- Email: [email protected]
HPLC method for the concentration determination of ipriflavone and its seven metabolites in human plasma
Workshop 2. Air Pollution and Allergy
14
-9"] DEVELOPMENT OF ANALYTICAL METHODOLOGY FOR THE DETECTION OF MUTAGENIC HALOGENATED FURANONES IN DRINKING WATER Isabel Ramos *, Marfa-Pilar Marco, Angel Messeguer. Dpt. of Biological Organic Chemistry, C1D-CSIC. J. Girona, 18. 08034 Barcelona, Spain The occurrence of halogenated furanones in drinking water is a matter of concern due to the mutagenicity exhibited by these compounds. The most important representative of this family of toxins is 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, a compound known also as MX. This furanone has been widely detected by research groups from Northern Europe and Japan in samples from chlorinated drinking and humic water. Epidemiological studies have suggested that there is a dose-dependent association between mutagenicity of drinking water and kidney and bladder cancer in humans. Consequently, levels of halogenated furanone derivatives should be adequately monitored and minimized. In this context, immunochemical methods constitute an easy, cheap and rapid way to analyze large numbers of samples. However, MX and its derivatives are molecules too small to be immunogenic by themselves; therefore, they need to be conjugated to a protein. The strategy of conjugation includes the insertion of a spacer which could link MX to the protein. In our case, we envisaged the insertion of the spacer at position C-5 of the furanone ring. By this procedure, antibodies raised against the conjugate will face a more characteristic part of MX. In the present communication results along this line will be presented. In addition, studies directed to resolve the pair of enantiomers of MX will be also reported. [1] Hemming, J, Holbom, B, Reunanen, M, Kronberg, L, Chemosphere, 15, 549-556, 1986.
Keywords: mutagenicity; halogenated furanones; water; immunoas-
W2. Air Pollution and Allergy [--W-~-]
POLLUTION AND THE DEVELOPMENT OF ALLERGY: THE EAST AND WEST GERMANY STORY
Thomas Nicolai. Universitiitskinderklinik, Miinchen, Germany Allergic diseases have a strong genetic background, but environmental factors have a major influence in determining which genetically predisposed subjects will develop allergic symptoms. Air pollution and social conditions have received widespread attention. The unification of Germany provided a unique opportunity to study the impact of radically different environmental and social conditions on the development of allergies in two genetically homogenous populations. Whereas motorization and heavy NO2 exposure were the characteristic of many West German cities, heavily polluting industrialization with scanty motorization and private coal burning for heating purposes was typical of East German cities. We assessed the prevalence of asthma and allergic disorders in 9-11 year old children in Leipzig and Halle in East Germany, as well as in Munich, West Germany. Both East German cities were heavily polluted due to private coal burning and industrial emissions, whereas Munich has moderate industry but heavy car traffic. All fourth grade pupils in Munich (n = 7,445) were compared with those in Leipzig 1990 (n = 1,429) and Leipzig and Halle 1991 (n = 3,105). The prevalence of hay fever and skin test reactivity to common aeroallergens was considerably higher in West Germany as compared to East Germany. Furthermore, the prevalence of asthma was also higher in the West German study area. However, when atopy was taken into account, there was no longer a significant difference in the prevalence of asthma between the two parts of the country.
Keywords: allergy; children; air pollution; East/West Germany
say NASAL LAVAGE BIOMARKERS IN AIR POLLUTION -'~ HPLC METHOD FOR THE CONCENTRATION DETERMINATION OF IPRIFLAVONE AND ITS SEVEN METABOLITES IN HUMAN PLASMA Ferenc Ltvai, Istv~in Szatm~iri. Chinoin Pharmaceutical and
Chemical Works Co. Ltd. H-1045 Budapest, T6 utca 1-5., Hungary
Peter A. Steerenberg * i, Paul H. Fischer 2, L. van Bree 3, Henk Van Loveren 1.1 Lab. of Pathology and lmmunobiology, 2 Dept. of
Chronic Diseases and Environmental Epidemiology, 3 Lab. of Health Effects Research, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands
Keywords: ipriflavone; metabolites; HPLC; SPE; human clinical
It is widely accepted that humans exposed to known concentrations of ozone under controlled conditions exhibit reversible changes that affect the large and small airways as well as the alveolar region of the lung. Among the reversible changes, the induction of inflammatory responses in the lung are of major concern. The nose is the first region of the respiratory tract that comes into contact with airborne pollutants. It has been suggested that the cellular responses to toxicants in the nose are similar to those in the lower airway. It has been shown in experimental studies in which volunteers were exposed to ozone that the number of polymorphonuclear leukocytes (PMN's), and to a lower extent eosinophils were increased in nasal lavage (NAL) as well as in bronchial lavage fluid (BAL). In epidemiological studies positive associations were found between ozone levels and the numbers of PMN's, the concentration myeloperoxidase (MPO), eosinophil cationic protein (ECP), interleukin-8 (IL-8) and some markers for exudation in the NAL. The inter-variation, expressed as coefficient of variation (CV), appeared much higher than the intra-individual variation. The sensitivity was calculated, and it was estimated that ozone effects with a studyprotocol of 6 repeated measurements among 35 children and an assumed 26% increase in cells or mediators per 100/zg O3/m 3 could be detected. Due to the high CV for the number of leukocytes, to measure increase in leukocyte number more individuals are needed.
samples
Keywords: nasal lavage; biomarkers; airpollution; immunology; epi-
Ipriflavone, an anti-osteoporosis agent, is already on the market in Hungary, Japan and Italy. Along with new registrations in progress clinical investigations are going on in several countries. For the evaluation of safety and the clinical efficacy the determination of plasma levels of the drug and its metabolites were necessary. Ipriflavone metabolism is mainly resulting in oxidative, hydroxyl and carboxyl metabolites among them. The metabolites form glucuronide-conjugates. In order to follow the total conversion processes of ipriflavone the plasma samples obtained from patients were digested with bovine fl-glucuronidase enzyme. For sample preparation solid phase extraction used, for the concentration determinations a reversed phase HPLC method was developed, applying UV detection (254 nm) and gradient elution. The separation was performed on Waters Nova-Pak C18 column (2 mm x 15 cm), with 0.4 ml/min flow rate, the column temperature was 50°C. Samples were run with a gradient system using 9/1, v/v mixture of acetonitrile and water (containing 0.1% phosphoric acid) as solvent 'A' and 9/1, v/v mixture of water (containing 0.1% phosphoric acid) and acetonitrile as solvent 'B'. The LOQ was 10 ng/ml for all the compounds. The validation parameters characterize a reliable and reproducible HPLC method.
demiology
14
-9"] DEVELOPMENT OF ANALYTICAL METHODOLOGY FOR THE DETECTION OF MUTAGENIC HALOGENATED FURANONES IN DRINKING WATER Isabel Ramos *, Marfa-Pilar Marco, Angel Messeguer. Dpt. of Biological Organic Chemistry, C1D-CSIC. J. Girona, 18. 08034 Barcelona, Spain The occurrence of halogenated furanones in drinking water is a matter of concern due to the mutagenicity exhibited by these compounds. The most important representative of this family of toxins is 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, a compound known also as MX. This furanone has been widely detected by research groups from Northern Europe and Japan in samples from chlorinated drinking and humic water. Epidemiological studies have suggested that there is a dose-dependent association between mutagenicity of drinking water and kidney and bladder cancer in humans. Consequently, levels of halogenated furanone derivatives should be adequately monitored and minimized. In this context, immunochemical methods constitute an easy, cheap and rapid way to analyze large numbers of samples. However, MX and its derivatives are molecules too small to be immunogenic by themselves; therefore, they need to be conjugated to a protein. The strategy of conjugation includes the insertion of a spacer which could link MX to the protein. In our case, we envisaged the insertion of the spacer at position C-5 of the furanone ring. By this procedure, antibodies raised against the conjugate will face a more characteristic part of MX. In the present communication results along this line will be presented. In addition, studies directed to resolve the pair of enantiomers of MX will be also reported. [1] Hemming, J, Holbom, B, Reunanen, M, Kronberg, L, Chemosphere, 15, 549-556, 1986.
Keywords: mutagenicity; halogenated furanones; water; immunoas-
W2. Air Pollution and Allergy [--W-~-]
POLLUTION AND THE DEVELOPMENT OF ALLERGY: THE EAST AND WEST GERMANY STORY
Thomas Nicolai. Universitiitskinderklinik, Miinchen, Germany Allergic diseases have a strong genetic background, but environmental factors have a major influence in determining which genetically predisposed subjects will develop allergic symptoms. Air pollution and social conditions have received widespread attention. The unification of Germany provided a unique opportunity to study the impact of radically different environmental and social conditions on the development of allergies in two genetically homogenous populations. Whereas motorization and heavy NO2 exposure were the characteristic of many West German cities, heavily polluting industrialization with scanty motorization and private coal burning for heating purposes was typical of East German cities. We assessed the prevalence of asthma and allergic disorders in 9-11 year old children in Leipzig and Halle in East Germany, as well as in Munich, West Germany. Both East German cities were heavily polluted due to private coal burning and industrial emissions, whereas Munich has moderate industry but heavy car traffic. All fourth grade pupils in Munich (n = 7,445) were compared with those in Leipzig 1990 (n = 1,429) and Leipzig and Halle 1991 (n = 3,105). The prevalence of hay fever and skin test reactivity to common aeroallergens was considerably higher in West Germany as compared to East Germany. Furthermore, the prevalence of asthma was also higher in the West German study area. However, when atopy was taken into account, there was no longer a significant difference in the prevalence of asthma between the two parts of the country.
Keywords: allergy; children; air pollution; East/West Germany
say NASAL LAVAGE BIOMARKERS IN AIR POLLUTION -'~ HPLC METHOD FOR THE CONCENTRATION DETERMINATION OF IPRIFLAVONE AND ITS SEVEN METABOLITES IN HUMAN PLASMA Ferenc Ltvai, Istv~in Szatm~iri. Chinoin Pharmaceutical and
Chemical Works Co. Ltd. H-1045 Budapest, T6 utca 1-5., Hungary
Peter A. Steerenberg * i, Paul H. Fischer 2, L. van Bree 3, Henk Van Loveren 1.1 Lab. of Pathology and lmmunobiology, 2 Dept. of
Chronic Diseases and Environmental Epidemiology, 3 Lab. of Health Effects Research, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands
Keywords: ipriflavone; metabolites; HPLC; SPE; human clinical
It is widely accepted that humans exposed to known concentrations of ozone under controlled conditions exhibit reversible changes that affect the large and small airways as well as the alveolar region of the lung. Among the reversible changes, the induction of inflammatory responses in the lung are of major concern. The nose is the first region of the respiratory tract that comes into contact with airborne pollutants. It has been suggested that the cellular responses to toxicants in the nose are similar to those in the lower airway. It has been shown in experimental studies in which volunteers were exposed to ozone that the number of polymorphonuclear leukocytes (PMN's), and to a lower extent eosinophils were increased in nasal lavage (NAL) as well as in bronchial lavage fluid (BAL). In epidemiological studies positive associations were found between ozone levels and the numbers of PMN's, the concentration myeloperoxidase (MPO), eosinophil cationic protein (ECP), interleukin-8 (IL-8) and some markers for exudation in the NAL. The inter-variation, expressed as coefficient of variation (CV), appeared much higher than the intra-individual variation. The sensitivity was calculated, and it was estimated that ozone effects with a studyprotocol of 6 repeated measurements among 35 children and an assumed 26% increase in cells or mediators per 100/zg O3/m 3 could be detected. Due to the high CV for the number of leukocytes, to measure increase in leukocyte number more individuals are needed.
samples
Keywords: nasal lavage; biomarkers; airpollution; immunology; epi-
Ipriflavone, an anti-osteoporosis agent, is already on the market in Hungary, Japan and Italy. Along with new registrations in progress clinical investigations are going on in several countries. For the evaluation of safety and the clinical efficacy the determination of plasma levels of the drug and its metabolites were necessary. Ipriflavone metabolism is mainly resulting in oxidative, hydroxyl and carboxyl metabolites among them. The metabolites form glucuronide-conjugates. In order to follow the total conversion processes of ipriflavone the plasma samples obtained from patients were digested with bovine fl-glucuronidase enzyme. For sample preparation solid phase extraction used, for the concentration determinations a reversed phase HPLC method was developed, applying UV detection (254 nm) and gradient elution. The separation was performed on Waters Nova-Pak C18 column (2 mm x 15 cm), with 0.4 ml/min flow rate, the column temperature was 50°C. Samples were run with a gradient system using 9/1, v/v mixture of acetonitrile and water (containing 0.1% phosphoric acid) as solvent 'A' and 9/1, v/v mixture of water (containing 0.1% phosphoric acid) and acetonitrile as solvent 'B'. The LOQ was 10 ng/ml for all the compounds. The validation parameters characterize a reliable and reproducible HPLC method.
demiology