Drug monitoring during ciprofloxacin prophylaxis of allogeneic stem cell transplant patients: associations with bacterial infections through a monocentric observational prospective study.

BACKGROUND: Bacterial infection ranks amongst the most common causes of morbidity and mortality in patients undergoing allogeneic haematopoietic stem cell transplantation (alloHSCT). Although ciprofloxacin (CIP) prophylaxis is recommended, information on serum levels and clinical course is lacking. AIM: To investigate relationships between CIP level and failure of prophylaxis, particularly in terms of whether different pharmacokinetic (PK) indices [area under the concentration-time curve (AUC0-24h) vs single time samples] correlate differently with the outcome. METHODS: This prospective observational monocentric study was conducted at a 1500-bed teaching hospital (March 2018-March 2019), including 63 adult patients with alloHSCT receiving CIP prophylaxis. Blood samples were drawn at three sampling times (1, 6 and 12 h post-administration), twice per week, and measured via high performance liquid chromatography. The onset of febrile episodes (FEBs) indicated suspected failure of CIP prophylaxis. Positive blood cultures [bloodstream infection (BSI)] indicated confirmed failure of prophylaxis. FINDINGS: Seven of 63 patients died without significant differences in their average CIP levels compared with survivors, with patients experiencing FEBs (54/63) displaying a 13% [95% confidence interval (CI) 4-22%] lower probability of survival. In total, 225 sets of three values (triplets) were obtained from 58 primary CIP episodes. Triplets preceding BSI with Gram-negative bacteria (GNB-BSI) showed lower AUC0-24h on average, but similar single time sample indices. An AUC0-24h of ≤21.61 mgh/L resulted in four-fold higher odds of GNB-BSI (adjusted odds ratio 3.96, 95% CI 1.21-13.00). These results were independent of the administration route, patient demographics or sampling protocol deviations, indicating reduced CIP exposure upon GNB-BSI events. CONCLUSION: Monitoring CIP levels, using multiple sampling times, may be useful to reduce alloHSCT-associated bacterial infections. Further analysis is needed to investigate causality.

Exploring the overlap between alopecia areata and major depressive disorder: Epidemiological and genetic perspectives.

BACKGROUND: Research suggests that Alopecia areata (AA) and Major Depressive Disorder (MDD) show substantial comorbidity. To date, no study has investigated the hypothesis that this is attributable to shared genetic aetiology. OBJECTIVES: To investigate AA-MDD comorbidity on the epidemiological and molecular genetic levels. METHODS: First, epidemiological analyses were performed using data from a cohort of adult German health insurance beneficiaries (n = 1.855 million) to determine the population-based prevalence of AA-MDD comorbidity. Second, analyses were performed to determine the prevalence of MDD in a clinical AA case-control sample with data on psychiatric phenotypes, stratifying for demographic factors to identify possible contributing factors to AA-MDD comorbidity. Third, the genetic overlap between AA and MDD was investigated using a polygenic risk score (PRS) approach and linkage disequilibrium score (LDSC) regression. For PRS, summary statistics from a large MDD GWAS meta-analysis (PGC-MD2) were used as the training sample, while a Central European AA cohort, including the above-mentioned AA patients, and an independent replication US-AA cohort were used as target samples. LDSC was performed using summary statistics of PGC-MD2 and the largest AA meta-analysis to date. RESULTS: High levels of AA-MDD comorbidity were reported in the population-based (MDD in 24% of AA patients), and clinical samples (MDD in 44% of AA patients). MDD-PRS explained a modest proportion of variance in AA case-control status (R2  = 1%). This signal was limited to the major histocompatibility complex (MHC) region on chromosome 6. LDSC regression (excluding MHC) revealed no significant genetic correlation between AA and MDD. CONCLUSIONS: As in previous research, AA patients showed an increased prevalence of MDD. The present analyses suggest that genetic overlap may be confined to the MHC region, which is implicated in immune function. More detailed investigation is required to refine understanding of how the MHC is involved in the development of AA and MDD comorbidity.

Epigenetic signatures in antidepressant treatment response: a methylome-wide association study in the EMC trial.

Although the currently available antidepressants are well established in the treatment of the major depressive disorder (MDD), there is strong variability in the response of individual patients. Reliable predictors to guide treatment decisions before or in an early stage of treatment are needed. DNA-methylation has been proven a useful biomarker in different clinical conditions, but its importance for mechanisms of antidepressant response has not yet been determined. 80 MDD patients were selected out of >500 participants from the Early Medication Change (EMC) cohort with available genetic material based on their antidepressant response after four weeks and stratified into clear responders and age- and sex-matched non-responders (N = 40, each). Early improvement after two weeks was analyzed as a secondary outcome. DNA-methylation was determined using the Illumina EPIC BeadChip. Epigenome-wide association studies were performed and differentially methylated regions (DMRs) identified using the comb-p algorithm. Enrichment was tested for hallmark gene-sets and in genome-wide association studies of depression and antidepressant response. No epigenome-wide significant differentially methylated positions were found for treatment response or early improvement. Twenty DMRs were associated with response; the strongest in an enhancer region in SORBS2, which has been related to cardiovascular diseases and type II diabetes. Another DMR was located in CYP2C18, a gene previously linked to antidepressant response. Results pointed towards differential methylation in genes associated with cardiac function, neuroticism, and depression. Linking differential methylation to antidepressant treatment response is an emerging topic and represents a step towards personalized medicine, potentially facilitating the prediction of patients' response before treatment.

Titanium wear from magnetically controlled growing rods (MCGRs) for the treatment of spinal deformities in children.

Magnetically controlled growing rods (MCGRs) are an effective treatment method for early-onset scoliosis (EOS). In recent years, increasing titanium wear was observed in tissue adjacent to implants and in blood samples of these patients. This study aims to investigate the potential correlation between amount of metal loss and titanium levels in blood during MCGR treatment as well as influencing factors for metal wear. In total, 44 MCGRs (n = 23 patients) were retrieved after an average of 2.6 years of implantation and analyzed using a tactile measurement instrument and subsequent metal loss calculation. Titanium plasma levels (n = 23) were obtained using inductively coupled plasma-mass spectrometry (ICP-MS). The correlation of both parameters as well as influencing factors were analyzed. Titanium abrasion on MCGRs was observed in the majority of implants. There was no correlation of metal implant wear or titanium plasma values to the duration of MCGR implantation time, number of external lengthening procedures, patient's ambulatory status, gender, weight or height. Material loss on the MCGRs showed a positive correlation to titanium blood plasma values. The present study is one of the first studies to analyze retrieved MCGRs using high-precision metrological techniques and compare these results with ICP-MS analyses determining blood titanium values.

Prenatal maternal stress is associated with lower cortisol and cortisone levels in the first morning urine of 45-month-old children.

Prenatal stress (PS) has been related to altered hypothalamic-pituitary-adrenal (HPA) axis activity later in life. So far, studies in children assessing HPA axis functioning have focused on salivary cortisol, reflecting daytime activity. The present work is part of a prospective study and aims to extend knowledge about the association between PS and HPA axis regulation in children. To do so, we investigated cortisol, cortisone, and the ratio cortisone/(cortisone + cortisol) in the first morning urine of 45-month-old children in relation to several measures of maternal stress during pregnancy. Urinary cortisol and cortisone were measured by online turbulent flow chromatography coupled with high performance liquid chromatography-tandem mass spectrometry. PS was defined as: perceived stress for aim 1 (Perceived Stress Scale; n = 280); presence of self-reported (n = 371) and expert-rated psychopathology for aim 2 (Mini International Neuropsychiatric Interview; n = 281); continuous measures of anxiety and depression for exploratory aim 3 (State-Trait Anxiety Inventory and Edinburgh Postnatal Depression Scale; n = 280). Aim 1: Perceived maternal PS showed negative associations with cortisol and cortisone levels. Aim 2: The presence of expert-rated maternal psychopathology was associated with reduced morning cortisone. Aim 3: Continuous measures of anxiety and depression showed negative associations with cortisol and cortisone levels. After correcting for multiple testing, perceived maternal PS (aim 1) and prenatal level of anxiety (aim 3) were significant predictors of children's urinary cortisol and cortisone in the morning (and, in the case of cortisone, also prenatal level of depression). The ratio cortisone/(cortisone + cortisol) as a global marker for the balance between the enzymes metabolizing cortisol to cortisone and vice versa (11ß-hydroxysteroid dehydrogenases type 1 and 2; 11ß-HSD1 and 2) was not associated with any measure of maternal PS (aims 1-3). The present study provides insight into possible programming effects of PS on nocturnal HPA axis activity and a proxy of 11ß-HSD in a large sample. The results suggest that the nocturnal rate of cortisol production is lower in children exposed to PS, but do not support the hypothesis of divergent 11ß-HSD activity.

Stress reactivity in preschool-aged children: Evaluation of a social stress paradigm and investigation of the impact of prenatal maternal stress.

Prenatal maternal stress is an established risk factor for somatic and psychological health of the offspring. A dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in offspring has been suggested as an important mechanism. However, the impact of prenatal stress on stress reactivity in preschool-aged children is not yet well understood. This is partly due to the fact that for this age group there is no stress test as well established as for older children and adults. In the present work a previously published stress test (Kryski et al., 2011) was evaluated in a large sample of 45-month-old children (n = 339). Furthermore, the relation between measures of prenatal maternal stress and cortisol reactivity was investigated. Prenatal stress was defined as psychopathology (self-report available for n = 339; expert-rating available for a subsample of n = 246) and perceived stress (n = 244) during pregnancy. The stress paradigm elicited significant increases in salivary cortisol 30 and 40 min after the test, and 60.8% of the children were classified as responders. Lower cortisol levels after the stress test were observed in the group of children with prenatal stress defined as maternal psychopathology (both self-reported and expert-rated). Maternal perceived stress as a continuous measure was not significantly associated with cortisol levels. However, when comparing children in the highest quartile of maternal perceived stress to all other children, significantly lower cortisol values were observed in the prenatally stressed group. The present study confirms the paradigm by Kryski et al. as an effective stress test for preschool-aged children. Moreover, it provides further evidence that prenatal stress impacts HPA axis reactivity. Future studies should target the timing, nature, and intensity of prenatal stressors and their effect on the stress response in offspring at different developmental stages.

Endogenous cortisol in keratinized matrices: Systematic determination of baseline cortisol levels in hair and the influence of sex, age and hair color.

The measurement of hair cortisol is increasingly used to measure long-term cumulative cortisol levels and investigate its role as an important stress mediator. In this study a comparative statistical analysis of five independent studies (all analyzed in our laboratory) was performed to investigate baseline ranges of cortisol values in hair and evaluate potential influences of sex, age and hair color. Cortisol concentrations in hair of 554 subjects were measured and a comparative statistical analysis was performed. The analysis showed that cortisol levels significantly differ depending on age. The toddler group (7 months (0.6 years) to 3 years) showed significantly higher values (median 10pg/mg, p-value<0.0001, d=0.78) than the adolescent group. The adolescent groups showed significantly lower (p-value<0.0001, d=0.58 and p<0.0001, d=0.13) values (median 2.4pg/mg and 2.8pg/mg) than the adult group (median 5.8pg/mg). Furthermore, in the adult group men showed significantly higher cortisol values than women (p-value<0.05, d=0.17). This effect could not be seen in the adolescent group. Black hair showed higher cortisol concentrations than blond hair (p-value<0.0001, d=1.3). In addition, two rounds of interlaboratory comparisons for hair cortisol samples between four laboratories revealed very consistent results. Our results demonstrate that baseline cortisol levels are generally low in hair thus making a standardized and well-elaborated analytical method indispensable for accurate determination. Age-dependent normative baseline cortisol levels (toddlers, adolescents and adults) are highly recommended based on the comparative analysis comprising five independent studies.

Genome-wide association study of borderline personality disorder reveals genetic overlap with bipolar disorder, major depression and schizophrenia.

Borderline personality disorder (BOR) is determined by environmental and genetic factors, and characterized by affective instability and impulsivity, diagnostic symptoms also observed in manic phases of bipolar disorder (BIP). Up to 20% of BIP patients show comorbidity with BOR. This report describes the first case-control genome-wide association study (GWAS) of BOR, performed in one of the largest BOR patient samples worldwide. The focus of our analysis was (i) to detect genes and gene sets involved in BOR and (ii) to investigate the genetic overlap with BIP. As there is considerable genetic overlap between BIP, major depression (MDD) and schizophrenia (SCZ) and a high comorbidity of BOR and MDD, we also analyzed the genetic overlap of BOR with SCZ and MDD. GWAS, gene-based tests and gene-set analyses were performed in 998 BOR patients and 1545 controls. Linkage disequilibrium score regression was used to detect the genetic overlap between BOR and these disorders. Single marker analysis revealed no significant association after correction for multiple testing. Gene-based analysis yielded two significant genes: DPYD (P=4.42 × 10-7) and PKP4 (P=8.67 × 10-7); and gene-set analysis yielded a significant finding for exocytosis (GO:0006887, PFDR=0.019; FDR, false discovery rate). Prior studies have implicated DPYD, PKP4 and exocytosis in BIP and SCZ. The most notable finding of the present study was the genetic overlap of BOR with BIP (rg=0.28 [P=2.99 × 10-3]), SCZ (rg=0.34 [P=4.37 × 10-5]) and MDD (rg=0.57 [P=1.04 × 10-3]). We believe our study is the first to demonstrate that BOR overlaps with BIP, MDD and SCZ on the genetic level. Whether this is confined to transdiagnostic clinical symptoms should be examined in future studies.

Genome-wide association study of pathological gambling.

BACKGROUND: Pathological gambling is a behavioural addiction with negative economic, social, and psychological consequences. Identification of contributing genes and pathways may improve understanding of aetiology and facilitate therapy and prevention. Here, we report the first genome-wide association study of pathological gambling. Our aims were to identify pathways involved in pathological gambling, and examine whether there is a genetic overlap between pathological gambling and alcohol dependence. METHODS: Four hundred and forty-five individuals with a diagnosis of pathological gambling according to the Diagnostic and Statistical Manual of Mental Disorders were recruited in Germany, and 986 controls were drawn from a German general population sample. A genome-wide association study of pathological gambling comprising single marker, gene-based, and pathway analyses, was performed. Polygenic risk scores were generated using data from a German genome-wide association study of alcohol dependence. RESULTS: No genome-wide significant association with pathological gambling was found for single markers or genes. Pathways for Huntington's disease (P-value=6.63×10(-3)); 5'-adenosine monophosphate-activated protein kinase signalling (P-value=9.57×10(-3)); and apoptosis (P-value=1.75×10(-2)) were significant. Polygenic risk score analysis of the alcohol dependence dataset yielded a one-sided nominal significant P-value in subjects with pathological gambling, irrespective of comorbid alcohol dependence status. CONCLUSIONS: The present results accord with previous quantitative formal genetic studies which showed genetic overlap between non-substance- and substance-related addictions. Furthermore, pathway analysis suggests shared pathology between Huntington's disease and pathological gambling. This finding is consistent with previous imaging studies.

Duplications in RB1CC1 are associated with schizophrenia; identification in large European sample sets.

Schizophrenia (SCZ) is a severe and debilitating neuropsychiatric disorder with an estimated heritability of ~80%. Recently, de novo mutations, identified by next-generation sequencing (NGS) technology, have been suggested to contribute to the risk of developing SCZ. Although these studies show an overall excess of de novo mutations among patients compared with controls, it is not easy to pinpoint specific genes hit by de novo mutations as actually involved in the disease process. Importantly, support for a specific gene can be provided by the identification of additional alterations in several independent patients. We took advantage of existing genome-wide single-nucleotide polymorphism data sets to screen for deletions or duplications (copy number variations, CNVs) in genes previously implicated by NGS studies. Our approach was based on the observation that CNVs constitute part of the mutational spectrum in many human disease-associated genes. In a discovery step, we investigated whether CNVs in 55 candidate genes, suggested from NGS studies, were more frequent among 1637 patients compared with 1627 controls. Duplications in RB1CC1 were overrepresented among patients. This finding was followed-up in large, independent European sample sets. In the combined analysis, totaling 8461 patients and 112 871 controls, duplications in RB1CC1 were found to be associated with SCZ (P=1.29 × 10(-5); odds ratio=8.58). Our study provides evidence for rare duplications in RB1CC1 as a risk factor for SCZ.

Aqueous humor concentrations of topically administered caspofungin in rabbits.

BACKGROUND: The echinocandin caspofungin (CAS) is a novel antifungal drug with fungicidal in vitro activity against all Candida spp., which are the most frequent cause of fungal keratitis. Penetration of CAS through the cornea into the aqueous humor after topical administration was investigated. METHODS: A CAS solution with a concentration of 7 mg/ml was applied onto each rabbit's cornea. Drug application after corneal epithelium abrasion was processed in different time intervals: single application with aqueous humor sampling after 1 and 2 h. In addition, after continuous application of CAS every 30 min, aqueous humor concentrations of CAS after 1, 2 and 5 h were analyzed by liquid-chromatography tandem mass spectrometry. RESULTS: Topical administration of CAS without corneal epithelium abrasion resulted in no detectable amounts of the drug in the aqueous humor. However, with corneal abrasion, after a single application, levels of 2.16 +/- 1.57 microg/ml (n = 6) were reached after 1 h and then decreased to 1.76 +/- 0.88 microg/ml (n = 2) after 2 h. After serial application every 30 min, the following intracameral levels of CAS were detected: after 1 h, 2.11 +/- 1.09 microg/ml (n = 6); after 2 h, 4.94 +/- 1.80 microg/ml (n = 5), and after 5 h, 3.45 +/- 2.11 microg/ml (n = 6). CONCLUSION: In the aqueous humor, therapeutic drug levels can be reached that cover the MICs of most fungi after epithelial abrasion. To achieve a sustained high level of CAS as an effective antifungal therapy for corneal keratitis, CAS should be administered topically every 30 min after removal of the corneal epithelium.

Acid adaptation of Lactobacillus delbrueckii subsp. bulgaricus induces physiological responses at membrane and cytosolic levels that improves cryotolerance.

AIMS: This work aimed at clarifying the physiological responses of Lactobacillus delbrueckii subsp. bulgaricus CFL1 cells after exposure to acidification at the end of fermentation, in relation to their cryotolerance. METHODS AND RESULTS: Cells acidified at the end of the fermentation (pH 5.25 for 30 min) had their cryotolerance improved as compared to the reference condition (pH 6.0). By analyzing the cytosolic proteome, it was established that changes occurred in the synthesis of 21 proteins, involved in energy metabolism, nucleotide and protein synthesis and stress response. Acidification also induced a slight decrease in unsaturated to saturated and cyclic to saturated membrane fatty acid ratios. CONCLUSIONS: Lactobacillus bulgaricus CFL1 was able to develop a combined physiological response at both membrane and cytosolic levels. This acid adaptation was referred as a cross-protection phenomenon as it allowed the cells to become more tolerant to cold stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increased knowledge concerning the physiological mechanisms that explained the cross-protection by acid adaptation. It may be useful for improving cryotolerance of lactic acid bacteria, either in cells banks or in an industrial context.

Aqueous humor concentration of voriconazole after topical administration in rabbits.

OBJECTIVES: Voriconazol is a triazole antifungal drug with in vitro fungicidal activity against all Candida spp., Fusarium spp. and Aspergillus spp. which are frequent causes of fungal keratitis depending on geographic location. We investigated the penetration of voriconazole through the cornea into the aqueous humor (AH) after topical administration. METHODS: A 1% voriconazole solution was applied onto each rabbit's cornea. Topical drug application was processed at different time intervals: single drug application with AH sampling after 30 min, 1 h, 2 h, 3 h and 6 h. In addition, we evaluated AH samples after repeated topical application of voriconazole every 30 min after 1, 2, 4 and 6 h. Furthermore, after repeated drug application every hour, we analyzed voriconazole concentration after 2, 3, 4 and 6 h. All samples were analyzed by high-performance liquid chromatography (HPLC)-UV. RESULTS: A single application showed a maximum peak in AH of 3.58 microg/ml (N = 9) after 30 min. Within 3 h the concentration decreased to 0.04 microg/ml (N = 11). Application of voriconazole every half an hour revealed a peak value of 6.73 microg/ml (N = 10) after 2 h; after 4 h the value decreased to 6.19 microg/ml (N = 10) and was constant after 6 h (6.12 microg/ml, N = 6). When administrated every hour, only lower AH concentrations of voriconazole were reached with a maximum level of 2,06 microg/ml (N = 8) after four hours. CONCLUSION: In AH, therapeutic drug levels that cover the minimum inhibitory concentrations (MIC) of most fungi can be reached. To achieve a sustained high level of voriconazole as an effective antifungal therapy for corneal keratitis, voriconazole should be topically administered every 30 min.

Multi-center evaluation of analytical performance of the microparticle enzyme immunoassay for sirolimus.

OBJECTIVES: This study evaluated the analytical characteristics of the new Abbott microparticle enzyme immunoassay (MEIA) for sirolimus. DESIGN AND METHODS: The protocol consisted of nine sections: evaluation of antibody specificity, linearity, detection limit, quantification limit, endogenous interferents, exogenous interferents, precision, proficiency testing panel, and method comparison. RESULTS: The mean analytical detection limit was 0.68 microg/L. The sirolimus concentration corresponding to a total CV of 20% was 1.5 microg/L. Linearity of response was demonstrated across the dynamic range of the assay. Total precision (CVs) at QC control levels from 5 to 22 microg/L ranged from 5.7 to 12.6%. Assay standardization was found to be in good agreement with LC/MS/MS as compared with target values for spiked sirolimus proficiency samples from an international sirolimus proficiency testing program. Good correlations (R values) of the immunoassay were observed in comparisons to LC/MS/MS. R values tended to be lower in comparisons with LC/UV methods. Across both LC-based methods and all study sites, there was approximately 25% overall positive slope bias due to cross reactivity of the MEIA antibody to metabolites of sirolimus. The assay cross-reactivity to metabolites of sirolimus parent drug ranged from 6 to 63%. Assay interferences were minimal with the exception of hematocrit, which presented a negative relationship to measured sirolimus concentration. CONCLUSIONS: The MEIA demonstrated acceptable analytical characteristics for use for routine monitoring of sirolimus immunosuppressive therapy, and is a viable alternative to HPLC-based methods for sirolimus monitoring.

WITHDRAWN: Multi-center evaluation of analytical performance of the microparticle enzyme immunoassay for sirolimus.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Clin. Biochem. 39 (2006) 378-386, doi:10.1016/j.clinbiochem.2006.01.017. The duplicate article has therefore been withdrawn. This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The Publisher apologizes for any inconvenience this may cause.

Splanchnic oxygen transport, hepatic function and gastrointestinal barrier after normothermic cardiopulmonary bypass.

BACKGROUND: The effect of non-pulsatile, normothermic cardiopulmonary-bypass (CPB) on the splanchnic blood-flow and oxygen-transport, the hepatic function and the gastrointestinal barrier were observed in a prospective observational study in 31 adults undergoing cardiac valve replacement surgery. METHODS: The splanchnic (i.e. hepatic) blood-flow (HBF) was measured by the constant infusion of indocyanine-green (ICG) using a hepatic-venous catheter. Liver function was examined by calculation of lactate uptake, ICG extraction and the monoethylglycinexylidide (MEGX) test. A day before and after surgery the gastrioduodenal and intestinal permeability was measured by determination of sucrose and lactulose/mannitol excretion. RESULTS: Splanchnic blood flow and oxygen delivery did not decrease during and after surgery while splanchnic oxygen consumption (P < 0.0125) and arterial lactate concentrations increased. The splanchnic lactate uptake paralleled the lactate concentration. After but not during CPB an increase of systemic oxygen consumption was observed. The MEGX test values decreased on the first day after surgery. The ICG extraction was attenuated during the operation. The gastroduodenal and the intestinal permeability increased significantly postoperatively (P < 0.002, respectively, P < 0.001). There was no correlation between these findings and the duration of CPB. There was a significant correlation of the intestinal permeability but not of the gastroduodenal permeability between the prior and after surgery values (P < 0.001). CONCLUSION: Increased oxygen consumption during CPB may indicate an inflammatory reaction due to the pump beginning in the splanchnic area or a redistribution of the splanchinc blood flow during the CPB. Normothermic CPB does not lead to a significant or prolonged reduction of liver function. Normothermic CPB causes an increase of gastrointestinal permeability. The intestinal barrier function prior to surgery was accountable for the degree of loss of intestinal barrier function following surgery.

Glucuronide and glucoside conjugation of mycophenolic acid by human liver, kidney and intestinal microsomes.

Mycophenolic acid (MPA) is primarily metabolized to a phenolic glucuronide (MPAG) as well as to two further minor metabolites: an acyl glucuronide (AcMPAG) and a phenolic glucoside (MPAG1s). This study presents investigations of the formation of these metabolites by human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as by recombinant UDP-glucuronosyltransferases. HLM (n=5), HKM (n=6), HIM (n=5) and recombinant UGTs were incubated in the presence of either UDP-glucuronic acid or UDP-glucose and various concentrations of MPA. Metabolite formation was followed by h.p.l.c. All microsomes investigated formed both MPAG and AcMPAG. Whereas the efficiency of MPAG formation was greater with HKM compared to HLM, AcMPAG formation was greater with HLM than HKM. HIM showed the lowest glucuronidation efficiency and the greatest interindividual variation. The capacity for MPAGls formation was highest in HKM, while no glucoside was detected with HIM. HKM produced a second metabolite when incubated with MPA and UDP-glucose, which was labile to alkaline treatment. Mass spectrometry of this metabolite in the negative ion mode revealed a molecular ion of m/z 481 compatible with an acyl glucoside conjugate of MPA. All recombinant UGTs investigated were able to glucuronidate MPA with K:(M:) values ranging from 115.3 to 275.7 microM l(-1) and V(max) values between 29 and 106 pM min(-1) mg protein(-1). Even though the liver is the most important site of MPA glucuronidation, extrahepatic tissues particularly the kidney may play a significant role in the overall biotransformation of MPA in man. Only kidney microsomes formed a putative acyl glucoside of MPA.