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Background: Fibrosing interstitial pneumonias (IPs) include idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP). It has been suggested that oxidative damage plays a role in the pathophysiology of idiopathic interstitial pneumonias. Diffuse alveolar hemorrhage (DAH) can cause oxidative stress. Accordingly, we hypothesized that episodes of DAH might trigger fibrosing IP development., Methods: Patients using coumarins with confirmed DAH were retrospectively gathered during a 9 year period and reviewed for the development of IPF or fibrosing NSIP., Results: A total of 65 patients with DAH could finally be included, 31 (48 %) of whom subsequently developed a fibrosing IP. The majority of these 31 patients developed the fibrosing IP within 3 years after DAH confirmation. A total of 41 (63 %) patients died within 3.0 ± 0.9 (range 1.3-4.7) years after the DAH diagnosis had been confirmed. Twenty-two of the deceased (54 %) had finally developed fibrosing IP., Conclusions: Almost half of the patients with established episodes of DAH developed fibrosing IP; therefore it seems that DAH might be a trigger for the development of fibrosing IP. This observation warrants prospective studies to further evaluate the clinical impact of these findings.

Serum lipase is a biochemical marker used for diagnosing acute pancreatitis. Lipase has largely replaced amylase in terms of diagnostic value, although both markers are still commonly used. In this publication, we discuss the diagnostic superiority of lipase compared to amylase in acute pancreatitis. Two cases are discussed; both patients presented with elevated lipase values but due to different causes. The first patient, who had recently experienced cholecystitis, presented to the emergency room with abdominal pain and high lipase levels. The second patient, who had received a renal transplant a few years earlier, visited the gastroenterological outpatient clinic with abdominal pain and only a slightly elevated lipase level.

Objective: We investigated suspected longitudinal interaction effects of apolipoprotein E (APOE) genotype and educational attainment on cognitive decline in normal aging., Method: Our sample consisted of 571 healthy, nondemented adults aged between 49 and 82 years. Linear mixed-models analyses were performed with four measurement time points: baseline, 3-year, 6-year, and 12-year follow-up. Covariates included age at baseline, sex, and self-perceived physical and mental health. Dependent measures were global cognitive functioning (Mini-Mental State Examination; Folstein, Folstein, & McHugh, 1975), Stroop performance (Stroop Color-Word Test; Van der Elst, Van Boxtel, Van Breukelen, & Jolles, 2006a), set-shifting performance (Concept Shifting Test; Van der Elst, Van Boxtel, Van Breukelen, & Jolles, 2006b), cognitive speed (Letter-Digit Substitution Test; Van der Elst, Van Boxtel, Van Breukelen, & Jolles, 2006c), verbal learning (Verbal Learning Test: Sum of five trials; Van der Elst, Van Boxtel, Van Breukelen, & Jolles, 2005), and long-term memory (Verbal Learning Test: Delayed recall)., Results: We found only faint evidence that older, high-educated carriers of the APOE-ε4 allele (irrespective of zygosity) show a more pronounced decline than younger, low-educated carriers and noncarriers (irrespective of educational attainment). Moreover, this outcome was confined to concept-shifting performance and was especially observable between 6- and 12-year follow-ups. No protective effects of higher education were found on any of the six cognitive measures., Conclusions: We conclude that the combination of APOE-ε4 allele and high educational attainment may be a risk factor for accelerated cognitive decline in older age, as has been reported before, but only to a very limited extent. Moreover, we conclude that, within the cognitive reserve framework, education does not have significant protective power against age-related cognitive decline.

Background: The hemolytic products cell-free oxyhemoglobin (FHb) and arginase-1 reduce nitric oxide (NO) bioavailability by scavenging NO and by degrading the NO precursor arginine to ornithine, respectively. In this study we evaluated the relevance of hemolysis to NO-dependent blood flow in patients undergoing cardiovascular surgery., Methods: Plasma FHb, arginase-1, and amino acid concentrations were measured perioperatively. Forearm blood flow (FBF) responses to the intra-arterial administered NO-donor sodium nitroprusside (SNP) and the endothelium-dependent vasodilator acetylcholine (ACh) were measured by venous occlusion plethysmography., Results: When peak values plasma FHb and arginase-1 were found, vascular dilatation to SNP, but not ACh, was significantly reduced compared with 1 day postoperatively, when FHb had returned to baseline levels (p < 0.05). Interestingly, plasma FHb concentration was inversely correlated to FBF responses to SNP (r -0.93, p < 0.001). In contrast, the increase in arginase-1 was not biologically relevant as the ratio of plasma arginine to ornithine remained constant., Conclusion: We conclude that hemolysis with concomitant release of FHb during cardiovascular surgery is associated with impaired NO-dependent forearm blood flow., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Cardiac troponins (cTn) are the preferred markers for the diagnosis of acute myocardial infarction (AMI). The guidelines recommend the use of the 99th percentile upper reference concentration of a healthy population as the diagnostic cut-off for AMI. However, a broad range of upper reference limits is still employed, complicating the diagnosis of AMI. This overview is meant to assist laboratory specialists to define an appropriate cut-off value for the diagnosis of AMI. Therefore, we provide an overview of the analytical performance and upper reference limits of seven (high-)sensitivity cTn assays: Roche high-sensitivity cTnT and ADVIA Centaur, Stratus CS, Dimension Vista, Vitros ECi, Access and Architect cTnI assays. It is shown that none of the reference populations completely met the guidelines, including those in package inserts. Forty percent of the studies collected less than the advised minimum of 300 subjects. Many studies (50%) did not report their inclusion criteria, while lower 99th percentile limits were observed when more stringent selection criteria were applied. Higher troponin cut-offs were found in men and elderly subjects, suggesting sex- and age-specific cut-offs would be considered. Therefore, there is still need for a large, rigorously screened reference population to more accurately establish cTn upper reference limits.

Background: Cardiac troponin T measured by a high-sensitivity assay (hs-cTnT) recently proved to be of prognostic value in several populations. The hs-cTnT assay may also improve risk stratification in acute dyspnea., Methods: We prospectively studied the prognostic value of hs-cTnT in 678 consecutive patients presenting to the emergency department with acute dyspnea. On the basis of conventional cardiac troponin T assay (cTnT) and hs-cTnT assay measurements, patients were divided into 3 categories: (1) neither assay increased (cTnT<0.03 μg/L, hs-cTnT<0.016 μg/L), (2) only hs-cTnT increased≥0.016 μg/L (cTnT<0.03 μg/L), and (3) both assays increased (cTnT≥0.03 μg/L, hs-cTnT≥0.016 μg/L). Moreover, the prognostic value of hs-cTnT was investigated if cTnT was not detectable (<0.01)., Results: One hundred seventy-two patients were in the lowest, 282 patients in the middle, and 223 patients in the highest troponin category. Patients in the second and third categories had significantly higher mortality compared to those in the first category (90-day mortality rate 2%, 10%, and 26% in groups 1, 2, and 3, respectively, P<0.001; 1-year mortality rate 9%, 21%, and 39%, P<0.001). Importantly, in patients with undetectable cTnT (n=347, 51%), increased hs-cTnT indicated worse outcome [90-day mortality, odds ratio 4.26 (95% CI 1.19-15.21); 1-year mortality, hazard ratio 2.27 (1.19-4.36), P=0.013], whereas N-terminal pro-brain-type natriuretic peptide (NT-proBNP) was not predictive of short-term outcome., Conclusions: hs-cTnT is associated with mortality in patients presenting with acute dyspnea. hs-cTnT concentrations provide additional prognostic information to cTnT and NT-proBNP testing in patients with cTnT concentrations below the detection limit. In particular, the hs-cTnT cutoff of 0.016 μg/L enables identification of low-risk patients.

Objective: To evaluate early-pregnancy changes in lipid profile in recurrent preeclampsia., Methods: In this retrospective observational study, blood samples were obtained from 41 normotensive women with a history of early-onset preeclampsia preconceptionally and at 12 and 16 weeks in the next pregnancy. We assessed triglycerides (TGs), total cholesterol (TC), and high- and low-density lipoprotein cholesterol (HDL-C and LDL-C, respectively). We analyzed differences in longitudinal patterns between normal and recurrent preeclamptic next pregnancy using mixed-design repeated measurements analysis of covariance (ANCOVA)., Results: Eleven (28%) women developed recurrent preeclampsia. Eighteen (45%) women had a normal pregnancy. In normal pregnancy, LDL-C declines transiently in the first trimester (P < .01). In women who develop recurrent preeclampsia later on this decline was absent. Moreover, from 12 weeks onward the elevating levels of HDL-C stagnates in women who subsequently develop recurrent preeclampsia (P = .02)., Conclusion: These observations point to an abnormal early adaptation of lipid metabolism to pregnancy preceding clinical manifestation of preeclampsia.

The present study examined the associations between genetic variation in folate metabolism on the one hand and cognitive functioning and mood on the other in healthy individuals. Two independent population-based samples were used, including 777 participants, aged 24-82 years, from the Maastricht Aging Study (MAAS); and 818 participants, aged 50-70 years, from the Folic Acid and Carotid Intima-Media Thickness (FACIT) study. Thymidylate synthase (TS) 2R→3R and serine hydroxymethyltransferase (SHMT1) 1420C→T polymorphisms were determined in both populations. In addition, the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C→T polymorphism was determined in the MAAS population. Cognitive performance was assessed in both populations using a neuropsychological test battery. In the MAAS population only, cognitive performance was retested after 12years of follow-up (n=612), and mood was measured at baseline (n=772) and 12-year follow-up (n=565) by means of the depression subscale of the Symptom Checklist 90. We found that in both study populations, cognitive performance was not associated with TS 2R→3R or SHMT1 1420C→T polymorphisms at baseline, after correction for age, sex, and level of education. The MTHFR 677C→T polymorphism was not associated with cognitive performance in the MAAS population. None of the polymorphisms in the MAAS population were related to mood at baseline or over 12 years. In conclusion, our findings do not support the involvement of genetic variation in folate metabolism in cognitive performance or mood in healthy individuals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Often, the connection between drug use and the development of related inflammatory damage or idiosyncratic toxicities is hard to recognize and objectify. The presence of cytochrome P450 (CYP) variant genotypes appears to be a substantial susceptibility risk factor in the development of drug-induced pulmonary adverse events. We hypothesized that the presence of variant alleles may be associated with serious complications of illicit drug use., Case Report: We report the cases of two cocaine users who developed a 'flu-like' syndrome with diffuse interstitial infiltrates after cocaine abuse. Genotyping for CYP (CYP2C9, CYP2C19) and vitamin K epoxide reductase complex 1 (VKORC1) allelic variants (-1639G/A and 1173C/T) was performed in these two patients. Both cases were heterozygous for VKORC1 variant alleles, and both possessed a CYP2C polymorphism (case 1: CYP2C19*1/*2; case 2: CYP2C9*1/*3)., Conclusions: The described drug abuse cases suggest that an association between the presence of CYP2C and VKORC1 allelic variants and cocaine-induced interstitial lung damage is highly likely. It is assumed that these polymorphisms contribute to intra-individual variability in drug response and toxicity, including cocaine response and toxicity. Moreover, the importance of including pharmacogenomics in the work-up of patients with suspected drug-induced (lung) toxicity, such as alveolar hemorrhage, is highlighted by these cases.

Background and Aims: Studies evaluating the effect of arginine supplementation in human wound healing are inhomogeneous with conflicting results. This study aims to clarify the role of arginine supplementation in the healing of human skin graft donor sites., Methods: 35 subjects undergoing skin autografting were randomly assigned to receive intravenous arginine (n = 16) or placebo (n = 19) for 5 days in a dose of 30 g of arginine or an isovolumetric amount of placebo (25.2g of alanine). Wound healing was evaluated at the donor sites by objectifying angiogenesis, reepithelialization and neutrophil influx. Plasma amino acid concentrations were measured to evaluate our intervention., Results: The two groups were comparable in age, morbidity and nutritional, metabolic and inflammatory state. Plasma arginine and alanine levels increased significantly upon supplementation in the two groups, respectively. No differences were found between the arginine supplementation group and the placebo group in the studied parameters. Placebo vs. arginine; mean ± SD: neutrophil influx on day 2: 6.67 ± 3.0 vs. 6.57 ± 3.3, p = 0.66; angiogenesis on day 10: 8.0 ± 2.8 vs. 8.9 ± 3.1; reepithelialization in % on day 10: 81 ± 8.5 vs. 85 ± 7.1., Conclusion: Intravenous arginine supplementation does not improve angiogenesis, reepithelialization or neutrophil influx in healing of human skin graft donor sites., (Copyright © 2010 Elsevier Ltd and ISBI. All rights reserved.)

The aims of this study were to assess the association of BTNL2G16071A with the course of pulmonary sarcoidosis and to verify the association with disease predisposition. In addition, the linkage between BTNL2G16071A and certain human leukocyte antigen (HLA)-DRB1/DQB1 types was investigated. In a retrospective case-control study BTNL2G16071A, HLA-DQB1, and HLA-DRB1 were typed in 632 sarcoidosis patients. These patients were classified into 304 patients with persistent sarcoidosis and 328 patients with nonpersistent sarcoidosis. The BTNL2 16071A variant allele was present significantly more often in patients with persistent disease (92.4%; 281/304) compared with patients demonstrating a nonpersistent course (86.6%; 284/328; odds ratio (OR) = 1.89 with 95% confidence interval (95% CI) 1.11-3.22). Furthermore, BTNL2 16071A variant allele carriers have an increased risk (OR = 1.85, 95% CI 1.19-2.88) of developing sarcoidosis. Moreover, the strong linkage between variant allele and HLA-DRB1*15 presence (OR = 8.43, 95% CI 3.02-23.5) was confirmed. The presence of a BTNL2G16071A variant allele almost doubles the risk of progressing to persistent pulmonary sarcoidosis in addition to increasing the risk of developing sarcoidosis. Presumably, these increased risks are caused by the strong linkage between BTNL2G16071A and DRB1*15. The choice between determining BTNL2G16071A SNP or the HLA-DRB1 type depends on the ability and/or availability to perform either test., (Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Purinergic receptors are important for the regulation of inflammation, muscle contraction, neurotransmission and nociception. Extracellular ATP and its metabolites are the main ligands for these receptors. Occasional reports on beneficial results of ATP administration in human and animal studies have suggested the bioavailability of oral ATP supplements. We investigated whether prolonged daily intake of oral ATP is indeed bioavailable. Thirty-two healthy subjects were randomised to receive 0, 250, 1250 or 5000 mg ATP per d for 28 d by means of enteric-coated pellets. In addition, on days 0 and 28, all thirty-two subjects received 5000 mg ATP to determine whether prolonged administration would induce adaptations in the bioavailability of ATP. ATP supplementation for 4 weeks did not lead to changes in blood or plasma ATP concentrations. Of all ATP metabolites, only plasma uric acid levels increased significantly after the administration of 5000 mg of ATP. Prolonged administration of ATP was safe as evidenced from liver and kidney parameters. We conclude that oral administration of ATP only resulted in increased uric acid concentrations. On the basis of these findings, we seriously question the claimed efficacy of oral ATP at dosages even lower than that used in the present study.

Chorioamnionitis frequently induces a fetal inflammatory response syndrome (FIRS), characterized by an elevation of proinflammatory mediators and systemic inflammation. Although there is increasing evidence that inflammation and lipid metabolism influence each other, the effects of chorioamnionitis-induced FIRS on fetal lipid homeostasis are currently not known. Accordingly, we hypothesize that chorioamnionitis induces an inflammatory response in the fetal liver, consequently leading to metabolic disturbances. Chorioamnionitis was induced by intra-amniotic injection of 10 mg endotoxin (control) for 2 d or 2 wk before delivery. Saline injections were given to controls. The effect of chorioamnionitis on hepatic inflammation and metabolic parameters was analyzed in ovine fetuses at the GA of 125 d (normal GA = 150 d). We found that 2 d after the endotoxin injections, inflammatory markers were significantly higher compared with controls. In addition, lipid and glucose metabolism were disturbed in response to endotoxin. Moreover, the antioxidant state capacity was reduced, and hepatic damage was apparent. Two weeks after the endotoxin injections, the fetal livers were still inflamed and had higher glucose concentrations in the blood. In addition, the levels of markers for hepatic damage (alanine aminotransferase and aspartate aminotransferase) were increased. In conclusion, chorioamnionitis induces liver inflammation leading to metabolic disturbances in the fetus.

Objective: To characterize the influence of Gelofusine (succinylated gelatin) on the performance of point-of-care testing (POCT) for human chorionic gonadotropin (hCG) devices., Design and Methods: Three brands of urine and urine/serum hCG POCT devices were verified., Results: Succinylated gelatin affected the performance of the control band in the QuickVue hCG POCT devices. Alternative devices were not affected. The hCG test performance is not influenced., Conclusions: Gelofusine affects specifically the quality control performance of the QuickVue hCG POCT devices., (Copyright © 2010. Published by Elsevier Inc.)

Matrix γ-carboxyglutamate (Gla) protein (MGP) is an important local inhibitor of vascular calcification, which can undergo two post-translational modifications: vitamin K-dependent γ-glutamate carboxylation and serine phosphorylation. While carboxylation is thought to have effects upon binding of calcium-ions, phosphorylation is supposed to affect the cellular release of MGP. Since both modifications can be exerted incompletely, various MGP species can be detected in the circulation. MGP levels were measured with two commercially available competitive and two novel sandwich assays in healthy controls, in patients with rheumatic disease, aortic valve disease, and end-stage renal disease, as well as in volunteers after vitamin K supplementation (VKS) and treatment with vitamin K antagonists (VKA). Major differences were found between the MGP assays, including significantly different behaviour with regard to vascular disease and the response to VKA and VKS. The dual-antibody assay measuring non-phosphorylated, non-carboxylated MGP (dp-ucMGP) was particularly sensitive for these changes and would be suited to assess the vascular vitamin K status. We conclude that the different assays for particular circulating MGP species allows the assessment of various aspects of the MGP system.

Purpose: This multicenter, phase II study evaluates the efficacy and safety of erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, plus sorafenib, a multityrosine kinase inhibitor against vascular endothelial growth factor receptors, in patients with previously untreated advanced non-small cell lung cancer (NSCLC)., Experimental Design: Chemotherapy-naïve patients with stage IIIB/IV NSCLC received erlotinib (150 mg once a day) and sorafenib (400 mg twice a day) until disease progression or unacceptable toxicity. The primary end point was the rate of nonprogression at 6 weeks. Secondary end points included objective response rate (ORR), time to progression, overall survival, and adverse events. Exploratory end points included pretreatment EGFR and KRAS mutation status, pharmacokinetics, and cytochrome P450 polymorphisms., Results: Fifty patients initiated therapy. The nonprogression rate at 6 weeks was 74%: 12 (24%) partial response and 25 (50%) stable disease. Ultimately, the ORR was 28%. Median time to progression was 5.0 months [95% confidence interval (95% CI), 3.2-6.8 months]. Median overall survival was 10.9 months (95% CI, 3.8-18.1 months). Grade 3/4 adverse events included fatigue (16%), hand-foot skin reaction (16%), rash (16%), diarrhea (14%), and hypophosphatemia (42%). There was one treatment-related fatal pulmonary hemorrhage. Patients with wild-type EGFR had a higher ORR (19%) than previously reported for single-agent erlotinib/sorafenib. Erlotinib levels were lowered. This was associated with CYP3A4 polymorphism and was possibly due to sorafenib., Conclusion: Despite a possible drug interaction, sorafenib plus erlotinib has promising clinical activity in patients with stage IIIB/IV NSCLC and has an acceptable safety profile. Further evaluation of this combination as potential salvage therapy in EGFR mutation-negative patients and the possible drug interaction is warranted., (Copyright 2010 AACR.)

Background: Diffuse alveolar hemorrhage (DAH) is a life-threatening bleeding complication that can occur as a result of oral anticoagulation therapy., Objective: We hypothesized that in patients treated with coumarins, alveolar hemorrhage is associated with vitamin K epoxide reductase (VKORC1) and cytochrome P450 (CYP) 2C9 (CYP2C9) variant alleles. In addition, in the case of acenocoumarol use, CYP2C19 allelic variants also play a role., Methods: During a 7-year period, data on patients using coumarins with confirmed DAH were gathered. Of 173 confirmed DAH cases, 75 received oral anticoagulants, and 63 (84%) of these 75 patients were included because their DNA was available. For genotyping the CYP2C9*2 (430C>T), CYP2C9*3 (1075A>C), CYP2C19*2 (681G>A), CYP2C19*3 (636G>A), VKORC1 (-1639G>A), and VKORC1 (1173C>T) single nucleotide polymorphisms (SNPs), real-time PCRs were performed., Results: In 62 (98.4%) of 63 patients with DAH, variant alleles were found. In 51 (81.0%) of the 63 patients, VKORC1 allelic variants (20 homozygotes and 31 heterozygotes) were present. In 31 (49.2%) of the 63 DAH cases, CYP2C9 allelic variants (three homozygotes, 26 heterozygotes, and two compound heterozygotes) were observed, and in 20 (32.0%) of the 63 patients, variant alleles of both genes were observed., Conclusion: Genotyping of four SNPs for VKORC1 and CYP2C9 polymorphisms is useful in predicting a high probability of the occurrence of DAH in patients receiving oral anticoagulants. Early and timely use of genotyping is recommended to prevent a fatal outcome and to provide safer and more individualized anticoagulant therapy.

Erlotinib is increasingly being used for the treatment of non-small cell lung cancer. The recommended dose is 150 mg/day and no efficacy data is available for lower doses. We describe a case of dramatic tumor response to 50 mg erlotinib in a patient with EGFR mutation positive NSCLC who developed a severe rash on full dose erlotinib. Rash is known to correlate with response and survival in patients treated with erlotinib. Our case suggests that in the presence of rash, dose reductions to "subtherapeutic" levels remain effective and may prevent unnecessary early treatment termination.

Aim: To evaluate whether the quality and quantity of DNA isolated from noninvasively obtained buccal swab (BS) samples, using the previously described isolation method for dried blood spot (DBS) samples was satisfactory., Materials & Methods: From 25 healthy volunteers, DBS samples were obtained by the capillary finger prick method and BS samples were obtained by rubbing a sterile, dry cotton swab against the inside of their cheek. Thereafter, DNA was isolated. In addition, the quantity of the obtained DNA was measured and melting curve analyses for both sampling methods were performed to establish the quality of the obtained DNA from both the DBS and BS samples., Results: The derivative melting curves of the DNA samples obtained from the capillary blood and BS were comparable and highly reproducible. The mean DNA concentrations measured were 16.0 ng/microl (12.6-19.4 ng/microl) and 70.2 ng/microl (57.3-83.1 ng/microl), respectively, for the DBS and BS samples (p < 0.001)., Conclusion: The DBS DNA isolation method appeared to be extremely useful to discriminate between genotypes. This expands the possibilities of this quick and easy DNA isolation procedure. In particular, the noninvasive BS sampling method appeared to be a good alternative to invasive sampling methods.

Background: Diagnosis of ventilator-associated pneumonia (VAP) is difficult. The usefulness of high-sensitivity procalcitonin (ProCa-S) and high-sensitivity C-reactive protein (CRPH) in bronchoalveolar lavage (BAL) fluid and serum in the prediction of VAP was determined., Methods: The study was conducted over a 28-month period (November 1999-June 2002) at the University Hospital Maastricht. BAL fluid samples were collected from patients admitted to the intensive care unit. Differential cell count and quantitative culture of BAL fluid were performed. C-reactive protein (CRP) and procalcitonin (PCT) on BAL fluid were determined by means of two high-sensitivity kits (CRPH and ProCa-S, respectively). Since both kits were designed for use on serum, validation for use on BAL fluid was performed., Results: A total of 117 patients were included. 43.6% (51/117) had microbiologically confirmed VAP. Both CRPH and ProCa-S showed good matrix effect, linearity and intra- and inter-assay variation. No significant differences in PCT and CRP concentrations in serum and BAL fluid were found between the VAP and the non-VAP group., Conclusions: Both the ProCa-S and the CRPH kits can be used for assessing the concentration of PCT and CRP in BAL fluid, respectively. PCT and CRP concentrations in BAL fluid appeared to be of no additional value in the diagnosis of VAP.

Several commercial DNA isolation kits are available for extracting the genomic DNA from the ethylene diamine tetra-acetic acid (EDTA) whole blood samples. To obtain DNA from whole blood these DNA isolation procedures require quite some hands on time and are rather expensive. An alternative technique could be dried blood spot (DBS) sampling, with which DNA isolation is faster, cheaper and logistics are easier. We have developed a non-commercial DBS method and examined its performance in practice. DNA isolation from EDTA blood samples and made blood spots on filter paper from 106 renal transplant recipients were compared. Additionally, DNA isolation with a column method and two different DBS method was performed for 10 healthy volunteers and compared. Also DNA isolation with only capillary blood using both DBS methods from another 100 healthy volunteers has been investigated. Real-time PCR FRET assays for the CYP3A4 A-392G, CYP3A5 A6986G, ABCB1 C1236T, G2677T/A and C3435T polymorphisms were used and the melting curves of both DNA isolation methods were compared. In all cases DNA extracted with the column method corresponded completely with the results of the DNA isolated with the DBS procedure. Hence, DNA isolation from filter paper appears to be a useful alternative for the commercially available DNA isolation kits.

Background: Familial combined hyperlipidemia (FCHL) is a genetic form of dyslipidemia, which is characterized by an increased cardiovascular risk. The current study was conducted to investigate the relation of endothelial, inflammatory and fibrinolysis markers with the presence of hypertriglyceridemia and fatty liver in FCHL, in order to advance insight in their contribution to the cardiovascular risk profile., Materials and Methods: Key plasma markers of low-grade inflammation, endothelial dysfunction and fibrinolysis were measured in 38 hypertriglyceridemic FCHL patients and 38 age and sex-matched spouses. The presence of fatty liver was determined with ultrasound., Results: hsCRP, vWF, PAI-1, tPA and tPA/PAI-1 complex levels were significantly higher in hypertriglyceridemic FCHL patients compared to spouses (p<0.05). Subsequent analyses revealed that these increased levels were confined to FCHL patients with the fatty liver phenotype (n=25). Only PAI-1 and tPA levels were also elevated in the hypertriglyceridemic FCHL patients without fatty liver (n=13). Of interest, 11 hypertriglyceridemic non-FCHL patients with the E2/E2 genotype displayed significantly lower PAI-1 levels when compared to the overall FCHL population (p=0.001), implicating a role for apolipoprotein E in the relation of PAI-1 with plasma triglycerides., Conclusion: Markers of fibrinolysis were increased in all hypertriglyceridemic FCHL patients, whereas an increased state of endothelial dysfunction and inflammation was particularly observed in those hypertriglyceridemic FCHL patients who also have fatty liver. These results demonstrate the complex genesis of the unfavourable cardiovascular risk profile that is present in FCHL, and illustrate the potential risk of fatty liver above, and beyond hypertriglyceridemia per se.

Background: Most drugs currently used in clinical practice are effective in only 25% to 60% of patients, while adverse drug reactions (ADRs) as a consequence of treatment are estimated to cost billions of US dollars and tens of thousands of deaths., Aim: To review the prevalence and clinical significance of cytochrome P450 polymorphisms., Results: The cytochrome P450 enzyme families 1-3 are responsible for 70 to 80% of all phase I dependent drug metabolisms. In 90% metabolic activity dependents on six enzymes: CYP1A2, CYP3A, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Polymorphisms in the CYP450 gene can influence metabolic activity of the subsequent enzymes. A poor metabolizer (PM) has no or very poor enzyme activity. A consequence of PM is drug toxicity if no other metabolic route is available, or when multiple drugs are metabolized by the same cytochrome. In that case dose reduction is an option to prevent toxic effects., Conclusions: In the future genotyping should be considered to identify patients who might be at risk of severe toxic responses, in order to guide appropriate individual dosage. Medical therapy should be a close cooperation between clinicians, pharmacologists and laboratory specialists, leading to reduced therapeutic errors, ADRs and health care costs.

Objective: In literature, a great diversity of limited sampling strategies (LSS) have been recommended for tacrolimus monitoring, however proper validation of these strategies to accurately predict the area under the time concentration curve (AUC0-12) is limited. The aim of this study was to determine whether these LSS might be useful for AUC prediction of other patient populations., Methods: The LSS from literature studied were based on regression equations or on Bayesian fitting using MWPHARM 3.50 (Mediware, Groningen, the Netherlands). The performance was evaluated on 24 of these LSS in our population of 37 renal transplant patients with known AUCs. The results were also compared with the predictability of the regression equation based on the trough concentrations C0 and C12 of these 37 patients. Criterion was an absolute prediction error (APE) that differed less than 15% from the complete AUC0-12 calculated by the trapezoidal rule., Results: Thirteen of the 18 (72%) LSS based on regression analysis were capable of predicting at least 90% of the 37 individual AUC0-12 within an APE of 15%. Additionally, all but three LSS examined gave a better prediction of the complete AUC0-12 in comparison with the trough concentrations C0 or C12 (mean 62%). All six LSS based on Bayesian fitting predicted <90% of the 37 complete AUC0-12 correctly (mean 67%)., Conclusions: The present study indicated that implementation of LSS based on regression analysis could produce satisfactory predictions although careful evaluation is necessary.

Tacrolimus, an immunosuppressant used after organ transplantation, has a narrow therapeutic range and its pharmacokinetic variability complicates its daily dose assessment. P-glycoprotein (P-gp), encoded by the adenosine triphosphate-binding cassette B1 (ABCB1) and the cytochrome (CYP) 3A4 and 3A5 enzymes appears to play a role in the tacrolimus metabolism. In the present study, two different renal transplant recipient groups were used to examine the influence of ABCB1 and CYP3A polymorphisms on the daily tacrolimus dose and several pharmacokinetic parameters. In total 63 Caucasian renal transplant recipients divided into 26 early [median (range) of the days since transplantation - 16 (3-74)] and 37 late [median (range) of the days since transplantation - 1465 (453-4128)] post-transplant recipients were genotyped for ABCB1 and CYP3A polymorphisms. The pharmacokinetic parameters of tacrolimus were determined for all renal transplant recipients and correlated with their corresponding genotypes. A significant difference in allele frequencies of the CYP3A4*1B (P = 0.028) and CYP3A5*1 (P = 0.022) alleles was observed between the early and late post-transplant recipient groups. Significantly higher dose-normalized trough levels (dnC(0)), dose-normalized area under the curve (dnAUC(0-12)), and dose-normalized maximum concentration (dnC(max)) were observed for carriers of the CYP3A5*3 variant allele in both renal transplant patient groups. Except for the daily tacrolimus dose (P = 0.025) no significant differences were observed for carriers of the CYP3A4*1B variant allele. Neither the individual ABCB1 polymorphisms nor the ABCB1 haplotypes were associated with any pharmacokinetic parameter. We noticed that patients carrying a CYP3A5*1 allele require a twofold higher tacrolimus dose compared with homozygous carriers of the CYP3A5*3 variant allele to maintain the target dnAUC(0-12). Therefore, genotyping for the CYP3A5*3 variant allele can contribute to a better and more individualized immunosuppressive therapy in transplant patients.

Tacrolimus has a narrow therapeutic window and a wide interindividual variation in its pharmacokinetics. The cytochrome P450 3A (CYP3A) and the ATP-binding cassette B1 (ABCB1) genes play an important role in the tacrolimus disposition. Therefore, the aim of this study was to evaluate whether CYP3A and ABCB1 polymorphisms are associated with the area under the time concentration curve (AUC0-12) calculated using a two time point sample strategy. The CYP3A and ABCB1 genotypes were determined by real-time polymerase chain reaction (RT-PCR) fluorescence resonance energy transfer (FRET) assays in 103 Chinese renal transplant recipients and consequently related to their dose-normalized (dn)AUC0-12. A significant allele-dependent effect (Kruskal-Wallis; p < 0.001) was observed between the CYP3A5*3 polymorphism and the dnAUC0-12. Multiple regression analysis showed that the CYP3A5*3 polymorphism is the most significant independent variable and explained 35% of the dose requirement variability in relation to tacrolimus use. Regarding the ABCB1 G2677T/A and C3435T polymorphisms, a trend was observed between the different genotypes and the dnAUC0-12. In conclusion, the CYP3A5*3 polymorphism may be an important factor in determining the dose requirement for tacrolimus and genotyping can help determine the initial daily dose required by individual patients for adequate immunosuppression.

Background: Accumulation of larger molecular weight uraemic toxins molecules may have a negative effect on the cardiovascular and nutritional state of dialysis patients and influence uraemic symptomatology. Their clearance can be enhanced by the use of haemofiltration (HF)., Methods: The effects of low-flux haemodialysis (HD) (ultrapure dialysate; polyamide membranes) and pre-dilution on-line HF (1:1 blood/substitution ratio; target filtration volume: 1.2 times body weight) on cardiovascular and nutritional parameters, interdialytic levels of uraemic toxins and quality of life (QOL; Laupacis questionnaire) were assessed during 1 year follow-up. Forty patients were randomized., Results: After 1 year, 27 patients were eligible for analysis (HF: 13 patients; HD: 14 patients). Left ventricular mass index did not change in the HF patients (127+/-33 --> 131+/-36 g/m(2) after 12 months) or in the HD group (135+/-34 --> 138+/-32 g/m(2)). Also, there were no changes in pulse wave velocity, and 48 h systolic and diastolic blood pressures. Lean body mass, assessed by dual-energy X-ray absorptiometry, increased in the HF group (44.8+/-8.9 --> 46.2+/-9.6 kg; P<0.05), but not in the HD group (49.4+/-9.2 --> 50.6+/-8.8 kg), although differences between groups were not significant. Insulin-like growth factor-1 levels remained stable in the HF patients, but decreased in the HD group (P<0.05 between groups). QOL for physical symptoms improved in the HF group (4.2+/-1.2 --> 5.0+/-1.1; P<0.05 within the HF group and P = 0.06 between groups), but not in the HD group (4.0+/-1.0 --> 4.4+/-1.4). beta2-microglobulin, complement factor D and homocysteine decreased significantly in the HF but not in the HD group, whereas l-ADMA, leptin and advanced glycation end-products-related fluorescence did not change., Conclusions: No changes in cardiovascular parameters were observed during pre-dilution on-line HF compared with low-flux HD. Treatment with on-line HF resulted in marked changes in the uraemic toxicity profile, an improvement in physical well-being and a small improvement in nutritional state.

The polymorphisms (OATP)1B1 A388G and T521C of the solute carrier organic anion-transporter family member 1B1 gene (SLCO1B1), previously known as OATP-C, have potential impacts on drug metabolism. In order to establish a fast and consistent assay for these polymorphisms, rapid speed polymerase chain reaction (PCR) fluorescence resonance energy transfer (FRET) assays on the LightCycler were developed for both OATP1B1 polymorphisms. A locked nucleic acid (LNA) on the polymorphic location within the sensor probe was necessary to discriminate both alleles of the OATP1B1 T521C polymorphism. To confirm the reliability of both real-time PCR FRET assays, these new methods were validated by genotyping 120 samples using a PCR restriction fragment length polymorphism (RFLP) assay and an allele-specific PCR. The results of the real-time PCR FRET assays were completely in line with conventional PCR methods, indicating that the real-time PCR FRET assays are appropriate for clinical settings.

Background: A vast amount of scientific research is directed towards the beneficial effects of antioxidants on health. For this reason, several assays have been developed to determine the total antioxidant capacity of blood., Methods: In this study two procedures based on the use of the green-blue 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS(*+)) were compared. In the first (commercially available) procedure, ABTS(*+) was generated in the presence of the blood sample. In the second procedure, referred to as the decolorization assay, antioxidants react with preformed ABTS(*+)., Results: It was found that the first procedure leads to greater underestimation of the actual antioxidant capacity and is more prone to artifacts than the second procedure. Therefore, only the latter procedure was evaluated in detail and it appeared that (i) plasma is preferred over serum, (ii) the high background produced by albumin can be circumvented by deproteination, (iii) samples can be stored at -80 degrees C for 12 months, and (iv) the assay has high precision. Due to poor linearity, the procedure has to be standardized to allow sample comparison., Conclusions: The decolorization assay is a reliable and robust assay that can be applied routinely to predict the antioxidant capacity of blood.

Background: An important advantage of convective therapies is improved vascular reactivity. However, it is not well known whether the vascular response during convective therapies remains superior when compared to haemodialysis (HD) with an adjusted temperature of the dialysate. It has also been suggested that convective therapies may impair small electrolyte removal through an effect on the Donnan equilibrium. In the present study, we compared the haemodynamic response and small electrolyte removal between pre-dilution on-line haemofiltration (HF) and HD procedures., Methods: Cardiac output (CO), central blood volume (CBV) and peripheral vascular resistance (PVR) were assessed, using the saline dilution technique, in 12 stable patients during HF and HD with two different temperatures of the dialysate [36.5 and 35.5 degrees C (HD(36.5) and HD(35.5))]. Balances for sodium, potassium, calcium and conductivity were assessed using total dialysate/filtrate collections. Target filtration volume for HF was 1.2 times body weight. The temperature of the infusate was 36.5 degrees C., Results: The change (Delta) in CBV was less during HD with a dialysate temperature of 35.5 degrees C (-0.03+/-0.14 l; P<0.05) compared to HF (-0.16+/-0.05 l) and HD(36.5) (-0.11+/-0.14 l), but the other haemodynamic parameters did not differ between the studied techniques. DeltaPVR was significantly related to DeltaCBV (r = -0.46; P<0.01), whereas DeltaCBV was related to ultrafiltration rate (r = -0.34; P = 0.05). DeltaCO was related to DeltaCBV (r = 0.62; P<0.001). Solute balances did not differ between HF and HD., Conclusion: Using the saline dilution method, no difference in the change in CO and PVR was observed between on-line HF vs HD(36.5) and HD(35.5). Only CBV declined to a significantly lesser degree during HD(35.5), although absolute differences were small. Changes in the other haemodynamic variables appeared more dependent upon the degree and rapidity of fluid removal than upon the treatment modality. No difference in small electrolyte balance was observed between HF and HD, suggesting that ionic removal is not impaired during on-line HF.

The hypothesis was tested that thyroid function, as indicated by serum thyroid-stimulating hormone (TSH) level, is associated with cognitive performance in a healthy aging population. In a random sample of 120 participants recruited from the Maastricht Aging Study (MAAS), aged between 49 and 71 years, we assessed TSH level, mood state (Symptom Check List, subscale depression), and three domains of cognitive function: verbal memory, general sensorimotor speed, and complex flexibility. After correction for age, sex, and educational level, a negative association between TSH and memory function was apparent: higher levels of TSH predicted lower levels of memory performance. Exclusion of individuals with TSH levels suspect for thyroid disorder (n=2) or who were on thyroid replacement (n=3) attenuated this association. Furthermore, additional control for mood status reduced the association below the significance level. No interaction between age and TSH on cognition was found, which indicated that the TSH-memory association was independent of age group level. We conclude that the association between TSH level and memory performance was small and dependent on mood status and the presence of (possible) thyroid disease in this relatively healthy population based sample. Prospective studies are needed to address the role of thyroid function in age-related cognitive decline.

Background: During hemodialysis, vascular reactivity is impaired, which can be corrected by lowering dialysate temperature. It has also been shown that nitric oxide (NO) is related to intradialytic hypotension. As NO synthesis may be temperature-dependent, this study addressed the influence of dialysate temperature on the NO synthetic capacity of plasma., Methods: NO synthetic capacity was studied during hemodialysis with a dialysate temperature of 37.5 degrees C (dialysis-37.5 degrees C) and programmed extracorporeal blood cooling (cool dialysis; Blood Temperature Monitor; Fresenius C) in 12 stable patients. NO synthetic capacity was assessed ex vivo by [3H]L-citrulline formation from [3H]L-arginine in cultured endothelial cells after incubation with plasma samples obtained during the respective sessions., Results: Core temperature decreased (-0.32 +/- 0.10 degrees C) and energy transfer rate was significantly lower (-27.5 +/- 2.8 W; p < 0.05) during cool dialysis compared to dialysis-37.5 degrees C (0.19 +/- 0.06 degrees C and -0.8 +/- 1.2 W respectively; p < 0.05). Systolic blood pressure decreased during dialysis-37.5 degrees C (-19 +/- 4 mm Hg; p < 0.05), but not during cool dialysis (-6 +/- 5 mm Hg). NO synthetic capacity increased during dialysis-37.5 degrees C (55.5 +/- 9.3 to 73.5 +/- 10.2 pmol/10(5) cells; p < 0.05), in contrast to cool dialysis (67.3 +/- 11.1 to 66.2 +/- 10.8 pmol/10(5) cells)., Conclusion: The stimulatory effect of uremic plasma on endothelial NO synthesis was augmented during dialysis-37.5 degrees C but not during cool dialysis., (Copyright 2004 S. Karger AG, Basel)

Background: The implementation of a high-sensitivity C-reactive protein (hs-CRP) assay as a routine laboratory parameter may be necessary. It would be most practical to use one CRP method giving reliable results for the whole concentration range. We report here the evaluation of two new hs-CRP methods, which cover both the low and the high concentration ranges., Methods: The BN ProSpec hs-CRP (Dade Behring) and Synchron LX 20 PRO hs-CRP methods were compared with the existing hs-CRP IMMAGE method (taken as a reference) and, for the high concentration range, also with the routine Synchron LX 20 CRP method (all from Beckman). Agreement among methods was examined for 521 samples. Reference values were estimated in 291 blood donors. Additionally, the influence of sample turbidity, a major problem of the present Synchron LX20 CRP method, was evaluated., Results: Measurement of CPR by the BN ProSpec was linear down to 0.2 mg/L, whereas the linearity of Synchron LX20 PRO showed some systematic discrepancies. Over the whole measured range (0.2-250 mg/L), precision (coefficient of variation, CV) was < or =3.7% for the BN ProSpec and < or =6.1% for the LX20 PRO. The Synchron LX20 PRO hs-CRP method was found to be superior to the current routine Synchron LX20 CRP method with regard to precision in the low concentration range and the influence of sample turbidity. Both in the low concentration range and especially in the high concentration range, large discrepancies between methods were observed., Conclusion: Although acceptable performance was found for the Synchron LX20 PRO hs-CRP method, overall the performance of the BN ProSpec hs-CRP method was superior. However, standardization among assays needs further improvement in both the low and the high concentration ranges.

Two cases of interstitial pneumonia with cardiac failure developing in patients treated with the new antidepressant venlafaxine are presented. A strong relationship between the development of the patients' illness and the initiation of venlafaxine treatment was identified. The cytochrome P (CYP) 450 system is involved in the metabolism of venlafaxine, suggesting that alterations in the drug metabolic clearance might be, at least in part, responsible for the development of drug-induced damage in these cases. This might occur either as a consequence of a genetic factor or concomitant drug therapy with an inhibitor of the related CYP system. After identifying the causative agent in the first case, withdrawal of the antidepressant together with corticosteroid treatment led to a favorable outcome. In the other case, the multiorgan failure became fatal. These cases highlight a hitherto undescribed association of an adverse lung reaction and heart failure due to venlafaxine.

An adequate iron management is important in the treatment of anemia and in hemodialysis (HD) patients. Serum ferritin and transferrin saturation (TS) may be influenced by the presence of inflammation. Recently, the soluble transferrin receptor (s-TfR) has been advocated as a parameter of iron status in HD patients. The aim of the present study was to assess firstly the relation between serum ferritin, TS, and s-TfR in HD patients and to predict their agreement (assessed by kappa) in the diagnosis of iron deficiency, and, secondly, to assess the influence of inflammation on the relation between the parameters of iron state. Iron deficiency by either marker was respectively defined as ferritin <100 microg/l, TS <20%, or s-TfR >2.4 microg/ml. In the overall group of patients, TS and s-TfR were significantly related (r = -0.38), whereas s-TfR and serum ferritin were not. Both serum ferritin and TS were related to CRP (r = 0.50 and -0.34; p < 0.05), whereas s-TfR was not. The kappa value for agreement between serum ferritin and TS in the diagnosis of iron deficiency was 0.24 (p = 0.07), 0.12 (p = NS) for the agreement between TS and s-TfR and 0 for that between serum ferritin and s-TfR. In patients with CRP levels

We describe the application of capillary electrophoresis to detect DNA fragments, obtained after amplifying a part of the apolipoprotein E (apoE) gene with polymerase chain reaction (PCR). Compared to conventional agarose slab gel electrophoresis (AGE), CE appears the method of choice with regard to resolution and sensitivity, to detect DNA fragments in the range of 20-100 base pairs. Especially discrimination between apoE2/E2 and apoE2/E3 genotypes is more reliable with CE than with AGE, this being of great clinical value in the diagnosis of familiary dysbetalipoproteinemia.

The well-known Kjeldahl method for the determination of faecal nitrogen is rather complex, time-consuming and expensive. Therefore, the use of near-infrared spectroscopy in determining the amount of nitrogen in faeces has been studied. To our knowledge we are the first to present the calibration equation for the determination of nitrogen with near-infrared spectroscopy. A good correlation (r = 0.96) was found between results from near-infrared spectroscopy and the Kjeldahl method. The imprecision of both methods was comparable. Once the rather laborious calibration has been performed, near-infrared spectroscopy is shown to be a very simple and rapid method for measuring nitrogen in faeces.

The applicability of near-infrared spectroscopy to determine the amount of fat in faeces has been investigated. Near-infrared spectroscopy was favourably compared with the well known titrimetric method (Van de Kamer et al., J Biol Chem 1948; 177:347-55). A good correlation between near-infrared spectroscopy and the titrimetric method was found. The measurement of faecal fat by near-infrared spectroscopy is found to be more precise than the manual method. Moreover, near-infrared spectroscopy is shown to be a very simple and rapid method for measuring fat in faeces. However, it was shown that performing one's own calibration curve is necessary. Due to this necessity and the costs of the apparatus application of near-infrared spectroscopy is especially advantageous in laboratories with a substantial amount of samples to be analysed.

The chemical stability of 2',3'-dideoxyinosine has been studied over a wide pH range (0-12). A stability-indicating HPLC method was used to separate the degradation product from the parent drug. The effects of temperature, ionic strength and buffer components on the degradation kinetics were investigated. Furthermore, the influence of some cyclodextrins (alpha-, beta-, HP-beta-, DM-beta- and gamma-cyclodextrin) on the drug stability have been studied.

The effects of cyclodextrins on the chemical stability of several mitomycin antibiotics in an alkaline medium have been investigated. A stability-indicating high-performance liquid chromatographic method was used to determine the overall degradation rate constants. The influence of various parameters such as structural variations of the cyclodextrins and mitomycins, temperature and pH was studied. It appears that complexation is most favourable with gamma-cyclodextrin. All mitomycin-gamma-cyclodextrin complexes degrade at lower rates than those of the free drugs. Moreover, it was shown that gamma-cyclodextrin influences the equilibrium between mitomycin C and its zwitterion mesomer.

The interaction of alpha-, beta- and gamma-cyclodextrins with the anthracycline antibiotics doxorubicin and daunorubicin was investigated by LC, circular dichroism (CD) and absorption spectroscopy. All studies were performed in aqueous media at different temperatures and pH values. The anthracyclines complex only with gamma-cyclodextrin. Lineweaver-Burk and Scott's plots were used to calculate the stability constants of the anthracycline-gamma-cyclodextrin inclusion complexes.

The effect of cyclodextrins on the stability of six anthracyclines in acidic medium at 50 degrees C has been investigated using a stability-indicating high pressure liquid chromatographic method. The influences of various parameters, such as the structure of cyclodextrins (alpha-cyclodextrin, beta-cyclodextrin, dimethyl-beta-cyclodextrin and gamma-cyclodextrin) and anthracyclines, cyclodextrin concentration, the pH and the presence of a co-solvent, are investigated. Lineweaver-Burk plots were used to calculate the stability constants of the various inclusion complexes as well as the rate constants for degradation of the anthracycline guest molecules in the complexes with the host cyclodextrins. Anthracyclines complexate only with gamma-cyclodextrin to a substantial extent. On complexation the stability of the guest molecule increases, however, the degradation pattern does not alter. The influence of the pH on the degradation of the included molecule is identical to that of the free drug. Addition of co-solvents, such as acetonitrile, causes decomposition of the complex.

LL-D49194 alpha 1 is a recently discovered compound, produced by Streptomyces vinaceus-drappus. This micro-organism produces a number of antibiotics, all showing antibacterial and antitumour activity, of which LL-D49194 alpha 1 is one of the main compounds. The compounds' antitumour effectiveness has been proven in vitro and the drug is undergoing further tests. For the assay of the drug in plasma a high-performance liquid chromatographic (HPLC) system has been developed, preceded by a clean-up step. The drug is extracted from the biological matrix with ethyl acetate followed by direct HPLC analysis of the organic layer via an analytical RP8 column preceded by a guard column to retain endogenous plasma compounds. Detection of drug and metabolites was carried out by fluorescence with reference to a non-fluorescent internal standard detected by UV absorption. The detection limit was 1 ng ml-1 plasma (using 1 ml sample; signal-to-noise ratio, 3), i.e. 1 ng on column. The method has been utilized in a preliminary pharmacokinetic study in rat.