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ObjectivesHealth professions education (HPE) students are often not representative of the populations they will serve. The underrepresentation of non-traditional students is problematic because diversity is essential for promoting excellence in health education and care. This study aimed to understand the perceptions of traditional and non-traditional students regarding facilitators and barriers in preparing for HPE selection procedures, and to determine the role of social networks in their decision-making and preparations to apply.MethodsA qualitative study was conducted with 26 Dutch youth who were interested in university-level HPE programmes. Semistructured interviews and sociograms were analysed using thematic analysis, adopting a constructivist approach.ResultsTwenty-six high school students participated, with traditional and non-traditional backgrounds, with and without social networks in healthcare and higher education. Two themes were constructed. First, four high-impact facilitators helped to overcome barriers to apply and in preparation for selection: access to a social network connection working or studying in healthcare, to correct information, to healthcare experience and to a social network connection in higher education. Lack of information was the main barrier while access to social network connections in healthcare was the main facilitator to overcome this barrier. However, this access was unevenly distributed. Second, access alone is not enough: the need for agency to make use of available facilitators is also essential.ConclusionsThe themes are discussed using intersectionality. Traditional students with access to facilitators develop their self-efficacy and agency within social structures that privilege them, whereas non-traditional students must develop those skills without such structures. Our findings provide recommendations for the ways in which universities can remove barriers that cause unequal opportunities to prepare for the selection of HPE programmes. Along with equitable admissions, these recommendations can help to achieve a more representative student population and subsequently a better quality of health education and care.

Introduction Using a self-determination theory framework, we investigated burnout and engagement among PhD students in medicine, and their association with motivation, work-life balance and satisfaction or frustration of their basic psychological needs. Method This cross-sectional study was conducted among PhD students at a university medical centre (n = 990) using an electronic survey on background characteristics and validated burnout, engagement, motivation and basic psychological needs questionnaires. Cluster analysis was performed on the burnout subscale scores to find subgroups within the sample which had similar profiles on burnout. Structural equation modelling was conducted on a hypothesized model of frustration of basic psychological needs and burnout. Results The response rate was 47% (n = 464). We found three clusters/subgroups which were composed of PhD students with similar burnout profiles within the cluster and different profiles between the clusters. Cluster 1 (n = 199, 47%) had low scores on burnout. Clusters 2 (n = 168, 40%) and 3 (n = 55, 13%) had moderate and high burnout scores, respectively, and were associated with low engagement scores. Cluster 3, with the highest burnout scores, was associated with the lowest motivational, engagement, needs satisfaction and work-life balance scores. We found a good fit for the “basic psychological needs frustration associated with burnout” model. Discussion The most important variables for burnout among PhD students in medicine were lack of sleep and frustration of the basic psychological needs of autonomy, competence and relatedness. These add to the factors found in the literature.

Background Concerns exist about the role of selection in the lack of diversity in health professions education (HPE). In The Netherlands, the gradual transition from weighted lottery to selection allowed for investigating the variables associated with HPE admission, and whether the representativeness of HPE students has changed. Method We designed a retrospective multi-cohort study using Statistics Netherlands microdata of all 16-year-olds on 1 October 2008, 2012, and 2015 (age cohorts, N > 600,000) and investigated whether they were eligible students for HPE programs (n > 62,000), had applied (n > 14,000), and were HPE students at age 19 (n > 7500). We used multivariable logistic regression to investigate which background variables were associated with becoming an HPE student. Results HPE students with >= 1 healthcare professional (HP) parent, >= 1 top-10% income/wealth parent, and women are overrepresented compared to all age cohorts. During hybrid lottery/selection (cohort-2008), applicants with >= 1 top-10% wealth parent and women had higher odds of admission. During 100% selection (cohort-2015) this remained the case. Additionally, applicants with >= 1 HP parent had higher odds, those with a migration background had lower odds. Conclusions Odds of admission are increasingly influenced by applicants' backgrounds. Targeted recruitment and equitable admissions procedures are required to increase matriculation of underrepresented students.

Health professions education (HPE) research is dominated by variable-centred analysis, which enables the exploration of relationships between different independent and dependent variables in a study. Although the results of such analysis are interesting, an effort to conduct a more person-centred analysis in HPE research can help us in generating a more nuanced interpretation of the data on the variables involved in teaching and learning. The added value of using person-centred analysis, next to variable-centred analysis, lies in what it can bring to the applications of the research findings in educational practice. Research findings of person-centred analysis can facilitate the development of more personalized learning or remediation pathways and customization of teaching and supervision efforts. Making the research findings more recognizable in practice can make it easier for teachers and supervisors to understand and deal with students. The aim of this article is to compare and contrast different methods that can be used for person-centred analysis and show the incremental value of such analysis in HPE research. We describe three methods for conducting person-centred analysis: cluster, latent class and Q‑sort analyses, along with their advantages and disadvantage with three concrete examples for each method from HPE research studies. Electronic supplementary material The online version of this article (10.1007/s40037-020-00633-w) contains supplementary material, which is available to authorized users.

Indoxyl sulfate (IxS), a highly albumin-bound uremic solute, accumulates in chronic kidney disease (CKD) due to reduced renal clearance. This study was designed to specifically investigate the role of human serum albumin (HSA) in IxS renal secretion via organic anion transporter 1 (OAT1) in a microfluidic system and subsequently apply quantitative translation of

Objectives To describe the design, contents, and student evaluation of an Inquiry-Based (IB) elective course on Drug discovery and preclinical drug development in an undergraduate Pharmacy curriculum. Methods The course ‘Development of New Drugs’ is an elective course for third-year Bachelor of Pharmacy and Life Science students at Utrecht University, Department of Pharmaceutical Sciences. The course is a 7.5-credit hour course dedicated to an introduction into drug discovery and preclinical development. To stimulate, challenge, and motivate students into deep learning the course is designed according to the principles of Inquiry-Based Learning (IBL). The course consists of four group assignments and one individual assignment. Results The student evaluations of three consecutive years, 2008–2010, show a high appreciation of the course (7.7±0.7 on a 10-point scale (90% respondents, n = 47)). Furthermore, students have spent on average 18.5±6.0 h of the expected 20 hours per week on the course (87% respondents, n = 45). The students are highly motivated for the course and stimulated into critical thinking and problem solving. Conclusion The course is a successful way of introducing students to preclinical Drug Discovery and Development and underpins and supports the use of IBL in Pharmacy education.

The objective of the PHAR-QA (Quality assurance in European pharmacy education and training) project was to investigate how competence-based learning could be applied to a healthcare, sectoral profession such as pharmacy. This is the first study on evaluation of competences from the pharmacists’ perspective using an improved Delphi method with a large number of respondents from all over Europe. This paper looks at the way in which hospital pharmacists rank the fundamental competences for pharmacy practice. European hospital pharmacists (n = 152) ranked 68 competences for pharmacy practice of two types (personal and patient care), arranged into 13 clusters. Results were compared to those obtained from community pharmacists (n = 258). Generally, hospital and community pharmacists rank competences in a similar way. Nevertheless, differences can be detected. The higher focus of hospital pharmacists on knowledge of the different areas of science as well as on laboratory tests reflects the idea of a hospital pharmacy specialisation. The difference is also visible in the field of drug production. This is a necessary competence in hospitals with requests for drugs for rare diseases, as well as paediatric and oncologic drugs. Hospital pharmacists give entrepreneurship a lower score, but cost-effectiveness a higher one than community pharmacists. This reflects the reality of pharmacy practice where community pharmacists have to act as entrepreneurs, and hospital pharmacists are managers staying within drug budgets. The results are discussed in the light of a “hospital pharmacy” specialisation.

Traditionally, mast cells were regarded as key cells orchestrating type I hypersensitivity responses. However, it is now recognized that mast cells are widely involved in nonallergic (non-IgE) chronic diseases. Also, in inflammatory bowel disease (IBD), a disease not associated with increased IgE concentrations, clear signs of activation of mast cells have been found. In this study, we investigated if Ig-free L chain-induced hypersensitivity-like responses through activation of mast cells could contribute to the pathophysiology of IBD. As a mast cell-dependent model for IBD, mice were skin-sensitized with dinitrofluorobenzene followed by intrarectal application of the hapten. In this murine IBD model, F991 prevented mast cell activation and also abrogated the development of diarrhea, cellular infiltration, and colonic lymphoid follicle hyperplasia. Furthermore, passive immunization with Ag-specific Ig-free L chains (IgLCs) and subsequent rectal hapten challenge elicited local mast cell activation and increased vascular permeability in the colon of mice. Clinical support is provided by the observation that serum concentrations of IgLCs of patients suffering from Crohn’s disease are greatly increased. Moreover, increased presence of IgLCs was evident in tissue specimens from colon and ileum tissue of patients with IBD. Our data suggest that IgLCs may play a role in the pathogenesis of IBD, which provides novel therapeutic means to prevent or ameliorate the adverse gastrointestinal manifestations of IBD.

Immunoglobulin-free light chains (IgLC) secreted by B lymphocytes, have been shown to mediate hypersensitivity by inducing antigen-specific mast cell activation. Although both mast cells and sensory neurons contribute to the hypersensitivity response, the role of IgLC in relation to sensory neurons is unknown. We therefore aimed to investigate the effects of IgLC on cultures of murine dorsal root ganglion (DRG) neurons. Immunohistochemistry demonstrated that IgLC and IgE could specifically bind to DRG neurons, on which the presence of FceRI, the specific receptor for IgE, was demonstrated by western blotting. Further, optical recordings with Fluo-4 showed that application of the corresponding antigen to IgLC- or IgE-sensitized DRG neurons induces a sustained increase in intracellular Ca2+ in about half of these neurons. These results show that IgLC and IgE can mediate antigen-specific responses in murine neurons. Our findings present a novel way of antigen-specific neuronal activation.

This paper presents the results of the second European Delphi round on the ranking of competences for pharmacy practice and compares these data to those of the first round already published. A comparison of the numbers of respondents, distribution by age group, country of residence, etc., shows that whilst the student population of respondents changed from Round 1 to 2, the populations of the professional groups (community, hospital and industrial pharmacists, pharmacists in other occupations and academics) were more stable. Results are given for the consensus of ranking and the scores of ranking of 50 competences for pharmacy practice. This two-stage, large-scale Delphi process harmonized and validated the Quality Assurance in European Pharmacy Education and Training (PHAR-QA) framework and ensured the adoption by the pharmacy profession of a framework proposed by the academic pharmacy community. The process of evaluation and validation of ranking of competences by the pharmacy profession is now complete, and the PHAR-QA consortium will now put forward a definitive PHAR-QA framework of competences for pharmacy practice.

Inflammatory bowel disease (IBD) describes chronic inflammatory conditions of the gastrointestinal tract, and TNF-alpha plays a pivotal role in mediating the response. The proinflammatory cytokine TNF-alpha is rapidly released by mast cells after degranulation. In the present study, we hypothesized TNF-alpha to be an important player in our recently described mast cell-dependent murine model for IBD. The effect of neutralizing anti-TNF-alpha MAb was studied on colonic hypersensitivity in mice induced by a skin application of dinitrofluorobenzene (DNFB) followed by an intrarectal challenge with dinitrobenzene sulfonic acid. Features of the colonic hypersensitivity response included diarrhea, mast cell infiltration and activation, infiltration of inflammatory cells in the colon, colonic patch hypertrophy, and increased mast cell-derived TNF-alpha levels in the colon. Anti-TNF-alpha MAb could effectively abrogate diarrhea in DNFB-sensitized mice 72 h after the challenge. The numbers of colonic patches and total tissue damage scores were reduced by anti-TNF-alpha MAb treatment in DNFB-sensitized mice 72 h after the challenge. Mast cell infiltration and activation remained unaffected by neutralizing anti-TNF-alpha MAb. Treatment with the corticosteroid dexamethasone, a frequently used therapeutic treatment in IBD, resulted in a reduction of diarrhea, cellular infiltration, and total tissue damage scores to the same extent as anti-TNF-alpha MAb. Additionally, dexamethasone treatment could also reduce total TNF-alpha levels in the colon, mast cell numbers, and mast cell activation in both vehicle- and DNFB-sensitized mice 72 h after the challenge. These findings suggest that TNF-alpha can play an instrumental role in causing inflammatory responses in the present murine model for IBD downstream from mast cell activation.

The immunological mechanisms underlying the role of mast cells in the pathogenesis of inflammatory bowel disease (IBD) are poorly defined. In this study, non-IgE mediated colonic hypersensitivity responses in BALB/c mice induced by skin sensitization with dinitrofluorobenzene (DNFB) followed by an intrarectal challenge with dinitrobenzene sulfonic acid featured as a model to study the role of mast cells in the development of IBD. Vehicle- or DNFB-sensitized mice were monitored for clinical symptoms and inflammation 72 h after dinitrobenzene sulfonic acid challenge. DNFB-sensitized mice developed diarrheic stool, increased colonic vascular permeability, hypertrophy of colonic lymphoid follicles (colonic patches), and showed cellular infiltration at the microscopic level. Increased numbers of mast cells were found in the colon of DNFB-sensitized mice located in and around colonic patches associated with elevated levels of mouse mast cell protease-1 in plasma indicating mast cell activation. Colonic patches of DNFB mice, stimulated in vitro with stem cell factor indicated that an increase in TNF-α levels in the colon is mainly mast cell originated. Finally, neutrophil infiltration was observed in the colon of DNFB-sensitized mice. Induction of this model in mast cell-deficient WBB6F1 W/Wv mice shows a profound reduction of characteristics of the colonic hypersensitivity reaction. Reconstitution with bone marrow-derived mast cells in WBB6F1 W/Wv mice fully restored the inflammatory response. This study demonstrates the importance of mast cells in the development of clinical symptoms and inflammation in the presented murine model for IBD.

The contribution of reactive nitrogen species to the development of airway hyperresponsiveness in a mouse model of allergic inflammation was investigated by the use of selective inhibitors of nitric oxide and superoxide formation. Sensitized mice, repeatedly challenged with ovalbumin showed a significant (P

Release of allergic mediators from mast cells is enhanced by very late Ag (VLA)-5-mediated interaction of these cells with fibronectin. In this report, we show that VLA-5-mediated adhesion of bone marrow-derived mast cells to fibronectin can be induced by two different pathways: first, FcεRI clustering, which depends on calmodulin activation and extracellular Ca2+, and, second, by Mn2+ stimulation, which is independent of calmodulin activation and antagonized by Ca2+. Previous studies have shown the presence of several cation-binding domains in VLA-5 that are homologous to the calcium-binding EF-hands of calmodulin. To show a role for EF-hands of different proteins in VLA-5-mediated adhesion, we used calcium-like peptides (CALP), CALP1 and CALP2, designed to bind to EF-hands based on inverted hydropathy. CALP1 and, more potently, CALP2 inhibited FcεRI-induced adhesion to fibronectin via different mechanisms. The target for the effects of CALP1 and 2 on FcεRI-induced adhesion and degranulation was intracellular and likely involved calmodulin. Interestingly only CALP2 was able to inhibit Mn2+-induced calmodulin-independent adhesion by interfering with an extracellular target, which is probably VLA-5. We conclude that CALP1 and 2 can inhibit VLA-5-mediated adhesion of mast cells to fibronectin through binding to EF-hands of multiple proteins, and that these peptides can be used as lead compounds for the development of future therapy against allergy.

Peroxynitrite (ONOO(-)) the highly reactive coupling product of nitric oxide and superoxide, has been implicated in the pathogenesis of an increasing number of (inflammatory) diseases. At present, however, selective peroxynitrite antagonizing agents with therapeutic potential are not available. Therefore, the NADPH-oxidase inhibitor apocynin (4-hydroxy-3-methoxy-acetophenone) was tested for its ability to inhibit peroxynitrite formation in vitro The murine macrophage cell-line J774A.1, stimulated with IFNgamma/LPS, was used as a model. Conversion of 123-dihydrorhodamine (123-DHR) to its oxidation product 123-rhodamine was used to measure peroxynitrite production. Stimulated peroxynitrite formation could be completely inhibited by apocynin, by the superoxide scavenger TEMPO as well as by the nitric oxide synthase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhibited superoxide and nitric oxide formation respectively as confirmed by measuring lucigenin enhanced chemiluminescence and nitrite accumulation. It is concluded that J774A.1 macrophages produce significant amounts of peroxynitrite, which is associated with nitric oxide production and NADPH-oxidase dependent superoxide formation. The NADPH-oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite formation by macrophages, which may be of future therapeutic significance in a wide range of inflammatory disorders.

Previously, it was shown that depletion and stabilization of the mucosal mast cell around the time of challenge were very effective in reducing delayed-type hypersensitivity (DTH) reactions in the small intestine of the rat. The role of mucosal mast cells in the early component of intestinal DTH reaction was further investigated in this study. In vivo small intestinal vascular leakage and serum levels of rat mast cell protease II (RMCP II) were determined within 1 h after intragastric challenge of rats that had been sensitized with dinitrobenzene 5 days before. A separate group of rats was used to study vasopermeability in isolated vascularly perfused small intestine after in vitro challenge. To investigate the effects of mast cell stabilization on the early events of the DTH reaction, doxantrazole was used. The influence of sensory nerves was studied by means of neonatal capsaicin-induced depletion of sensory neuropeptides. Within 1 h after challenge, a significant increase in vascular permeability was found in vivo as well as in vitro. This was associated with a DTH-specific increase in RMCP II in the serum, indicating mucosal mast cell activation. In addition, doxantrazole treatment and caspaicin pretreatment resulted in a significant inhibition of the DTH-induced vascular leakage and an increase in serum RMCP II. These findings are consistent with an important role for mucosal mast cells in early vascular leakage changes of intestinal DTH reactions. In addition, sensory nervous control of mucosal mast cell activation early after challenge is demonstrated.

The bradykinin-induced rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ) and the bradykinin receptor involved in this response were characterized in bovine pulmonary artery endothelial cells. It was found that bradykinin induces an intracellular biphasic Ca 2+ response, consisting of a transient peak followed by an elevated plateau phase. Both bradykinin and the bradykinin B 1 receptor agonist, des-Arg 9 -bradykinin, induced a concentration-dependent increase in [Ca 2+ ] i , but the bradykinin-induced rise was much greater. Moreover, the bradykinin-induced [Ca 2+ ] i rise could be inhibited by the bradykinin B 2 receptor antagonists, d -Arg 0 [Hyp 3 , Thi 5,8 , d -Phe 7 ]bradykinin and Hoe 140 ( d -Arg[Hyp 3 , Thi 5 , d -Tic 7 , Oic 8 ]bradykinin), but not by the bradykinin B 1 receptor antagonist, des-Arg 9 -[Leu 8 ]bradykinin. From these results it can be concluded that a bradykinin B 2 receptor is involved in this response. Furthermore, we found that the tachykinin NK 1 receptor antagonist, RP67580 ([imino 1 (methoxy-2-phenyl)-2 ethyl]-2 diphenyl 7,7 perhydroisoindolone-4 (3aR, 7aR)), and its negative enantiomer, RP68651 (2-[1-imino 2-(2 methoxy phenyl) ethyl] 7,7 diphenyl 4-perhydroisoindolone (3aS–7aS)), could inhibit the bradykinin-induced [Ca 2+ ] i response, although no functional tachykinin NK 1 receptors were found. Binding studies evidenced no binding of RP67580 or RP68651 to the bradykinin receptor. We conclude that RP67580 inhibits the bradykinin-induced rise in [Ca 2+ ] i via a bradykinin B 2 receptor-independent mechanism.

In this study, we investigated the importance of the beta 2-integrins for the development of tracheal hyperreactivity in a murine model for nonallergic asthma. The response was induced by skin sensitization with dinitrofluorobenzene (DNFB) followed by an intranasal challenge with the same hapten. Twenty-four hours after the challenge, tracheal hyperreactivity, a decrease in T cells in the blood, and increased neutrophil numbers in bronchoalveolar lavage fluid (BALF) and blood were observed. Monoclonal antibodies (mAbs) directed against the alpha-chains of LFA-1 (FD441.8) and Mac-1 (M1/70) were injected intravenously 2 h before and 2 h after the challenge. Treatment with anti-LFA-1 mAb totally inhibited the development of tracheal hyperreactivity measured 24 h after the challenge, whereas anti-Mac-1 mAb had only a partial effect on this response. The decrease in T cells in the blood, which was also evident 24 h after the challenge, was totally inhibited by treatment with anti-LFA-1, whereas anti-Mac-1 had little effect. The increase in the number of neutrophils in BALF at this time point was completely inhibited by both anti-LFA-1 and anti-Mac-1. In summary, evidence presented in this report highlights the possible importance of the adhesion molecule LFA-1 in the development of tracheal hyperreactivity. Our results suggest that LFA-1 present on T cells may play an integral role in this response.

Intestinal microvascular permeability was studied in the isolated vascularly perfused small intestine of the rat by arterial injection of tracer molecules and collection of venous samples. The injection mixture contained a rhodamine-labeled dextran and a fluorescein-labeled dextran or free fluorescein. Pharmacokinetic analysis, based on statistical moment theory, of the tracer outflow concentration-time curve and the application of either the well-stirred model (WSM) or parallel tube model (PTM) was used to assess vasopermeability. The results indicate that the experimental system cannot be considered a pure WSM or a PTM. No different intrinsic clearance (Clint,i) values were found by applying the two models: Clint,i (in ml/min) = 1.23 +/- 0.14 (radius 0.5 nm); 0.44 +/- 0.09 (radius 1.4 nm); 0.31 +/- 0.08 (radius 2.2 nm); 0.02 +/- 0.01 (radius 6.0 nm); and 0 (radius 20.8 nm). Infusion of histamine (10(-5)-10(-3) M) and destruction of the endothelium via perfusion with distilled water increased the permeability for the tracers. We have established a technique for measurement of microvascular permeability characteristics in the rat small intestine. Histamine-induced changes and destruction of the endothelium can be detected in a quantitatively reliable way.

This study was performed to establish relationships between the structure of 2,5-bis(1-aziridinyl)-1,4-benzoquinones (BABQs) bearing different substituents at the 3- and 6-position and their acute toxic effects in rat hepatocytes. The cell viability, loss of cellular glutathione (GSH+GSSG) and loss of ATP were followed during 4 h of incubation of freshly isolated hepatocytes. The toxicity of these compounds (100 microM) was predicted better by their reactivity with GSH than by their redox cycling in rat liver microsomes. The time of 50% loss of viability (LT50) correlated very well with the time of 50% depletion of ATP (AT50). LT50 could be adequately predicted by using the electronic field parameter (Ftotal) describing the electron withdrawing or donating properties for all the substituents on the quinone-nucleus. 7-(Di)halogen-substituted BABQs that all very rapidly depleted cellular glutathione showed significant differences in AT50 as well as in LT50. This suggests that alterations in ATP levels are important for explaining the differences in cytotoxicity of these compounds.

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.

The contribution of the rat small intestine to systemic and presystemic elimination of l-dopa was studied. When l-dopa was administered into the vascular perfusate, a systemic extraction ratio of 0·38 was found, the major part being decarboxylated to dopamine. The intestinal l-dopa clearance was estimated to be 171 mL min−1 kg−1. Thus, l-dopa intestinal clearance in rat represents up to at least 20% of the total body clearance. After luminal administration of l-dopa 83–88% of the administered dose was absorbed within 60 min. The total amount of l-dopa appearing in the vascular perfusate increased more than proportionally to the increase in the dose. In contrast, the amount of dopamine increased less than proportionally to the dose. As a result, the intestinal first pass appeared to be strongly dose-dependent. Since the total percentage absorbed from the lumen was independent of the administered dose and the total amount that appeared in the vascular perfusate increased linearly with the dose, the dose dependency was probably due to saturation of intestinal l-dopa decarboxylation.

Quinones can be metabolized by various routes: substitution or reductive addition with nucleophilic compounds (mainly glutathione and protein thiol groups), one-electron reduction (mainly by NADPH: cytochrome P-450 reductase) and two-electron reduction (by D,T-diaphorase). During reduction semiquinone radicals and hydroquinones are formed, which can transfer electrons to molecular oxygen, resulting in the formation of reactive oxygen intermediates and back-formation of the parent quinone (redox cycling). Reaction of semiquinones and reactive oxygen intermediates with DNA and other macromolecules can lead to acute cytotoxicity and/or to mutagenicity and carcinogenicity. The enhanced DNA-alkylating properties of certain hydroquinones are exploited in the bioreductive alkylating quinones. Acute cytotoxicity of quinones appears to be related to glutathione depletion and to interaction with mitochondria and subsequent disturbance of cellular energy homoeostasis and calcium homoeostasis. These effects can to a certain extent be predicted from the electron-withdrawing and electron-donating effects of the substituents on the quinone nucleus of the molecule. Prediction of cytostatic potential remains much more complicated, because reduction of the quinones and the reactivity of the reduction products with DNA are modulated by the prevailing oxygen tension and by the prevalence of reducing enzymes in tumour cells.

In this study the effect of metabolism of menadione (2-methyl-1,4-naphthoquinone) on ATP generation in isolated rat hepatocytes was investigated. Menadione-induced cytotoxicity correlated well with the depletion of ATP. Loss of viability lagged approximately 25 min behind the depletion of ATP. Our results suggest that depletion of ATP may be mediated by interference with glycolysis and protein breakdown, resulting in a lack of oxidizable substrates for ATP generation. (i) Menadione reduced proteolysis to 27% of control after 60 min of incubation. (ii) Increased glycogenolysis was not accompanied by accumulation of glycolytic end-products. The increased levels of glucose 6-phosphate were mainly metabolized to glucose. (iii) Menadione induced a time- and concentration-dependent inhibition of the glyceraldehyde-3-phosphate dehydrogenase activity, although no accumulation of glycolytic intermediates was found. The data presented suggest that glycolysis may be inhibited upstream of glyceraldehyde-3-phosphate dehydrogenase. (iv) Suppletion of metabolic substrates (pyruvate, oxaloacetate, and glutamine) postponed the menadione-induced ATP depletion and delayed the onset of cell killing. The protecting effect of these metabolic substrates could be reversed by atractyloside, an inhibitor of the ADP/ATP translocase. The temporary protection of metabolic substrates suggests that additional mechanisms (e.g., cofactor depletion, mitochondrial damage, enzyme inactivation) may play a role in menadione-induced ATP depletion. The present study substantiates the critical role of ATP depletion in menadione-induced cell death.

The gastro-intestinal tract is highly innervated by both intrinsic and extrinsic sensory nerves and this neuronal component is thought to play a role in local inflammatory responses. This in vivo study was designed to determine the function of substance P and the tachykinin NK1 receptor in the pathogenesis of inflammatory bowel disease by the use of the specific antagonist RP 67580. The dinitrofluorobenzene (DNFB)-induced colonic hypersensitivity model is associated with increased levels of substance P in the colon. The tachykinin NK1 receptor antagonist RP 67580 was used to investigate the role of substance P on the development of diarrhea, mast cell infiltration and activation, colonic tissue damage, hypertrophy of colonic lymphoid structures and leukocyte infiltration. The formation of watery diarrhea could completely be abrogated by treatment with RP 67580 in DNFB-sensitized animals 72 h after challenge. Antagonizing the tachykinin NK1 receptor in these animals also resulted in significantly reduced colonic patch hypertrophy, leukocyte recruitment and tissue damage. Total levels of substance P in the colon of DNFB-sensitized mice treated with the inactive enantiomer of the tachykinin NK1 receptor antagonist were significantly higher compared to DNFB-sensitized mice treated with RP 67580 72 h after challenge. Although RP 67580 was capable of reducing the total number of mast cells present in the colon, mast cell activation was not affected by this treatment. In conclusion, in this chemically-induced immunological model for inflammatory bowel disease we demonstrated an important role for tachykinin NK1 receptors, and its ligand substance P, in the development of colitis downstream from mast cell activation.

Immunoglobulin-free light chains elicit immediate hypersensitivity-like responses.Redegeld FA, van der Heijden MW, Kool M, Heijdra BM, Garssen J, Kraneveld AD, Van Loveren H, Roholl P, Saito T, Verbeek JS, Claassens J, Koster AS, Nijkamp FP.Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands. f.a.m.redegeld@pharm.uu.nlImmunoglobulin (Ig)-free light chains IgLC are present in serum and their production is augmented under pathological conditions such as multiple sclerosis, rheumatoid arthritis and neurological disorders. Until now, no (patho)physiological function has been ascribed to circulating Ig light chains. Here we show that IgLCs can confer mast cell dependent hypersensitivity in mice. Antigenic stimulation results in plasma extravasation, cutaneous swelling and mast-cell degranulation. We show that IgLCs have a crucial role in development of contact sensitivity, which could be completely prevented by a novel IgLC antagonist. Although IgE and IgG(1) are central to the induction of immediate hypersensitivity reactions, our results show that IgLCs have similar activity. IgLCs may therefore be a novel factor in the humoral immune response to antigen exposure. Our findings open new avenues in investigating the pathogenesis of autoimmune diseases and their treatments.

The toxicity of aziridinylbenzoquinones may occur by a number of mechanisms, including oxidative stress caused by redox cycling and the activation of the aziridine groups. Isolated hepatocytes were used to assess the relationship between the redox status of NADP(H) associated with oxidative stress, the level of NAD(H) closely linked with DNA repair and the cytotoxicity of three 2,5-bis(aziridinyl)-1,4-benzoquinones (BABQ). Exposure of hepatocytes to the BABQ TW13 (200 microM) and TW25 (100 microM), which are able to arylate and to redox cycle, resulted in increased intracellular NADP+ from0.3 nmol/mg protein to 1.5 nmol/mg protein within 60 min. The increase in intracellular NADP+ was followed by the onset of cell death by 180 min. In contrast, exposure to lower concentrations of TW13 (100 microM), TW25 (50 microM) and carboquone (100-200 microM) (which neither arylates nor redox cycles via a one-electron reduction) resulted in a less pronounced (1.0 nmol/mg) increase in NADP+ and there was no evidence of cell death within the 180 min incubation. BABQ had a concentration dependent effect on intracellular NAD+. Exposure of hepatocytes to TW13 (200 microM) and TW25 (100 microM) resulted in a decrease in intracellular NAD+ from2.7 to1.0 nmol/mg protein within 60 min. At concentrations of the BABQ where the level of NAD+ remained1.0 nmol/mg protein after 30 min, the hepatocytes remained viable at 180 min. These changes in intracellular pyridine nucleotides suggests two mechanisms may be involved in BABQ cytotoxicity. At high concentrations, aziridinylbenzoquines may cause cytotoxicity via oxidative stress following redox cycling. At lower concentrations however, the predominant pyridine nucleotide change is a prolonged depletion of NAD+, suggesting extensive DNA damage which may lead to delayed cell death.

1. This study investigates the effects of capsaicin-induced depletion of sensory neuropeptides and of neurokinin1 (NK1) receptor blockade on delayed-type hypersensitivity (DTH)-induced changes of vascular permeability in the small intestine of the mouse. 2. The DTH reaction in the small intestine was elicited by dinitrofluorobenzene (DNFB)-contact sensitization followed by oral dinitrobenzene sulphonic acid (DNBS) challenge. To assess vascular leakage the accumulation of the plasma marker, Evans blue (EB), was measured 2, 24 and 48 h after the challenge. 3. The small intestinal DTH reaction was characterized by a significant increase in vascular permeability 24 h after the challenge of previously sensitized mice when compared to vehicle-sensitized mice (P0.05, ANOVA). Capsaicin-induced depletion of sensory neuropeptides, two weeks before the sensitization, completely inhibited the DTH-induced increase in small intestinal vascular permeability at 24 h (P0.05, ANOVA). Vehicle/control: 108.2 +/- 8.6 ng EB mg-1 dry weight; vehicle/DTH 207.8 +/- 25.1 ng EB mg-1 dry weight; capsaicin/control: 65.8 +/- 11.9 ng EB mg-1 dry weight; capsaicin/DTH: 84.3 +/- 7.6 ng EB mg-1 dry weight. 4. The tachykinins, substance P and neurokinin A (1.5 to 50 x 10(-11) mol per mouse, i.v.), induced an increase in vascular leakage in the small intestine of naive mice. The specific NK1 receptor antagonist, RP67580 (10(-9) mol per mouse, i.v.) was the most effective in reducing the substance P-induced plasma extravasation when compared with other NK receptor antagonists, FK224 and FK888. 5. Treatment of DNFB-sensitized mice with RP67580 (10-9 mol per mouse, i.v.) immediately before and 1 h after the DNBS challenge resulted in a significant reduction of the DTH-induced increase in vascular permeability at 24 h (vehicle/control: 107.5 +/- 8.8 ng EB mg-1 dry weight; RP67580/control:95.4 +/- 5.4 ng EB mg-1 dry weight; vehicle/DTH: 206.6 +/- 22.6 ng EB mg-1 dry weight; RP67580/DTH:132.6 +/- 13.6 ng EB mg-1 dry weight, P0.05, ANOVA).6. These results suggest that sensory nerves are involved in the development of small intestinal DTH reactions in the mouse. NK1 receptors could play an important role in the initiation of the DTH-induced changes in vascular leakage.

The influence of variation of perfusion flow rate on the renal clearance of p-aminohippuric acid and 1-naphthol was studied with an isolated perfused rat kidney preparation. Kidney functions were well maintained at low perfusion flow rates by the use of a fluorocarbon emulsion to increase the oxygen capacity of the perfusion buffer. Renal extraction of p-aminohippuric acid decreased with increasing perfusion flow. Our data show that at high perfusion flow rates maximal extractable perfusion flow forms only a small part of the total perfusion flow. 1-Naphthol is rapidly metabolized to its glucuronide and sulfate conjugate in the isolated perfused rat kidney. Using PAH as a marker for the maximal extractable perfusion flow, 1-naphthol could be regarded as a high-extraction compound even at high perfusion flow rates. Our results suggest that p-aminohippuric acid clearance, rather than total perfusion flow rate, should be used as the measure of maximal extractable blood flow for the estimation of extraction ratio in the isolated perfused kidney of compounds excreted or metabolized by the proximal tubules.

The biochemical mechanism of cytotoxicity, induced by the quinoid compound 2-methyl 1,4-naphthoquinone (menadione), was investigated in hepatocytes freshly isolated from fasted and fed rats. Hepatocytes from fasted rats were significantly more vulnerable to the toxicity of menadione than hepatocytes from fed rats. Menadione (150 μ m ) induced a 50% loss of viability of cells (LT 50 ) from fasted rats after 55 min of incubation, whereas a LT 50 of 80 min was observed after exposure of hepatocytes from fed rats to menadione. Glutathione and NADPH levels were rapidly depleted by menadione metabolism. This depletion was sustained during the incubation period. No significant differences were found in the time course and extent of the menadione-induced glutathione and NADPH depletion in hepatocytes of both nutritional states. Menadione also affected the energy status of the hepatocytes. The ATP content of cells from fasted rats decreased to 50% (AT 50 ) within 18 min of exposure to menadione, whereas a 50% loss of ATP content of hepatocytes from fed rats was reached at 65 min. In contrast to depletion of glutathione and NADPH, the time course and extent of menadione-induced ATP depletion correlated well with the time of onset and rate of cell killing. Our results suggest that menadione metabolism may interfere with both mitochondrial and glycolytic ATP production. Depletion of ATP might be a critical step in menadione-induced cytotoxicity.

Glucuronidation and sulphation of 1-naphthol and 7-hydroxycoumarin was studied in isolated rat intestinal epithelial cells and in microsomes prepared from these cells. In the isolated cells formation of 1-naphthol sulphate could not be detected. Sulphate conjugates of 7-hydroxycoumarin constitute a minor portion of total conjugates formed. Maximum glucuronidation rates for 1-naphthol and 7-hydroxycoumarin do not differ significantly from each other (approximately 12.5 nmoles/min X g intestine). The intestinal microsomal UDP-glucuronosyltransferase, prepared from isolated cells, could be activated in vitro by Triton X-100 and MgCl2. Activation increased both Kappm and Vmax for 1-naphthol; Kappm for UDP-glucuronic acid was decreased by activation with MgCl2 but increased again by further addition of Triton X-100. In fully activated microsomes Kappm for 1 naphthol was 69.7 +/- 13.9 microM and Vmax was 70.0 +/- 3.9 nmoles/min X mg microsomal protein; Kappm for UDP-glucuronic acid was 0.67 +/- 0.06 mM. The glucuronidation rate (expressed as nmoles/min X g intestine) in microsomes is substantially higher than in isolated cells. It appears that glucuronidation in intact cells is limited by factors other than the extracellular substrate concn. Both cellular uptake of the substrate and availability of UDP-glucuronic acid can play a significant role. It is concluded that isolated mucosal cells are more suitable for predicting intestinal first-pass metabolism of phenolic xenobiotics than intestinal microsomes, because cellular substrate uptake and cosubstrate availability appear to be important determinants of the maximum glucuronidation rate.

Glucuronidation of various substrates in hepatic, intestinal and renal microsomes of control, phenobarbital (PB), 3-methylcholanthrene (3MC) and Aroclor-1254 (A1254) pretreated rats was investigated. UDPGT activities tested could be divided in four groups on the basis of their tissue distribution and induction by PB or 3MC in liver microsomes. GT1 activities (1-naphthol, benzo(a)pyrene-3,6-quinol) are induced by 3MC in liver microsomes and are present in all tissues investigated. GT2 activities (morphine, 4-hydroxybipheynl) are induced by PB in liver microsomes and appear to be restricted to the liver and the intestine. UDPGT activity towards bilirubin, although induced by PB, can be detected in hepatic, intestinal and renal microsomes. UDPGT activity towards fenoterol is restricted to the liver and intestine and is not induced by PB, 3MC or A1254. The presence of inducible immunoreactive UDPGT isoenzymes in microsomes of liver, intestine and kidney of control and induced rats was demonstrated by immunoblot analysis using rabbit anti-rat liver-GT1 antibodies. Induction of both 54 and 56 kDa polypeptides in hepatitis, intestinal and renal microsomes by 3MC or A1254 was observed. Purification of UDPGT (1-naphthol as substrate) from intestinal microsomes to apparent homogeneity yielded a polypeptide with an apparent molecular weight of 54-56 kDa. The results indicate that 54 and 56 kDa UDPGT polypeptides are the major A1254 inducible isoenzymes in intestinal and renal microsomes. An increase in immunoreactive protein is correlated with a biochemically measurable increase in glucuronidation capacity for GT1 substrates.

Two cell isolation procedures, i.e. a scraping/collagenase-treatment and a new vibration procedure in EDTA containing medium, were used to isolate intestinal epithelial cells. In both cell populations the metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was studied. Moreover, the time course and extent of induction of both steps in the biotransformation were investigated after oral 3-methylcholanthrene pretreatment of the rats. Twenty four hours after 3-methylcholanthrene pretreatment (20 mg kg-1) monooxygenase activity was induced about 6-fold and 2.5-fold when studied with cells of the vibratory and enzymic procedures, respectively. Control 7-ethoxycoumarin deethylase activity and 7-hydroxycoumarin glucuronidation were about the same when comparing both methods for cell-isolation. The formation of glucuronides in cells (both methods) is significantly lowered by 3-MC pretreatment, while sulphation remains unaffected. Results indicate that using enzymic treatment of mucosal scrapings, cell-populations are obtained containing relatively more differentiated (tip) cells. A number of advantages of the new (vibration) method are: better recovery, viability and reproducibility.

Several 2,5-bis(1-aziridinyl)-1,4-benzoquinones (BABQs) can be activated to alkylating species by reduction of the quinone moiety. On the other hand, cytotoxicity of these compounds can be induced by redox cycling. A series of BABQs and their methylated analogues (BMABQs) with different substituents at the 3- and 6-position was synthesized in order to investigate the influence of the substituents on the reduction of the quinone moiety and on the generation of superoxide anion radicals with rat liver microsomes. Superoxide anion production (SAP) ranged from 3.7 +/- 0.1 to 742 +/- 74 nmoles/min/mg protein with quinone concentrations of 10 nmoles/ml. NADPH-oxidation was measured under the same conditions and it correlated well (r = 0.88, P less than 0.001) with SAP. It ranged from 1.4 +/- 0.2 to 494 +/- 60 nmoles/min/mg protein. SAP for 22 B(M) ABQs showed a good correlation with the summated electronic substituent constant sigma para.total (r = 0.86, P less than 0.001). It can be concluded that superoxide anion production by 22 B(M)ABQs in rat liver microsomes can be predicted from structural features of the compounds.

The kinetic properties of the rat intestinal microsomal 1-naphthol: UDPglucuronosyltransferase (EC 2.4.1.17) were investigated in fully activated microsomes prepared from isolated mucosal cells. The enzyme appeared to follow an ordered sequential bireactant mechanism in which 1-naphthol and UDP-glucuronic acid (UDPGlcUA) are the first and second binding substrates and UDP and 1-naphthol glucuronide the first and second products, respectively. Bisubstrate kinetic analysis yielded the following kinetic fconstants: V max = 102 ± 6 nmol/min per mg microsomal protein, K m (UDPGlcUA) = 1.26 ± 0.10 mM, K m (1-naphtol) = 96 ± 10 μ M and K i (1-naphthol) = 25 ± 7 μ M. The rapid equilibrium random or ordered bireactant mechanisms, as well as the iso -Theorell-Chance mechanism, could be excluded by endproduct inhibition studies with UDP.UDP- N -acetylglucosamine (UDPGlcNAc), usually found to be an activator of UDP glucuronosyltransferase in liver microsomes, acted as a full competitive inhibitor towards UDPGlcUA in rat intestinal microsomes. With regard to 1-naphthol UDPGlcNAc exhibited a dual effect: both inhibition and activation was observed. The effect of activation by MgCl 2 and Triton X-100 on the kinetic constants and the inhibition patterns of UDP and UDPGlcNAc were investigated. The results obtained suggest that latency in rat intestinal microsomes may be due to endproduct inhibition by UDP. This endproduct inhibition could be abolished by in vitro treatment with MgCl 2 and Triton X-100.

UDP-glucuronyltransferase is one of the major enzymes responsible for phase II metabolism of xenobiotics [1]. Although the hepatic UDPGT seems to be of paramount importance for in vivo glucuronidation of xenobiotics, extrahepatic sites can also contribute to the total conjugative capacity [2]. The study of conjugation-reactions in the intestinal wall is particularly interesting because bioavailability of orally administered drugs can be affected to a large extent [3–5]. Predictions of the in vivo glucuronidation-capacity can be made by investigating microsomal preparations [1, 6, 7]. Since the microsomal UDPGT can be activated during isolation or in vitro -incubation, it is important to know if and to what extent the enzyme is activated in order to make valid extrapolations [1]. In contrast with the hepatic UDPGT, the intestinal UDPGT is considered to be inactivatable in vitro or, conversely, to be fully activated during the isolation procedure [1, 6–8]. We found, however, that the microsomal UDPGT, prepared from rat intestinal mucosal cells, could be activated in vitro by both the nonionic detergent Triton-X100 and UDPNAG. Some further experiments made clear that there is a qualitative similarity of the intestinal and hepatic microsomal UDPGT from the rat, of which evidence will be given below.

Rat intestinal epithelial cells were isolated by EDTA-chelation, combined with gentle shaking (modified Weiser procedure) or with strong longitudinal vibration (Harrison/Webster procedure). Both methods yield large numbers of viable cells and are relatively easy to use. Electronmicroscopical and biochemical data indicate that cell fractions from different levels of the villous region can be obtained only by the modified Weiser procedure. When strong mechanical forces are involved (Harrison-Webster procedure) the villus epithelium is released according to an all-or-nothing process. The biotransformational capacity of cell fractions, obtained from different levels of the villi by the modified Weiser procedure, was investigated. It was shown that the rate of metabolism of 7-ethoxycoumarin and 1-naphthol was substantially higher in lower villous cells than in cells isolated from the upper villous region. O-Deethylation of 7-ethoxycoumarin decreases from 145 +/- 13 pmole/min mg cell protein (72 +/- 4% conjugated) in lower villous cells to 62 +/- 12 pmole/min mg cell protein (37 +/- 6% conjugated) in tip cells. Glucuronidation of 1-naphthol decreased from 495 +/- 23 pmole/min mg cell protein (lower villous cells) to 137 +/- 13 pmole/min mg cell protein (tip cells).

Using the isolated vascularly fluorocarbon emulsion perfused rat small intestine some factors which determine the extent of the intestinal glucuronidation of 1-naphthol to 1-naphthol-beta-D-glucuronide were studied. Increasing the luminal 1-naphthol concentration resulted in a concomitant increase in the 1-naphthol appearance in the vascular perfusate. In contrast, the total appearance of 1-naphthol-beta-D-glucuronide increased less than proportional to the increase in the luminal 1-naphthol concentration. About 88% of the total amount of 1-naphthol-beta-D-glucuronide excreted was released into the vascular perfusate. The capacity-limited intestinal glucuronide efflux is most likely due to saturation of the excretory mechanism for 1-naphthol-beta-D-glucuronide. Decreasing the vascular flow rate influenced both the appearance of 1-naphthol and 1-naphthol-beta-D-glucuronide in the vascular perfusate, whereas the appearance of 1-naphthol-beta-D-glucuronide in the luminal perfusate was essentially flow-independent. A noradrenaline-induced change in the haemodynamic state of the vascular bed (with the total flow kept constant) resulted in a marked decrease in the naphthol vascular concentration. The vascular 1-naphthol-beta-D-glucuronide concentration was only slightly affected. These results indicate that changes in blood flow and blood flow distribution within the intestinal wall can affect the extent of presystemic intestinal metabolism by interfering with the absorption of the parent compound and the efflux of formed conjugates. These parameters can be of paramount importance for causing variable intestinal first-pass effects of drugs in vivo.

In order to clarify the pharmacological properties of some constituents of the essential oil of VALERIANA OFFICINALIS L. s. l. four pure compounds (valerenal, valerenic acid, valeranone and isoeugenyl-isovalerate), a hydrocarbon fraction, an oxygen fraction and the essential oil itself were submitted to a screening test. This test is an elaboration of the screening test of C AMPBELL and R ICHTER [11] to which several symptoms mentioned by I RWIN [12] and some other features have been added. From the results of this so-called Syndrome test valerenal and valerenic acid, which show some structural correspondence with the valepotriates, are pharmacologically active at the lowest dose levels. Together with valeranone, both compounds are likely to play a significant role in the depressive action of the essential oil whereas the activity of isoeugenyl-isovalerate is of relatively minor importance.

Drug metabolic capacity in liver microsomes of 14 rat inbred strains was investigated. Cytochrome P-450 content as well as the following enzyme activities were measured: NADPH cyt. c(P-450) reductase (Red.), aminopyrine N-demethylase (APDM), ethoxycoumarin O-deethylase (ECOD), 1-naphthol: UDP-glucuronosyltransferase (NGT) and hydrolysis of acetylsalicylic acid (ASA; measured at pH 5.5 and pH 7.4). All enzymes measured were found to exhibit statistically significant inter-strain differences. In males the enzyme activities varied over a 7.3-fold (ECOD) to 1.4-fold (cytochrome P-450) range. Other inter-strain differences were generally larger than 2-fold: ASA-hydrolysis at pH 5.5 and 7.4 (3.9- and 3.3-fold variation, respectively), NGT and Red. (2.1-fold variation) and APDM (1.8-fold variation). In females similar, but somewhat smaller inter-strain differences were observed. Correlations between different enzyme activities were generally poor (correlation coefficients r less than 0.7). An exception was the correlation between ASA-hydrolysis at pH 5.5 and pH 7.4 (r = 0.79). We conclude that ASA hydrolysis at pH 5.5 and 7.4 is mediated by the same enzyme or by coregulated enzymes and that all other activities are mediated by different or differentially regulated enzymes. Based on analysis of variance and subsequent inter-strain comparisons, all strains appear to express a unique profile of liver microsomal drug metabolism. No two strains are identical with respect to all activities measured. We suggest that differences between inbred rat strains and particularly the difference in balance between different enzymes in various strains can be used advantageously in pharmacological and toxicological experiments.

The glucoronidation of fenoterol (Berotec®, Partusisten®) in isolated rat hepatocytes and enterocytes was investigated. Two different glucuronides, fenoterol para -glucuronide and fenoterol meta -glucuronide, were formed in proportions, that were constant over the concentration range investigated (0–1 mM). The fraction of para -glucuronide formed was 0.40 ± 0.01 for hepatocytes and 0.54 ± 0.01 for enterocytes. Fenoterol consists of a racemic mixture of SS ′ (+)fenoterol and RR′ (−)fenoterol. The maximum glucuronidation rate of the (−)enantiomer ( V max = 3.6 ± 0.3 nmol/min/mg in hepatic microsomes and 3.4 ± 0.1 nmol/min/mg in intestinal microsomes) is significantly lower than the same values of the (+)isomer ( V max = 6.7 ± 0.8 nmol/min/mg in hepatic microsomes and 5.8 ± 0.4 nmol/min/mg in intestinal microsomes). K m app -values for the (−)enantiomer were lower than for the (+)enatiomer. Similar, but less pronounced, differences in V max were observed in isolated cells: V max = 148 ± 13 and 372 ± 50 pmol/min/mg [(−)fenoterol in hepatocytes and enterocytes], V max = 173 ± 12 and 444 ± 57 pmol/min/mg [(+)fenoterol in hepatocytes and enterocytes]. Calculation of intrinsic metabolic clearance (Cl int = V max /K m app ) from the cellular data suggests that the (+)enantiomer may be more efficiently eliminated by liver metabolism in vivo than the (−)enantiomer. This can result in stereoselective first-pass metabolism of the fenoterol enantiomers.

Using an isolated vasculary perfused rat small intestine we studied the role of luminal flow rate and intraluminal binding on the absorption of 1-naphthol (1-N) and the intestinal metabolism of 1-N to 1-naphthol-beta-D-glucuronide (1-NG). Raising the luminal perfusion rate resulted in a decrease in the luminal 1-N extraction ratio and an increase in the luminal 1-N clearance Cllum. The dependency of Cllum on flow rate appeared to conform to a convective diffusion model. A differential susceptibility of 1-N absorption and the total 1-NG appearance to the luminal flow rate resulted in a flow-dependent first-pass effect of 1-N. Next, the effect of intraluminal binding on 1-N disposition was studied in experiments in which albumin was added to the luminal perfusion fluid. The unbound concentration, as the driving force for the uptake of 1-N, seems not to be rate-limiting for the appearance of 1-NG. The total appearance of 1-NG in the presence of albumin was greater than would be anticipated from the free concentration of 1-N. As a result the extent of presystemic extraction increased with increasing albumin concentration. The precise mechanisms responsible for the phenomenona are not entirely clear. Consideration of the heterogeneity in the glucuronidation capacity along the rat small intestine and along the crypt-villus axis can help to explain the obtained results.