Transjugular intrahepatic portosystemic shunt: impact on systemic hemodynamics and renal and cardiac function in patients with cirrhosis.

Transjugular intrahepatic portosystemic shunt (TIPS) alleviates portal hypertension and possibly increases central blood volume (CBV). Moreover, renal function often improves; however, its effects on cardiac function are unclear. The aims of our study were to examine the effects of TIPS on hemodynamics and renal and cardiac function in patients with cirrhosis. In 25 cirrhotic patients, we analyzed systemic, cardiac, and splanchnic hemodynamics by catheterization of the liver veins and right heart chambers before and 1 wk after TIPS. Additionally, we measured renal and cardiac markers and performed advanced echocardiography before, 1 wk after, and 4 mo after TIPS. CBV increased significantly after TIPS (+4.6%, P < 0.05). Cardiac output (CO) increased (+15.3%, P < 0.005) due to an increase in stroke volume (SV) (+11.1%, P < 0.005), whereas heart rate (HR) was initially unchanged. Cardiopulmonary pressures increased after TIPS, whereas copeptin, a marker of vasopressin, decreased (-18%, P < 0.005) and proatrial natriuretic peptide increased (+52%, P < 0.0005) 1 wk after TIPS and returned to baseline 4 mo after TIPS. Plasma neutrophil gelatinase-associated lipocalin, renin, aldosterone, and serum creatinine decreased after TIPS (-36%, P < 0.005; -65%, P < 0.05; -90%, P < 0.005; and -13%, P < 0.005, respectively). Echocardiography revealed subtle changes in cardiac function after TIPS, although these were within the normal range. TIPS increases CBV by increasing CO and SV, whereas HR is initially unaltered. These results indicate an inability to increase the heart rate in response to a hemodynamic challenge that only partially increases CBV after TIPS. These changes, however, are sufficient for improving renal function. NEW & NOTEWORTHY For the first time, we have combined advanced techniques to study the integrated effects of transjugular intrahepatic portosystemic shunt (TIPS) in cirrhosis. We showed that TIPS increases central blood volume (CBV) through improved cardiac inotropy. Advanced echocardiography demonstrated that myocardial function was unaffected by the dramatic increase in preload after TIPS. Finally, renal function improved due to the increase in CBV. Recognition of these physiological changes significantly contributes to our clinical understanding of TIPS.

Neutrophil gelatinase-associated lipocalin and cystatin C in cirrhosis and portal hypertension: Relations to organ extraction and dysfunction.

BACKGROUND AND AIMS: Early detection of renal dysfunction in cirrhosis is important, and several renal biomarkers have been put forward. Neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C are markers of renal dysfunction, but relations to splanchnic and systemic hemodynamics and kinetics are sparsely studied in cirrhosis. In patients with cirrhosis and portal hypertension, we studied plasma levels and renal, hepatic, and peripheral extraction of NGAL and cystatin C and relations to patients characteristics, liver dysfunction, and hemodynamics. METHODS: Forty-five cirrhotic patients (Child class A/B/C:15/15/15) and 15 controls were evaluated with a full clinical, biochemical, and hemodynamic assessment. Urine and regional plasma concentrations of NGAL and cystatin C were measured. RESULTS: There was no significant difference in circulating or hepatic NGAL or cystatin C between all patients and controls but a trend towards increased levels with increasing Child class. In addition, there was a significant renal but no hepatic or systemic extraction of both NGAL and cystatin C (P < 0.001). Plasma NGAL correlated with glomerular filtration rate (r = -0.56, P < 0.0001), and hepatic venous pressure gradient (r = 0.34,P = 0.02) and urinary NGAL correlated with heart rate (r = 0.58, P= 0.007), blood pressure (r = -0.46, P < 0.05), cardiac output (r = 0.45, P < 0.05), and systemic vascular resistance (SVR) (r = -0.48, p < 0.05). Plasma cystatin C correlated with hepatic venous pressure gradient (r = 0.45, P < 0.005), blood pressure (-0.40, P < 0.01), and glomerular filtration rate (r = 0.98, P < 0.000). CONCLUSIONS: Extractions of NGAL and cystatin C levels seem largely unaffected by the severity of liver disease in cirrhosis with a renal extraction. These biomarkers therefore have the potential of being both valuable in diagnosing renal failure and reflecting the degree of portal hypertension and systemic haemodynamic changes.

Soluble urokinase plasminogen activator receptor (suPAR) in acute care: a strong marker of disease presence and severity, readmission and mortality. A retrospective cohort study.

OBJECTIVE: Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker associated with presence and progression of disease and with increased risk of mortality. We aimed to evaluate the unspecific biomarker suPAR as a prognostic marker in patients admitted to acute care. METHODS: This registry-based retrospective cohort study included 4343 consecutively admitted patients from the Acute Medical Unit at a large Danish university hospital. Time to readmission and death were analysed by multiple Cox regression. Results were reported as HRs for 30-day and 90-day follow-up. RESULTS: During 30-day follow-up, 782 patients (18.0%) were readmitted and 224 patients (5.2%) died. Comparing 30-day readmission and mortality between patients in the highest and lowest suPAR quartiles yielded HRs of 2.11 (95% CI 1.70 to 2.62) and 4.11 (95% CI 2.46 to 6.85), respectively, when adjusting for age, sex, Charlson score and C reactive protein. Area under the curve for receiver operating characteristics curve analysis of suPAR for 30-day mortality was 0.84 (95% CI 0.81 to 0.86). Furthermore, in the entire cohort, women had slightly higher suPAR compared with men, and suPAR was associated with age, admission time, admission to intensive care unit and Charlson score. CONCLUSIONS: In this large unselected population of acute medical patients, suPAR is strongly associated with disease severity, readmission and mortality after adjusting for all other risk factors, indicating that suPAR adds information to established prognostic indicators. While patients with low suPAR levels have low risk of readmission and mortality, patients with high suPAR levels have a high risk of adverse events.

Evaluation of NGAL TestTM on Cobas 6000.

BACKGROUND: Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a promising biomarker for acute kidney injury (AKI). Our objectives were to evaluate the NGAL Test(TM) from Bioporto for both urine NGAL and plasma NGAL on the Cobas 6000 c501 (Roche Diagnostics, Rotkreuz, Switzerland) with matched measurements run on Hitachi 917, the method's linearity on the Cobas 6000 in urine, EDTA and Lithium-Heparin (Li-Hep), the influence of using EDTA or Li-Hep tubes and, finally, the impact of freezing and thawing on the sample. METHODS: Forty matched samples of Li-Hep and EDTA plasma and 40 urine samples were analyzed for method, anticoagulant, and freeze-thaw comparisons. Linearity was assessed using high NGAL samples diluted in urine, EDTA, and Li-Hep plasma. Commercial internal controls were used for the imprecision study. RESULTS: The Cobas 6000 measured identically with the Hitachi 917, however, not in EDTA plasma (Median Difference = 17.50 µg/L, p < 0.0001). Freeze-thaw process reduced NGAL ((EDTA: Mean Difference = = 15.13 µg/L, p = 0.0014)(Li-Hep: Median Difference = = 6.5 µg/L, p = 0.0129)). NGAL results were higher in Li-Hep plasma than in EDTA plasma ((Non-thawed: Median Difference = = 14.5 µg/L, p < 0.0001), (Thawed: Median Difference = = 21.5 µg/L, p = 0.0003)). Linearity agreements were observed in all three specimens. Imprecision (CV%) was below 3%. CONCLUSION: The NGAL Test(TM) can be applied on the Cobas 6000 with acceptable performance, although the Cobas 6000 measured higher than the Hitachi 917 in EDTA plasma. Though clinically insignificant, we found that the freeze-thaw process had a reduced effect. NGAL results were higher in Li-Hep tubes than in EDTA tubes. Thus, for blood samples we recommend use of EDTA tubes for NGAL measurements.

Identifying the potential of changes to blood sample logistics using simulation.

Using simulation as an approach to display and improve internal logistics at hospitals has great potential. This study shows how a simulation model displaying the morning blood-taking round at a Danish public hospital can be developed and utilized with the aim of improving the logistics. The focus of the simulation was to evaluate changes made to the transportation of blood samples between wards and the laboratory. The average- (AWT) and maximum waiting time (MWT) from a blood sample was drawn at the ward until it was received at the laboratory, and the distribution of arrivals of blood samples in the laboratory were used as the evaluation criteria. Four different scenarios were tested and compared with the current approach: (1) Using AGVs (mobile robots), (2) using a pneumatic tube system, (3) using porters that are called upon, or (4) using porters that come to the wards every 45 minutes. Furthermore, each of the scenarios was tested in terms of what amount of resources would give the optimal result. The simulations showed a big improvement potential in implementing a new technology/mean for transporting the blood samples. The pneumatic tube system showed the biggest potential lowering the AWT and MWT with approx. 36% and 18%, respectively. Additionally, all of the scenarios had a more even distribution of arrivals except for porters coming to the wards every 45 min. As a consequence of the results obtained in the study, the hospital decided to implement a pneumatic tube system.

Danish doctors' reactions to 'internationalization' in clinical training in a public university hospital.

OBJECTIVE: From 2012 to 2015, two Departments of Obstetrics and Gynecology and two Departments of Pediatrics at the University of Copenhagen implemented an English medium international project. The project allowed international students to work in pairs with local Danish speaking students in a clinical setting. The student cohort was supported by Danish doctors who were responsible for student-pair supervision in English and, ultimately, patient care. Drawing on survey responses of 113 Danish doctors, this study considers the doctors' overall evaluation of the program and their perception of the international students' knowledge, skills and attitudes compared with local students. RESULTS: The Danish doctors rated the international and local students comparable in respect to professional commitment (p = 0.347), academic level (p = 0.134), and English proficiency (p = 0.080). The Danish doctors rated the international students significantly lower than the local students regarding communication with Danish doctors, other hospital staff, and patients (p < 0.001 in all cases). Ninety percent of the doctors involved in the project supported continuing working with internationalization if it included mixed pairs of students and a Danish doctor assigned each day to be exclusively responsible for student supervision. Language barriers for international medical students could be overcome but required substantial faculty support.

A low-gluten diet induces changes in the intestinal microbiome of healthy Danish adults.

Adherence to a low-gluten diet has become increasingly common in parts of the general population. However, the effects of reducing gluten-rich food items including wheat, barley and rye cereals in healthy adults are unclear. Here, we undertook a randomised, controlled, cross-over trial involving 60 middle-aged Danish adults without known disorders with two 8-week interventions comparing a low-gluten diet (2 g gluten per day) and a high-gluten diet (18 g gluten per day), separated by a washout period of at least six weeks with habitual diet (12 g gluten per day). We find that, in comparison with a high-gluten diet, a low-gluten diet induces moderate changes in the intestinal microbiome, reduces fasting and postprandial hydrogen exhalation, and leads to improvements in self-reported bloating. These observations suggest that most of the effects of a low-gluten diet in non-coeliac adults may be driven by qualitative changes in dietary fibres.

The pharmacokinetics and acute renal effects of oral microemulsion ciclosporin A in normal pigs.

BACKGROUND: The use of ciclosporin A (CsA) is limited by nephrotoxicity. Anatomic and physiologic similarities to humans make the pig a potential important animal model for research in effects and adverse events of CsA. In a long-term study CsA 10 mg/kg/day for 6 months did not cause deterioration in renal histology or function in pigs. For ideal CsA dosage in future long-term studies of nephrotoxicity we performed this study to describe pharmacokinetics and acute effects on renal function of CsA in normal pigs. MATERIALS AND METHODS: Three groups of 5 piglets comprised the material, a control group and 2 groups receiving 5 days treatment with CsA (Neoral) 15 or 30 mg/kg/day, respectively. CsA whole blood concentration (B-CsA) (hourly), renal blood flow, glomerular filtration rate (GFR) and serum creatinine were measured. The area under the curve from time 0 to 12 h was calculated using the Trapezoidal Rule. Pharmacokinetic calculations were performed using the KINFIT non-linear curve fitting module. RESULTS: B-CsA reached peak in median at 0.90 and 0.96 h (633 and 914 ng/ml) in the 15 and 30 mg/kg/day groups, respectively, and median AUC was 5055 and 6275 h ng/ml, respectively. Trough levels were 338 and 475 ng/ml. The distribution of CsA followed a 2-compartment model. Serum creatinine was 111 (control), 112 (15 mg/kg/day) and significantly increased to 168 ìmol/l in 30 mg/kg/day group, which also had a reduction in GFR compared to the other 2 groups. CONCLUSIONS: CsA causes acute nephrotoxicity in piglets. The distribution follows a 2-compartment model similar to humans, but higher doses are necessary in pigs.

Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays.

Cyclosporine exhibits a wide spectrum of metabolites that vary considerably in the extent to which they interfere with the various parent drug monitoring immunoassays. There is no consensus regarding the clinical significance of metabolites. Cyclosporine exerts its immunosuppressive action by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites in this perspective? The aim of the present study was to determine the concentration of cyclosporine (by means of three different assay methods) and the four most significant metabolites (AM1, AM4N, AM9, and AM1C) in relation to calcineurin phosphatase inhibition. Twelve randomly selected cyclosporine-treated renal transplant patients were included in the study. Blood samples were drawn before, 1, 2, 3, 4, 6, 8, and 12 hr after oral intake of cyclosporine. Parent drug and metabolites were determined by liquid chromatography/tandem mass spectrometry (LC/MSMS). Additionally, cyclosporine concentration was determined by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition correlates strongly with parent cyclosporine metabolites concentrations determined by all three assay methods. Determination methods that took metabolites into consideration exhibit stronger correlations with calcineurin phosphatase inhibition (sum of cyclosporin plus metabolites r=-0.93, LC/MSMS; pFPIA r=-0.94, P

A new model of paired clinical teaching of international and Danish medical students.

INTRODUCTION: Since 2006, one hospital has offered two clinical courses in obstetrics/gynaecology and paediatrics to international (I) students. However, as I-student enrolment increased, the hospital faced cut-backs. As from 2010, I-team course evaluations therefore dropped to unacceptable levels and more I- than Danish (DK) students failed exams. Therefore, in 2012 we started a three-year internationalisation project (I-project) at two hospitals. The primary intervention was to pair training for I- and DK-students at clinical contact, and to offer an exclusive daily lecturer for I-teams. METHODS: We compared the course evaluations and exam grades of I-teams and DK-teams for two years prior to (107 from I-teams - 211 participants from DK-teams) and during the I-project (245 participants from I-teams - 575 from DK-teams). RESULTS: During the I-project, the I-teams' course evaluations increased to acceptable values and to levels comparable to the evaluation scores of DK-teams. Furthermore, I-students now considered that their communication with the patients was acceptable. Before the I-project, I-students had lower exam grades (median = 10 (range: 0-12)) than DK-students (10 (4-12)) (p = 0.03), but during the I-project, exam grades increased to the levels achieved by DK-students (10 (2-12) - 10 (0-12) (p = 0.22), and no more I- than DK-students failed exams (p = 0.51). CONCLUSIONS: Pairing students for clinical training and allocating an exclusive lecturer for I-teams produced improved courses for internationalisation. Allocating an exclusive lecturer was associated with a cost of about 615 EUR per student per course when the team consisted of ten students. FUNDING: The Capital Region of Denmark and the University of Copenhagen. TRIAL REGISTRATION: not relevant.

[A targeted effort can secure a good and educating clinical stay for medical students].

The medical education at the University of Copenhagen introduces the student to clinical life through a number of clinical courses. In this article we describe measures taken to secure a good and educating stay on the department during a five-week course. We describe the process, procedures, planning, executing and evaluation of the five-week clinical course. The evaluation through direct feedback and subsequent electronic form is commented and essential learning points are discussed.

Plasma adiponectin in patients with active, early, and chronic rheumatoid arthritis who are steroid- and disease-modifying antirheumatic drug-naive compared with patients with osteoarthritis and controls.

OBJECTIVE: Rheumatoid arthritis (RA) is a systemic chronic inflammatory joint disease, whereas osteoarthritis (OA) is a local joint disease with low-level inflammatory activity. The pathogenic role of the adipocytokine adiponectin is largely unknown in these diseases. We hypothesized (1) that plasma adiponectin concentrations differ in healthy controls and patients with early disease-modifying antirheumatic drug (DMARD)-naive RA, chronic RA, and OA; (2) that changes in adiponectin are observed during methotrexate (MTX) treatment of chronic RA; and (3) that adiponectin correlates to disease activity measures in RA. METHODS: Plasma adiponectin was analyzed with a validated in-house immunoassay. We measured adiponectin in healthy controls (n = 45) and patients with early DMARD-naive RA (n = 40), chronic RA (n = 74), and OA (n = 35). In a subgroup of patients with chronic RA (n = 31), the longitudinal effect of MTX treatment on adiponectin (Week 0 vs Week 28) was investigated. RESULTS: Adiponectin differed significantly between healthy controls (mean 4.8 +/- SD 2.7 mg/l) and the 3 groups, with 8.9 +/- 4.8 mg/l in early RA, 11.6 +/- 5.6 mg/l in chronic RA, and 14.1 +/- 6.4 mg/l in OA. Longitudinally, MTX treatment increased adiponectin significantly from 9.7 +/- 4.5 mg/l at Week 0 to 11.0 +/- 4.5 mg/l at Week 28 in chronic RA. No correlations to disease activity measures were found. CONCLUSION: Both early DMARD-naive and chronic RA were associated with higher plasma adiponectin compared to healthy controls, but lower plasma adiponectin than OA. Adiponectin increased 13% during MTX treatment. In patients with RA and OA body mass index, age, sex, and disease activity measures failed to explain the findings.

Patients with rheumatoid arthritis treated with methotrexate (MTX): concentrations of steady-state erythrocyte MTX correlate to plasma concentrations and clinical efficacy.

OBJECTIVE: To investigate the accumulation of methotrexate (MTX) in circulating erythrocytes and the association with pharmacokinetic variables, weekly dose, and clinical efficacy in 2 cohorts of patients with chronic active rheumatoid arthritis (RA) undergoing MTX monotherapy. METHODS: Seventy-six patients with RA were included in this open prospective study: 40 were included before initiation of MTX therapy. Laboratory analyses, intracellular MTX concentrations in erythrocytes (Ery-MTX), and clinical examinations including toxicity data were performed prospectively for 52 weeks. Plasma concentrations of MTX were measured and area under the plasma concentration versus time curve (AUC) was estimated along with other pharmacokinetic variables in a population based software model. RESULTS: Ery-MTX rose after initiation of therapy and reached a steady state after 6-8 weeks. The correlation between steady-state Ery-MTX and dose was poor (r(2) = 0.16), whereas steady-state Ery-MTX levels correlated strongly with the estimated AUC (r(2) = 0.51, log-transformed variables). Both steady-state Ery-MTX levels and estimated AUC were significantly higher in patients responding to MTX therapy than in patients classified as nonresponders according to American College of Rheumatology core criteria and were similar to patients on longterm MTX therapy. CONCLUSION: Our results indicate that clinical efficacy and Ery-MTX may have a causal relation and that measurement of Ery-MTX or estimation of AUC in a software model provides useful guidelines to the clinician when starting MTX therapy in patients with RA. The latter can be performed immediately after initiation of therapy.

[Uniform reference intervals and harmonised results in clinical biochemistry in Scandinavia--light ahead]. / Ens referenceintervaller og harmoniserede resultater inden for klinisk biokemi i de nordiske lande--der er lys forude.

Danish laboratories are introducing identical reference intervals for a number of biochemical components in accordance with a Nordic agreement and recommendations during 2007. Danish doctors will consequently experience both changes in reference interval limits as well as adjustments of patients' levels of reported results for some components.

Plasma level of CXC-chemokine CXCL12 is increased in rheumatoid arthritis and is independent of disease activity and methotrexate treatment.

OBJECTIVE: Several actions of the chemokine CXCL12 have potential relevance for rheumatoid arthritis (RA). Interaction with CXCR4, the unique receptor for CXCL12, stimulates angiogenesis, mononuclear cell trafficking into the joints, lymphoid-tissue-like rearrangement of T cells within the synovium, and chondrocyte release of cartilage-degrading metalloproteinases. We investigated the level of CXCL12 in plasma (p-CXCL12) as a marker of RA diagnosis, RA disease activity, and response to methotrexate (MTX) treatment. METHODS: A prospective study including 36 patients with RA (ACR criteria) of at least 6 months' duration, and 50 sex and age matched healthy controls. ELISA for CXCL12 was performed on plasma prior to and after 16 and 28 weeks of MTX treatment in the patients with RA and once in controls. RESULTS: The p-CXCL12 was 1855 +/- 145 pg/ml in RA patients and 1273 +/- 79 pg/ml in controls (p < 0.001). During the 28 weeks of MTX treatment, the ACR disease activity variables decreased, whereas the p-CXCL12 level remained constant and increased. P-CXCL12 was not correlated to any ACR disease activity variable at any time (p > 0.05). CONCLUSION: Patients with RA had a significantly and constantly increased p-CXCL12 level compared to controls. The p-CXCL12 level was independent of any ACR disease activity variables, as well as response to MTX treatment.

The calcineurin activity profiles of cyclosporin and tacrolimus are different in stable renal transplant patients.

Cyclosporin and tacrolimus remain the cornerstone immunosuppressive drugs in organ transplantation. Dosing and monitoring these drugs is based on pharmacokinetic protocols, but measuring a pharmacodynamic parameter, calcineurin phosphatase (CaN) activity, could be a valuable supplement in determining optimal doses. Forty stable renal transplant patients were investigated three times in a 6-month period. Blood samples were drawn at 0, 1, 2, 3 and 4 h after oral intake of tacrolimus (FK) or cyclosporin at days 1 and 180. At day 90, one blood sample at trough level (FK) or C2 level (cyclosporin A, CsA) was drawn. CaN activity was determined in whole blood as the release of 32P from a phosphorylated peptide. Activity of the 32P was quantitated by liquid scintillation and results converted to Units CaN, utilizing a calibration curve with CaN. We demonstrated that calcineurin activity profiles at days 1 and 180 were the same for both drugs. Furthermore, we found that patients treated with tacrolimus or cyclosporin displayed different calcineurin activity profiles. We found that cyclosporin displayed greater calcineurin inhibition than tacrolimus. We have demonstrated that the two drugs exert significantly different effects on calcineurin activity in renal transplant patients with stable, well-functioning grafts and that tacrolimus-treated patients can maintain good, stable graft function with minimal CaN inhibition.

Role of metabolites and calcineurin inhibition on C2 monitoring in renal transplant patients.

BACKGROUND: Many transplantation centres have switched to C2 monitoring of cyclosporin-treated renal transplant patients. The rationale is that the C2 correlates best with AUC0-4 (area under the concentration-time curve), which again correlates with rejection and nephrotoxicity. It has also been demonstrated that calcineurin phosphatase is inhibited maximally 1-2 h after intake of cyclosporin in patients receiving their first dose. Cyclosporin is metabolized to many compounds, which may influence the results of immunoassays. Some metabolites may have immunosuppressive activity. METHODS: Cyclosporin metabolites were added to whole blood from healthy volunteers and the calcineurin phosphatase activity (CaN) was determined. Twenty renal transplant patients at varying times after transplantation had blood samples drawn in the morning before and 1, 2, 3 and 4 h after intake of their usual dose of cyclosporin microemulsion. Whole blood samples were analysed by liquid chromatography/tandem mass spectrometry for cyclosporin blood concentration and for the cyclosporin metabolites AM1, AM9, AM1c and AM4n. All samples were analysed for CaN utilizing a 32P-labelled 19 amino-acid peptide. RESULTS: The concentrations of AM1c and AM4n were very low and cannot contribute to CaN inhibition. The ratio of AM1 and AM9 to cyclosporin was high before intake of the drug, but it was much lower during the following 4 h. The 2-h values of cyclosporin were the best predictors of AUC0-4. Calcineurin phosphatase was most inhibited in the 2-h samples and the 2-h value of CaN was the best predictor of CaN AUC0-4. The correlation with calcineurin inhibition seemed better for cyclosporin plus metabolites than for cyclosporin. CONCLUSIONS: Samples collected at 2 h are the best predictors of AUC0-4 for both cyclosporin and calcineurin inhibition. The impact of metabolites appears to be small; however, the temporal profile of calcineurin inhibition seemed to follow cyclosporin plus metabolites better than cyclosporin alone.

Liquid chromatography tandem mass spectrometry assay for topiramate analysis in plasma and cerebrospinal fluid: validation and comparison with fluorescence-polarization immunoassay.

SUMMARY: The authors report the development and validation of a liquid chromatography tandem mass spectrometry assay (LC/MS/MS assay) for the analysis of topiramate (2,3:4,5-bis-o-(-1-methyl)-beta-D-fructopyranose sulfamate) in plasma and cerebrospinal fluid (CSF). Comparison is made with the commercially available fluorescence-polarization immunoassay (FPIA). LC/MS/MS ASSAY: Using the internal standard, 1,2:3,4-bis-o-(1-methylethylidene-alpha-D-galactopyranose sulfamate), a structural isomer, the calibration curve in plasma was linear in the concentration range of 0.02-20.0 mg/L (r(2) = 0.9998). The coefficients of variation in plasma were < or = 3%, and the accuracy ranged from 100% to 101% in the therapeutically relevant concentration range of 0.4-16.0 mg/L. In CSF, the mean recovery was 98%, and there was linearity between the nominal and the estimated concentration in the range of 1.5-20.0 mg/L (r(2)= 0.9996). FPIA: The calibration curve was linear in the concentration interval of 1.6-24.3 mg/L (r(2) = 0.9994), and the mean recovery was 96%. Accuracy in plasma was 99- 104%, and precision was 3.2-6.0%. In CSF, there was linearity between the nominal concentration and the estimated concentration in the range of 1.5-20.0 mg/L (r(2) = 0.9995), and the mean recovery was 100%. COMPARISON BETWEEN FPIA AND LC/MS/MS: There was a high correlation between the FPIA and the LC/MS/MS assay (r(2) = 0.9965 in plasma and r(2) = 0.9996 in CSF, P < 0.001 for both). In plasma and CSF, the two methods showed equal results, evaluated as the ratio between the two methods (plasma: median ratio = 1.00; 95% confidence interval [CI], 0.98-1.02, paired-sample test, P = 0.79; and CSF: median ratio = 1.00, 95% CI, 0.99-1.02, paired-sample test, P = 0.75). The coefficient of variation on the ratios between the two methods had similar levels: 5% in plasma and 3% in CSF. CONCLUSION: The new LC/MS/MS assay has favorable characteristics, being highly precise and accurate. FPIA also proved precise and accurate, and there was a high agreement with the LC/MS/MS assay in plasma and CSF. Either method displayed sufficient precision and accuracy and may thus be implemented in daily routine.

Randomized, concentration-controlled trial of topiramate in refractory focal epilepsy.

OBJECTIVE: To establish the concentration response of topiramate in patients with refractory focal epilepsy. METHODS: Sixty-five patients with more than eight seizures during an 8-week baseline were randomized to three prespecified plasma levels (low, 6 micromol/L [2 mg/L]; medium, 31 micromol/L [10.5 mg/L]; and high, 56 micromol/L [19 mg/L]). Topiramate treatment was titrated to one of the prespecified plasma levels during an 8-week titration period, followed by a 12-week observation period. RESULTS: The overall median (25th to 75th percentile) reduction in seizures during the observation compared with baseline was 50% (9.5 to 90%). In the individual groups, the median reduction was as follows: low, 39% (13 to 70%); medium, 85% (41 to 96%); and high, 39% (2.0 to 81%). The primary outcome of the trial was the comparison of seizure reduction (Mann-Whitney U test) between the low and the medium group (p = 0.03). Comparisons between the other groups were as follows: medium vs high (p = 0.05) and low vs high (p = 0.81). Psychiatric adverse events and adverse events related to the CNS were the most frequently encountered. Most adverse events showed concentration response, particularly between low and medium levels. CONCLUSIONS: Patients assigned to the medium plasma level (31 micromol/L [10.5 mg/L]) had the best seizure outcome. Patients in the medium and high groups experienced more adverse events than patients in the low group. Optimal treatment response is thus most likely found at plasma concentrations higher than 6 micromol/L (2 mg/L), but no further increase in efficacy seems to occur at concentrations above 31 micromol/L (10.5 mg/L).