodified peptide more than for the glycated peptide thereby improving the separation of all eight studied peptide pairs, even achieving baseline separations for some sequences. Thus the use of HFBA as ion-pair reagent provides a universally applicable eluent system in IP-RPLC to separate glycated peptides from their unmodified counterparts, even at the preparative scale required for synthetic peptides.
Keywords: Electrospray ionization (ESI); Glycation; Mass spectrometry (MS); Matrix-assisted laser desorption/ionization (MALDI); Solid-phase peptide synthesis (SPPS)
Development of a competitive lateral flow immunoassay for progesterone: influence of coating conjugates and buffer components by Geertruida A. Posthuma-Trumpie; Jakob Korf; Aart van Amerongen (pp. 1215-1223).
Several aspects of the development of competitive lateral flow immunoassays (LFIAs) are described. The quantitation of progesterone is taken as an example. The LFIA format consisted of a nitrocellulose membrane spotted with various progesterone conjugates as the test line. A mixture of primary antibody and secondary antibody adsorbed to colloidal carbon was used for signal generation. A digital scanner and dedicated software were used to quantitate the response. A reappraisal of the checkerboard titration, often used in the optimisation of immunoassays, is discussed. Surprisingly, the highest sensitivity of the LFIA format (IC50 of 0.6 µg L−1 progesterone in buffer) was achieved by using a high coating concentration of the analyte–protein conjugate and a high dilution of the antibody solution. Immediate addition of all reagents in LFIA was superior to premixing the components and allowing prereaction. Of several blocking agents tested bovine serum albumin was superior in performance, whereas the combination of ovalbumin and progesterone substantially influenced test results.
Keywords: Immunochromatography; Lateral flow immunoassay; Progesterone; Optimisation; Blocking agents
Development of a competitive lateral flow immunoassay for progesterone: influence of coating conjugates and buffer components by Geertruida A. Posthuma-Trumpie; Jakob Korf; Aart van Amerongen (pp. 1215-1223).
Several aspects of the development of competitive lateral flow immunoassays (LFIAs) are described. The quantitation of progesterone is taken as an example. The LFIA format consisted of a nitrocellulose membrane spotted with various progesterone conjugates as the test line. A mixture of primary antibody and secondary antibody adsorbed to colloidal carbon was used for signal generation. A digital scanner and dedicated software were used to quantitate the response. A reappraisal of the checkerboard titration, often used in the optimisation of immunoassays, is discussed. Surprisingly, the highest sensitivity of the LFIA format (IC50 of 0.6 µg L−1 progesterone in buffer) was achieved by using a high coating concentration of the analyte–protein conjugate and a high dilution of the antibody solution. Immediate addition of all reagents in LFIA was superior to premixing the components and allowing prereaction. Of several blocking agents tested bovine serum albumin was superior in performance, whereas the combination of ovalbumin and progesterone substantially influenced test results.
Keywords: Immunochromatography; Lateral flow immunoassay; Progesterone; Optimisation; Blocking agents
Quantification of refractory organic substances in freshwaters: further insight into the response of the voltammetric method by François Quentel; Montserrat Filella (pp. 1225-1230).
A recently published method for quantifying refractory organic matter (often referred to as humic substances) in freshwaters was applied to a wide range of International Humic Substance Society (IHSS) humic compounds in order to (i) gain a better understanding of the mechanism of the voltammetric response which is the basis of the analytical method and (ii) provide guidance on choosing the optimal standard to be used. At the same time, the sensitivity of the technique has been increased by switching from the pulse mode initially proposed to the square-wave mode. The results obtained show that (i) differences in adsorption onto the electrode rather than differences in complexation strength are responsible for the differences in the intensity of the signal obtained for the different humic compounds, (ii) carboxylate, N- and S-containing groups do not play a role in the voltammetric signal.
Keywords: Humics; Fulvics; Refractory organic matter; IHSS; SWV; Mo(VI); Freshwaters
Quantification of refractory organic substances in freshwaters: further insight into the response of the voltammetric method by François Quentel; Montserrat Filella (pp. 1225-1230).
A recently published method for quantifying refractory organic matter (often referred to as humic substances) in freshwaters was applied to a wide range of International Humic Substance Society (IHSS) humic compounds in order to (i) gain a better understanding of the mechanism of the voltammetric response which is the basis of the analytical method and (ii) provide guidance on choosing the optimal standard to be used. At the same time, the sensitivity of the technique has been increased by switching from the pulse mode initially proposed to the square-wave mode. The results obtained show that (i) differences in adsorption onto the electrode rather than differences in complexation strength are responsible for the differences in the intensity of the signal obtained for the different humic compounds, (ii) carboxylate, N- and S-containing groups do not play a role in the voltammetric signal.
Keywords: Humics; Fulvics; Refractory organic matter; IHSS; SWV; Mo(VI); Freshwaters
Evaluating the impact of extraction and cleanup parameters on the yield of total petroleum hydrocarbons in soil by Eija Saari; Paavo Perämäki; Jorma Jalonen (pp. 1231-1240).
Interlaboratory comparisons for the analysis of mineral oil in polluted soil using the GC–FID method indicate that extraction and cleanup conditions have significant effects on the analytical results. In this investigation a ruggedness test was performed on the extraction and cleanup method for the determination of total petroleum hydrocarbons in soil. A two-level Plackett–Burman design was utilized to study the effect of 11 different method parameters on the extraction recovery of total petroleum hydrocarbons (TPH) in soil. Both qualitative and quantitative factors were investigated. The results indicate that total petroleum hydrocarbons can be relatively reliably monitored through strict implementation of the ISO and CEN draft standards. However, variation in certain method parameters readily affects the validity of the results. The most critical factors affecting TPH recovery were the solvent and co-solvent used for extraction, the extraction time, adsorbent and its weight and sample TPH concentration. Because adaptation of the draft standards especially with respect to these factors easily leads to TPH recoveries higher than 200% or lower than 70%, the validity of the adapted method should always be verified. A proper estimate of the expanded uncertainty should also be appended to TPH results, because only then can the reliability of the results be guaranteed and further justification is gained to support the end-use of the data. This also supports the credibility of the analytical services and prevents the data end-users from drawing misleading conclusions concerning the environmental risks and potential remediation requirements.
Keywords: Total petroleum hydrocarbons; Extraction recovery; Plackett–Burman design; Ruggedness
Evaluating the impact of extraction and cleanup parameters on the yield of total petroleum hydrocarbons in soil by Eija Saari; Paavo Perämäki; Jorma Jalonen (pp. 1231-1240).
Interlaboratory comparisons for the analysis of mineral oil in polluted soil using the GC–FID method indicate that extraction and cleanup conditions have significant effects on the analytical results. In this investigation a ruggedness test was performed on the extraction and cleanup method for the determination of total petroleum hydrocarbons in soil. A two-level Plackett–Burman design was utilized to study the effect of 11 different method parameters on the extraction recovery of total petroleum hydrocarbons (TPH) in soil. Both qualitative and quantitative factors were investigated. The results indicate that total petroleum hydrocarbons can be relatively reliably monitored through strict implementation of the ISO and CEN draft standards. However, variation in certain method parameters readily affects the validity of the results. The most critical factors affecting TPH recovery were the solvent and co-solvent used for extraction, the extraction time, adsorbent and its weight and sample TPH concentration. Because adaptation of the draft standards especially with respect to these factors easily leads to TPH recoveries higher than 200% or lower than 70%, the validity of the adapted method should always be verified. A proper estimate of the expanded uncertainty should also be appended to TPH results, because only then can the reliability of the results be guaranteed and further justification is gained to support the end-use of the data. This also supports the credibility of the analytical services and prevents the data end-users from drawing misleading conclusions concerning the environmental risks and potential remediation requirements.
Keywords: Total petroleum hydrocarbons; Extraction recovery; Plackett–Burman design; Ruggedness
Static–dynamic sequential superheated liquid extraction of phenols and fatty acids from alperujo by J. A. Pérez-Serradilla; R. Japón-Luján; M. D. Luque de Castro (pp. 1241-1248).
Superheated liquids of different polarity have been used for sequential extraction of fatty acids and phenols from alperujo. Multivariate methodology has been used to optimise the static–dynamic extraction. Forty-two minutes are required to complete extraction (20 mg/kg of fatty acids and up to 2,200 mg/kg of hydroxytyrosol in the raw material used). The efficacy of the extraction has been demonstrated and compared with that of conventional methods (Folch and stirring-based methods for fatty acids and phenols, respectively), which needed 4.5 and 24 h for the extraction of fatty acids and phenols, respectively. The non-polar and polar extracts were injected into GC–MS and HPLC–MS–MS equipment, respectively, for individual separation–quantification of the target compounds. The simplicity of the experimental setup and the low costs of the raw material make the proposed method advisable when extraction of both fractions is required.
Keywords: Mass spectrometry/ICP-MS; Natural products; Agriculture; Extraction (SFE/SPE/SPME)
Static–dynamic sequential superheated liquid extraction of phenols and fatty acids from alperujo by J. A. Pérez-Serradilla; R. Japón-Luján; M. D. Luque de Castro (pp. 1241-1248).
Superheated liquids of different polarity have been used for sequential extraction of fatty acids and phenols from alperujo. Multivariate methodology has been used to optimise the static–dynamic extraction. Forty-two minutes are required to complete extraction (20 mg/kg of fatty acids and up to 2,200 mg/kg of hydroxytyrosol in the raw material used). The efficacy of the extraction has been demonstrated and compared with that of conventional methods (Folch and stirring-based methods for fatty acids and phenols, respectively), which needed 4.5 and 24 h for the extraction of fatty acids and phenols, respectively. The non-polar and polar extracts were injected into GC–MS and HPLC–MS–MS equipment, respectively, for individual separation–quantification of the target compounds. The simplicity of the experimental setup and the low costs of the raw material make the proposed method advisable when extraction of both fractions is required.
Keywords: Mass spectrometry/ICP-MS; Natural products; Agriculture; Extraction (SFE/SPE/SPME)
Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging
by Juan Feng; Marian Navratil; LaDora V. Thompson; Edgar A. Arriaga (pp. 1249-1249).
Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging
by Juan Feng; Marian Navratil; LaDora V. Thompson; Edgar A. Arriaga (pp. 1249-1249).