Defining pollen exposure times for clinical trials of allergen immunotherapy for pollen-induced rhinoconjunctivitis - an EAACI position paper.

BACKGROUND: Clinical efficacy of pollen allergen immunotherapy (AIT) has been broadly documented in randomized controlled trials. The underlying clinical endpoints are analysed in seasonal time periods predefined based on the background pollen concentration. However, any validated or generally accepted definition from academia or regulatory authorities for this relevant pollen exposure intensity or period of time (season) is currently not available. Therefore, this Task Force initiative of the European Academy of Allergy and Clinical Immunology (EAACI) aimed to propose definitions based on expert consensus. METHODS: A Task Force of the Immunotherapy and Aerobiology and Pollution Interest Groups of the EAACI reviewed the literature on pollen exposure in the context of defining relevant time intervals for evaluation of efficacy in AIT trials. Underlying principles in measuring pollen exposure and associated methodological problems and limitations were considered to achieve a consensus. RESULTS: The Task Force achieved a comprehensive position in defining pollen exposure times for different pollen types. Definitions are presented for 'pollen season', 'high pollen season' (or 'peak pollen period') and 'high pollen days'. CONCLUSION: This EAACI position paper provides definitions of pollen exposures for different pollen types for use in AIT trials. Their validity as standards remains to be tested in future studies.

Pollen-derived nonallergenic substances enhance Th2-induced IgE production in B cells.

BACKGROUND: B cells play a central role in IgE-mediated allergies. In damaged airway epithelium, they are exposed directly to aeroallergens. We aimed to assess whether direct exposure of B cells to pollen constituents affects allergic sensitization. METHODS: B cells from murine splenocytes and from blood samples of healthy donors were incubated for 8 days under Th2-like conditions with aqueous ragweed pollen extracts (Amb-APE) or its constituents. Secreted total IgM, IgG, and IgE was quantified by ELISA. Additionally, birch, grass, or pine-pollen extracts were tested. The number of viable cells was evaluated by ATP measurements. B-cell proliferation was measured by CFSE staining. IgE class switch was analyzed by quantitation of class switch transcripts. In an OVA/Alum i.p.-sensitization mouse model, Amb-APE was intranasally instilled for 11 consecutive days. RESULTS: Upon Th2 priming of murine B cells, ragweed pollen extract caused a dose-dependent increase in IgE production, while IgG and IgM were not affected. The low-molecular-weight fraction and phytoprostane E1 (PPE1) increased IgE production, while Amb a 1 did not. PPE1 enhanced IgE also in human memory B cells. Under Th1 conditions, Amb-APE did not influence immunoglobulin secretion. The IgE elevation was not ragweed specific. It correlated with proliferation of viable B cells, but not with IgE class switch. In vivo, Amb-APE increased total IgE and showed adjuvant activity in allergic airway inflammation. CONCLUSIONS: Aqueous pollen extracts, the protein-free fraction of Amb-APE, and the pollen-contained substance PPE1 specifically enhance IgE production in Th2-primed B cells. Thus, pollen-derived nonallergenic substances might be responsible for B-cell-dependent aggravation of IgE-mediated allergies.

Monitoring of occupational and environmental aeroallergens-- EAACI Position Paper. Concerted action of the EAACI IG Occupational Allergy and Aerobiology & Air Pollution.

Exposure to high molecular weight sensitizers of biological origin is an important risk factor for the development of asthma and rhinitis. Most of the causal allergens have been defined based on their reactivity with IgE antibodies, and in many cases, the molecular structure and function of the allergens have been established. Significant information on allergen levels that cause sensitization and allergic symptoms for several major environmental and occupational allergens has been reported. Monitoring of high molecular weight allergens and allergen carrier particles is an important part of the management of allergic respiratory diseases and requires standardized allergen assessment methods for occupational and environmental (indoor and outdoor) allergen exposure. The aim of this EAACI task force was to review the essential points for monitoring environmental and occupational allergen exposure including sampling strategies and methods, processing of dust samples, allergen analysis, and quantification. The paper includes a summary of different methods for sampling and allergen quantification, as well as their pros and cons for various exposure settings. Recommendations are being made for different exposure scenarios.

Influence of meteorological variables and air pollutants on measurements from automatic pollen sampling devices.

This study examines the influence of meteorological factors and air pollutants on the performance of automatic pollen monitoring devices, as part of the EUMETNET Autopollen COST ADOPT-intercomparison campaign held in Munich, Germany, during the 2021 pollen season. The campaign offered a unique opportunity to compare all automatic monitors available at the time, a Plair Rapid-E, a Hund-Wetzlar BAA500, an OPC Alphasense, a KH-3000 Yamatronics, three Swisens Polenos, a PollenSense APS, a FLIR IBAC2, a DMT WIBS-5, an Aerotape Sextant, to the average of four manual Hirst traps, under the same environmental conditions. The investigation aimed to elucidate how meteorological factors and air pollution impact particle capture and identification efficiency. The analysis showed coherent results for most devices regarding the correlation between environmental conditions and pollen concentrations. This reflects on one hand, a significant correlation between weather and airborne pollen concentration, and on the other hand the capability of devices to provide meaningful data under the conditions under which measurements were taken. However, correlation strength varied among devices, reflecting differences in design, algorithms, or sensors used. Additionally, it was observed that different algorithms applied to the same dataset resulted in different concentration outputs, highlighting the role of algorithm design in these systems (monitor + algorithm). Notably, no significant influence from air pollutants on the pollen concentrations was observed, suggesting that any potential difference in effect on the systems might require higher air pollution concentrations or more complex interactions. However, results from some monitors were affected to a minor degree by specific weather variables. Our findings suggest that the application of real-time devices in urban environments should focus on the associated algorithm that classifies pollen taxa. The impact of air pollution, although not to be excluded, is of secondary concern as long as the pollution levels are similar to a large European city like Munich.

Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine.

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66µM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26µM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

Airborne olive pollen counts are not representative of exposure to the major olive allergen Ole e 1.

Pollen is routinely monitored, but it is unknown whether pollen counts represent allergen exposure. We therefore simultaneously determined olive pollen and Ole e 1 in ambient air in Córdoba, Spain, and Évora, Portugal, using Hirst-type traps for pollen and high-volume cascade impactors for allergen. Pollen from different days released 12-fold different amounts of Ole e 1 per pollen (both locations P < 0.001). Average allergen release from pollen (pollen potency) was much higher in Córdoba (3.9 pg Ole e 1/pollen) than in Évora (0.8 pg Ole e 1/pollen, P = 0.004). Indeed, yearly olive pollen counts in Córdoba were 2.4 times higher than in Évora, but Ole e 1 concentrations were 7.6 times higher. When modeling the origin of the pollen, >40% of Ole e 1 exposure in Évora was explained by high-potency pollen originating from the south of Spain. Thus, olive pollen can vary substantially in allergen release, even though they are morphologically identical.

GSTM1, GSTT1 and GSTP1 gene polymorphism in polymorphous light eruption.

BACKGROUND: Polymorphous light eruption (PLE) is the most common chronic and idiopathic photodermatosis. PLE is assumed to represent an immunological hypersensitivity reaction to a radiation-induced cutaneous antigen involving reactive oxygen species (ROS) on the basis of a genetic predisposition. Among others, cellular protection against ROS is provided by glutathione S-transferases (GSTs). Different variants of the GST enzymes may influence the activity and efficiency of detoxification and biotransformation of unknown UV-induced skin-antigens and other factors that may play an important role in the pathogenesis of PLE. METHODS: In this study the relationship between isoenzymes of the GST genes GSTM1, GSTT1 and GSTP1 and possible protective or predisposing effects on PLE was examined in 29 patients and 144 controls. Diagnosis of PLE was based on the presence of characteristic clinical features. RESULTS: No association between the functional polymorphisms of the GST gene family and PLE was found. Prevalence of certain GST isoenzymes or polymorphisms in patients with PLE did not differ from healthy controls. CONCLUSION: Our data do not support prevalence of GST isoenzymes or polymorphisms as a protective effect against PLE. Especially a higher carrier frequency of GSTP1 Val(105) as a protective factor against PLE which has been published before could not be proved. The GST genotypes GSTM1, GSTT1 and GSTP1 (including SNPs) seem to have no relevant association with PLE.

Toxicity and elemental composition of particulate matter from outdoor and indoor air of elementary schools in Munich, Germany.

UNLABELLED: Outdoor particulate matter (PM(10)) is associated with detrimental health effects. However, individual PM(10) exposure occurs mostly indoors. We therefore compared the toxic effects of classroom, outdoor, and residential PM(10). Indoor and outdoor PM(10) was collected from six schools in Munich during teaching hours and in six homes. Particles were analyzed by scanning electron microscopy and X-ray spectroscopy (EDX). Toxicity was evaluated in human primary keratinocytes, lung epithelial cells and after metabolic activation by several human cytochromes P450. We found that PM(10) concentrations during teaching hours were 5.6-times higher than outdoors (117 ± 48 µg/m(3) vs. 21 ± 15 µg/m(3), P < 0.001). Compared to outdoors, indoor PM contained more silicate (36% of particle number), organic (29%, probably originating from human skin), and Ca-carbonate particles (12%, probably originating from paper). Outdoor PM contained more Ca-sulfate particles (38%). Indoor PM at 6 µg/cm(2) (10 µg/ml) caused toxicity in keratinocytes and in cells expressing CYP2B6 and CYP3A4. Toxicity by CYP2B6 was abolished with the reactive oxygen species scavenger N-acetylcysteine. We concluded that outdoor PM(10) and indoor PM(10) from homes were devoid of toxicity. Indoor PM(10) was elevated, chemically different and toxicologically more active than outdoor PM(10). Whether the effects translate into a significant health risk needs to be determined. Until then, we suggest better ventilation as a sensible option. PRACTICAL IMPLICATIONS: Indoor air PM(10) on an equal weight base is toxicologically more active than outdoor PM(10). In addition, indoor PM(10) concentrations are about six times higher than outdoor air. Thus, ventilation of classrooms with outdoor air will improve air quality and is likely to provide a health benefit. It is also easier than cleaning PM(10) from indoor air, which has proven to be tedious.

The allergen Bet v 1 in fractions of ambient air deviates from birch pollen counts.

BACKGROUND: Proof is lacking that pollen count is representative for allergen exposure, also because allergens were found in nonpollen-bearing fractions of ambient air. OBJECTIVE: We monitored simultaneously birch pollen and the major birch pollen allergen Bet v 1 in different size fractions of ambient air from 2004 till 2007 in Munich, Germany. METHODS: Air was sampled with a ChemVol high-volume cascade impactor equipped with stages for particulate matter (PM)>10 microm, 10 microm>PM>2.5 microm, and 2.5 microm>PM>0.12 microm. Allergen was determined with a Bet v 1-specific ELISA. Pollen count was assessed with a Burkard pollen trap. We also measured the development of allergen in pollen during ripening. RESULTS: About 93 +/- 3% of Bet v 1 was found in the PM > 10 microm fraction, the fraction containing birch pollen. We did not measure any Bet v 1 in 2.5 microm > PM > 0.12 microm. Either in Munich no allergen was in this fraction or the allergen was absorbed to diesel soot particles that also deposit in this fraction. Pollen released 115% more Bet v 1 in 2007 than in 2004. Also within 1 year, the release of allergen from the same amount of pollen varied more than 10-fold between different days. This difference was explained by a rapidly increasing expression of Bet v 1 in pollen in the week just before pollination. Depending on the day the pollen is released during ripening, its potency varies. CONCLUSION: In general, pollen count and allergen in ambient air follow the same temporal trends. However, because a 10-fold difference can exist in allergen potency of birch pollen, symptoms might be difficult to correlate with pollen counts, but perhaps better with allergen exposure.

Impact of urbanization on the proteome of birch pollen and its chemotactic activity on human granulocytes.

BACKGROUND: Epidemiologic studies reveal a dramatic increase in allergies in the last decades. Air pollution is considered to be one of the factors responsible for this augmentation. The aim of this study was to analyze the impact of urbanization on birch pollen. The birch pollen proteome was investigated in order to identify differences in protein abundance between pollen from rural and urban areas. The allergenicity of birch pollen from both areas was evaluated by assessing its chemotactic potency as well as its protein and allergen contents. METHODS: Difference gel electrophoresis (DIGE) was used to analyze the pollen proteome. The chemotactic activity of aqueous pollen extracts was determined by migration assays of human neutrophils. RESULTS: DIGE revealed 26 differences in protein spot intensity between pollen from urban and rural areas. One of these proteins was identified by de novo sequencing as the 14-3-3 protein, which resembles a stress-induced factor in other plant species. Furthermore, extracts from pollen collected in urban areas had higher chemotactic activity on human neutrophils compared to pollen from rural sites. CONCLUSIONS: The present study points to an impact of air pollution on allergen carrier proteome and release of chemotactic substances. The increment in proinflammatory substances such as pollen-associated lipid mediators might contribute to the described urban-rural gradient of allergy prevalence. Furthermore, our study suggests that allergenicity is determined by more than the sole allergen content.

Predicting the start, peak and end of the Betula pollen season in Bavaria, Germany.

Betula pollen is frequently found in the atmosphere of central and northern Europe. Betula pollen are health relevant as they cause severe allergic reactions in the population. We developed models of thermal requirements to predict start, peak and end dates of the Betula main pollen season for Bavaria (Germany). Betula pollen data of one season from 19 locations were used to train the models. Estimated dates were compared with observed dates, and the errors were spatially represented. External validation was carried out with time series datasets of 3 different locations (36years in total). RESULTS: The temperature requirements to detonate the main pollen season proved non-linear. For the start date model (error of 8,75days during external validation), daily mean temperatures above a threshold of 10°C from 28th of February onwards were the most relevant. The peak model (error of 3.58days) takes into account mean daily temperatures accumulated since the first date of the main pollen season in which the daily average temperature exceeded 11°C. The end model (error of 3.75days) takes into account all temperatures accumulated since the start of the main pollen season. CONCLUSION: These models perform predictions that enable the allergic population to better manage their disease. With the established relationship between temperatures and pollen season dates, changes in the phenological behaviour of Betula species due to climate change can be also estimated in future studies by taking into account the different climate scenarios proposed by previous climate change studies.

Pollen and spore monitoring in the world.

BACKGROUND: Ambient air quality monitoring is a governmental duty that is widely carried out in order to detect non-biological ("chemical") components in ambient air, such as particles of < 10 µm (PM10, PM2.5), ozone, sulphur dioxide, and nitrogen oxides. These monitoring networks are publicly funded and air quality data are open to the public. The situation for biological particles that have detrimental effects on health, as is the case of pollen and fungal spores, is however very different. Most pollen and spore monitoring networks are not publicly funded and data are not freely available. The information regarding which biological particle is being monitored, where and by whom, is consequently often not known, even by aerobiologists themselves. This is a considerable problem, as local pollen data are an important tool for the prevention of allergic symptoms. OBJECTIVE: The aim of this study was to review pollen monitoring stations throughout the world and to create an interactive visualization of their distribution. METHODS: The method employed to collect information was based on: (a) a review of the recent and historical bibliography related to pollen and fungal spore monitoring, and (b) personal surveys of the managers of national and regional monitoring networks. The interactive application was developed using the R programming language. RESULTS: We have created an inventory of the active pollen and spore monitoring stations in the world. There are at least 879 active pollen monitoring stations in the world, most of which are in Europe (> 500). The prevalent monitoring method is based on the Hirst principle (> 600 stations). The inventory is visualised as an interactive and on-line map. It can be searched, its appearance can be adjusted to the users' needs and it is updated regularly, as new stations or changes to those that already exist can be submitted online. CONCLUSIONS: The map shows the current situation of pollen and spore monitoring and facilitates collaboration among those individuals who are interested in pollen and spore counts. It might also help to improve the monitoring of biological particles up to the current level employed for non-biological components.

Cytochrome P4501B1 mediates induction of bone marrow cytotoxicity and preleukemia cells in mice treated with 7,12-dimethylbenz[a]anthracene.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) through many environmental pollutants, especially cigarette smoke. These chemicals cause a variety of tumors and immunotoxic effects, as a consequence of bioactivation by P-450 cytochromes to dihydrodiol epoxides. The recently identified cytochrome P4501B1 (CYP1B1) bioactivates PAHs but is also a physiological regulator, as evidenced by linkage of CYP1B1 deficiency to congenital human glaucoma. This investigation demonstrates that CYP1B1 null mice are almost completely protected from the acute bone marrow cytotoxic and preleukemic effects of the prototypic PAH 7,12-dimethylbenz[a]anthracene (DMBA). CYP1B1 null mice did not produce the appreciable amounts of bone marrow DMBA dihydrodiol epoxide DNA adducts present in wild-type mice, despite comparable hepatic inductions of the prominent PAH-metabolizing P-450 cytochrome, CYP1A1. Wild-type mice constitutively expressed low levels of bone marrow CYP1B1. These findings suggest that CYP1B1 is responsible for the formation of DMBA dihydrodiol epoxides in the bone marrow. Furthermore, this study substantiates the importance of DMBA dihydrodiol epoxide generation at the site of cancer initiation and suggests that tissue-specific constitutive CYP1B1 expression may contribute to cancer susceptibility in the human population.

Role of CYP1A2 in caffeine pharmacokinetics and metabolism: studies using mice deficient in CYP1A2.

We investigated the involvement of CYP1A2 in the pharmacokinetics and metabolism of caffeine using mice lacking its expression (CYP1A2 -/-). The half-life of caffeine elimination from blood was seven times longer in the CYP1A2 -/- than wild-type mice. The clearance was concomitantly eight times slower. No parameter that could affect the pharmacokinetics differed between CYP1A2-/-and wild-type mice such as creatinine for kidney function; alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and bilirubin for liver function; or albumin for protein binding. Other P450s CYP2A, 2B, 2C, 2EI, and 3A were also unchanged in the knockout animals. Caffeine 3-demethylated metabolites thought previously to be characteristic of CYP1A2 (especially 1-methylxanthine and I-methylurate) were also found in the urines of the CYP1A2-/-animals, although at 40% of the level found in wild-type mice. These data indicate that the clearance of caffeine in wild-type mice is primarily determined by CYP1A2.

Comparison of substrate metabolism by wild type CYP2D6 protein and a variant containing methionine, not valine, at position 374.

We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a three-fold lower turnover rate for (R)(+)-bufuralol 1'-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to alpha-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6-Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1'-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates.

Sex difference in antipyrine 3-hydroxylation. An in vivo-in vitro correlation of antipyrine metabolism in two rat strains.

Antipyrine metabolism depends on at least three isoenzymes of cytochrome P450 forming the main metabolites 3-OH-, 4-OH- and norantipyrine. We investigated to which extent antipyrine clearance and metabolite formation in vivo correlate with metabolite formation by microsomal fractions in vitro. The influence of sex was investigated in two rat strains. Antipyrine clearance in saliva was determined in 10-month-old Sprague-Dawley and Dark Agouti rats of either sex. Antipyrine and its metabolites in urine and microsomes were measured by a new HPLC method after solid phase or liquid extraction. Antipyrine clearance was 46% higher in males than in female rats. This was associated with a 40% higher urinary excretion of 3-OH-antipyrine in the male rats, the other metabolites being excreted to a similar extent. This higher production of 3-OH-antipyrine in vivo was paralleled by a higher intrinsic clearance in vitro while no sex difference in intrinsic clearance for the formation of the other metabolites was seen. The correlation between in vivo and in vitro metabolic clearance for 3-OH-antipyrine was good (r = 0.75) but unconvincing for 4-OH- (r = 0.49) and norantipyrine (r = 0.01). This could be due to further metabolism of 4-OH- and norantipyrine.

Metabolism of antipyrine in vivo in two rat models of liver cirrhosis. Its relationship to intrinsic clearance in vitro and microsomal membrane lipid composition.

Antipyrine metabolism depends on at least three isoenzymes of cytochrome P450 forming the main metabolites 3-OH-, 4-OH- and norantipyrine. We investigated to what extent antipyrine clearance and metabolite formation are impaired in two models of liver cirrhosis in the rat, namely micronodular cirrhosis induced by chronic exposure to phenobarbital/CCl4 and biliary cirrhosis induced by bile duct ligation. Salivary antipyrine clearance was decreased to a similar extent in cirrhosis induced by CCl4 and bile duct ligation (-35%). Clearance for production of 3-OH-antipyrine was decreased in both models, while 4-hydroxylation was maintained. Metabolic clearance of both 3-OH-antipyrine and 4-OH-antipyrine in vivo correlated with their clearance in vitro (r = 0.658 and r = 0.583) but not with that of norantipyrine. The microsomal cholesterol content was increased by 16% and 90% in CCl4 and bile duct-ligated cirrhotic rats (P < 0.001), respectively. Membrane fluidity, expressed as the ratio of phospholipids to cholesterol, correlated with the in vivo clearance for production of norantipyrine (r = 0.841) but not of 3-OH- or 4-OH-antipyrine, while clearance in vitro was not related to altered lipid composition. Our results demonstrate that the cytochrome P450 isoenzymes responsible for the different pathways of antipyrine metabolism are affected to different extents by cirrhosis. Alterations in intrinsic clearance explain only part of the loss of hepatocellular function. Altered lipid composition contributes to this loss of function but other factors, among them loss of hepatocytes and changes in microcirculation, could be more important determinants of the decrease in xenobiotic metabolism in cirrhosis.

A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin.

The O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC) by liver microsomes has been assessed as a method for monitoring the activity of cytochrome P450. The principle advantage of this substrate is the formation of a fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC) which can be assayed directly in the reaction medium. For rat microsomes the deethylated product was confirmed as the main metabolite, the reaction rate was linear with respect to both time and microsomal protein concentration and was independent of small changes in the added co-factors. A linear formation rate for the deethylated metabolite was also confirmed with dog and human microsomes. The intra-assay precision for rat, dog and human microsomes was 3, 5 and 4%, respectively. Hanes transformations of the dog and human data showed two phases, in contrast to a linear decline seen for the rat. Hybrid parameters for Vmax and Km, calculated from the apparently linear portions of these curves, gave interday SD for the Vmax of rat, dog and man of 2, 14 and 4%, respectively, and approximately 15% for the Km in all species. The Vmax in rat, dog and human microsomes was 1.4 +/- 0.2, 4.3 +/- 1.5 and 0.9 +/- 0.5 nmol HFC/min/nmol P450, respectively. The Km was 11.0 +/- 3.1, 67 +/- 19 and 6.8 +/- 2.5 microM, respectively. Direct evidence that at least two isoenzymes (cytochrome P450 1A2 and 2E1) metabolize EFC was obtained by experiments with competitive, suicide and immuno-inhibitors. Compared with ethoxycoumarin, the involvement of P450 2E1 in O-deethylation seemed similar in the rat. In conclusion, EFC provides a straightforward and reproducible assay for microsomal enzyme activity, requiring at most 25 pmol/mL of cytochrome P450.