Functional expression,inhibition and induction of CYP enzymes in HepaRG cells

Practically all human hepatocyte cell lines are deficient in major cytochrome P450 (CYP)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the recently developed HepaRG cell line to determine the spectrum of most important CYP enzyme activities involved in xenobiotic metabolism (CYP1A1/2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) and the effect of the prototypical CYP-inducer phenobarbital and a panel of known CYP-selective inhibitors on these activities. Comparison of these activities was carried out with two human primary hepatocyte populations. We show that excluding CYP2A6 and CYP2E1, HepaRG cells express high functional levels of most of the major xenobiotic metabolising CYPs. These activities were found to be selectively inhibited and induced by prototypical CYP-selective inhibitors and inducer at comparable levels to primary hepatocytes. In conclusion, HepaRG cells may be a promising cell line for various applications, which currently employ hepatic subcellular preparations or cultured primary hepatocytes.     

Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry

Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stable-isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether-ethyl acetate from 250 microL or 500 microL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode after separation on a reversed-phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d(2) testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2-100 nmol/L. Interassay CVs were 4.2-7.6 % at mean concentrations of testosterone of 3.3-45 nmol/L. Total measurement uncertainty (U, k = 2) was 12.9 % and 13.4 % at testosterone levels of 2.0 nmol/L and 20 nmol/L, respectively. The limit of detection was 0.05 nmol/L (signal-to-noise ratio = 3) and the overall method recovery of testosterone was 95 %. Correlation (r) with our in-house extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18-30, 31-50, 51-70 and over 70 years were established. Sensitivity and specificity of the LC-MS/MS method offer advantages over immunoassay and make it suitable for use as a high-throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.     

液相色谱-质谱联用技术在体外药物代谢物筛查中的应用

高效液相色谱与质谱联用技术已是现代药物发现中药物代谢产物筛查和鉴定最强大的分析手段,可以从品种繁多的样品中得到丰富的信息。本文关注这些技术在体外代谢研究中的应用,尤其是在筛查和鉴定生物转化形成的化学稳定代谢产物或反应性代谢产物方面的应用。为满足指定检测任务需审慎考虑选择合适的检测仪器,在代谢产物筛查中飞行时间质谱和静电场轨道阱质谱检测效率明显高于其他类型质谱。     

Predictive value of animal models for human cytochrome P450 (CYP)-mediated metabolism: a comparative study in vitro

One major challenge in drug development is defining of the optimal animal species to serve as a model of metabolism in man. The study compared the hepatic drug metabolism characteristics of humans and six widely used experimental animal species. Classical in vitro model enzyme assays with known human cytochrome P450 (CYP) enzyme selectivity were employed and optimized to target human hepatic CYP forms. The profile of CYP activities best resembling the human was seen in mouse followed by monkey, minipig, and dog liver microsomes, with rats displaying the most divergent. The widest interindividual variability was found in CYP3A-mediated midazolam -hydroxylase, and omeprazole sulphoxidase activities in human and monkey liver microsomes. These data demonstrate that if hepatic xenobiotic-metabolizing characteristics were to be the sole reason for the selection of animal species for toxicity studies, then the rat might not be the most appropriate model to mimic human CYP activity patterns.     

Effect of Physical Activity Counseling on Disability in Older People: A 2‐Year Randomized Controlled Trial

OBJECTIVES: To study the effect of a physical activity counseling intervention on instrumental activity of daily living (IADL) disability. DESIGN: Primary care–based, single‐blind, randomized controlled trial. SETTING: City of Jyväskylä, central Finland. PARTICIPANTS: Six hundred thirty‐two people aged 75 to 81 who were able to walk 500 meters without assistance, were at most moderately physically active, had a Mini‐Mental State Examination score greater than 21, had no medical contraindications for physical activity, and gave informed consent for participation. INTERVENTION: A single individualized physical activity counseling session with supportive phone calls from a physiotherapist every 4 months for 2 years and annual lectures on physical activity. Control group received no intervention. MEASUREMENTS: The outcome was IADL disability defined as having difficulties in or inability to perform IADL tasks. Analyses were carried out according to baseline IADL disability, mobility limitation, and cognitive status. RESULTS: At the end of the follow‐up, IADL disability had increased in both groups (P<.001) and was lower in the intervention group, but the group‐by‐time interaction effect did not reach statistical significance. Subgroup analyses revealed that the intervention prevented incident disability in subjects without disability at baseline (risk ratio=0.68, 95% confidence interval=0.47–0.97) but had no effect on recovery from disability. CONCLUSION: The physical activity counseling intervention had no effect on older sedentary community‐dwelling persons with a wide range of IADL disability, although it prevented incident IADL disability. The results warrant further investigation to explore the benefits of a primary care–based physical activity counseling program on decreasing and postponing IADL disability.     

Clustering of cardiovascular risk factors in type 2 diabetes mellitus: prognostic significance and tracking

Summary
Aim   Little attention has been paid to the prognostic significance and tracking effect of risk factor clusters characteristic of type 2 diabetes mellitus. We studied the clustering of eight cardiovascular risk factors (smoking, high body mass index, elevated systolic blood pressure, high serum, low density lipoprotein (LDL) cholesterol, high serum LDL triglycerides, low serum, high density lipoprotein (HDL) cholesterol, high fasting blood glucose and high plasma insulin concentration) and their effect on the prognosis and the tracking effect.
Methods   This study is a population-based prospective follow-up of newly diagnosed type 2 diabetic subjects (n = 133, aged 45–64 years) in Eastern Finland. The following end points were used: all-cause mortality, cardiovascular mortality, and incidences of first myocardial infarction and first stroke. Furthermore, we studied the 'tracking effect' of the risk factor clusters during the 10-year follow-up period.
Results   When the clustering of risk factors typical of type 2 diabetes mellitus was taken into account, all-cause mortality increased from 28.6% to 50.0% (p < 0.05) and cardiovascular disease mortality increased from 14.3% to 50.0% (p < 0.01) depending on the number of risk factors present. The incidence of first myocardial infarction increased from 0% to 40.0% (p < 0.05) as the number of risk factors increased from 0 to 5. In survivors, the proportion of individuals with no risk factors decreased and the proportion on individuals with three to four risk factors increased during the 10-year follow-up period despite the high mortality among the group with many risk factors.
Conclusions   The risk factor clusters among type 2 diabetic subjects are of great predictive value and when not aggressively treated, show a relentless increase despite selective mortality.     

Myocardial fatty acid oxidation in patients with impaired glucose tolerance

Abstract Aims/hypothesis. Fatty acids are an important source of energy in the myocardium. Abnormal myocardial fatty acid metabolism could contribute to the deterioration of cardiac function frequently observed in patients with Type II (non-insulin-dependent) diabetes mellitus. In our previous study, myocardial total uptake of non-esterified fatty acid (NEFA) was measured in patients with impaired glucose tolerance and found to be normal. This study aimed to investigate the subsequent metabolic steps and β-oxidation of NEFA. Methods. A total of 6 men with impaired fasting glucose (age 50 ± 2 years, BMI 29 ± 1 kg/m², means ± SEM) and 6 healthy men (50 ± 1 years, 25 ± 1 kg/m²) were studied in the fasting state. Myocardial blood flow was measured with [¹⁵O]H₂O and positron emission tomography and myocardial NEFA metabolism with [¹¹C]palmitic acid. Results. Myocardial blood flow was normal and not different between the impaired glucose tolerance and the control group (78 ± 6 vs 73 ± 13 ml/100 g/min, NS). The [¹¹C]palmitic acid uptake indices were similar between the groups (10.4 ± 0.5 vs 11.2 ± 0.8 ml/100 g/min, respectively, NS). The clearance of [¹¹C]-palmitate from the myocardium, an index of NEFA β-oxidation, was similar between the groups (half-times of activity 17.6 ± 1.6 vs 19.5 ± 2.3 min, respectively, NS) Conclusion/interpretation. The results indicate that myocardial NEFA uptake and β-oxidation are not altered in patients with IGT. Thus, it is not likely that altered NEFA metabolism contributes to the deterioration of the cardiac function in patients with IGT or Type II diabetes. [Diabetologia (2001) 44: 184–187] Received: 9 June 2000 and in revised form: 25 September 2000     

Physical and psychological functioning of daily living in relation to physical activity. A longitudinal study among former elite male athletes and controls

BACKGROUND AND AIMS: Physical exercise plays an important role in the prevention and reduction of disabilities in elderly people. The aim of this study was to determine the role of physical activity in the physical and psychological functioning of daily living in a cohort of former elite male athletes representing different sports, and controls of middle and old age. METHODS: Subjects were 664 former elite male athletes (mean age 64.4 years) and 500 controls (62.0 years) in middle and old age. Subjects were mailed "Physical activity and health survey" questionnaires in 1985 and 1995. The primary outcomes the physical and psychological functioning of daily living--were assessed in 1995 using items from the Mini-Finland Health Survey. Logistic regression was used for longitudinal as well as cross-sectional analyses to estimate odds ratios (OR) for poor physical and psychological functioning of daily living in relation to recreational physical activity adjusted for age, sport group, life-style, BMI, mood, chronic diseases, personality characteristics, life-events and socio-economic status. RESULTS: In the longitudinal analysis, low levels of physical activity (lowest MET quintile vs highest quintile) in 1985 (OR 4.91, 95% confidence interval (CI) 2.02-11.9), older age (> or =70 yrs vs under 60 yrs OR 9.93, 95% CI 4.90-20.2), depression (OR 2.03, 95% CI 1.01-4.09) and anxiety in 1995 (OR 2.67, 95% CI 1.34-5.32) increased the risk of poor physical functioning of daily living in 1995, whereas an increase in a physical activity between 1985-1995 (OR 0.89, 95% CI 0.83-0.95) protected against poor physical functioning of daily living. A history of participating in specific types of sports, especially among certain power sports (weight-lifting and track & field throwers) (OR 0.19, 95% CI 0.06-0.60) and team sports (OR 0.34, 95% CI 0.15-0.81) did reveal a significant protective effect against poor psychological functioning of daily living in the longitudinal analysis. CONCLUSIONS: This study suggests that an increase in physical exercise supports physical daily functionality. A specific history of sports participation promotes psychological well-being at an older age.