Therapeutic drug monitoring of everolimus using the dried blood spot method in combination with liquid chromatography–mass spectrometry

An assay of everolimus based on finger prick sampling and consecutive application as a blood spot on sampling paper has been developed. We explored several methods [K. Hoogtanders, J. van der Heijden, M. Christiaans, P. Edelbroek, J. van Hooff, L. Stolk, J. Pharm. Biomed. Anal. 44 (2006) 658–664; A. Allanson, M. Cotton, J. Tettey, et al., J. Pharm. Biomed. Anal. 44 (2007) 963–969] and developed a new method, namely the impregnation of sampling paper with a solution of plasma–protein, formic acid and ammonium acetate, in combination with the extraction of the blood spot by filter filtration. This kind of sample preparation provides new possibilities for blood spot sampling especially if analytes are adsorbed to the paper.The dried blood spot was analysed using the HPLC–electrospray-tandem mass spectrometry method, with 32-desmethoxyrapamycin as the internal standard. The working range of our study was 2–30 μg/l. Within this range, intra-and inter-assay variability for precision and accuracy was <15%. Everolimus blood spot samples proved stable for 3 days at 60 °C and for 32 days at 4 °C. Everolimus concentrations of one stable out-patient were compared after both blood spot sampling and conventional venous sampling on various occasions. Results indicate that this new method is promising for therapeutic drug monitoring in stable renal transplant patients.     

Performance of a predictive model to identify undiagnosed diabetes in a health care setting.

OBJECTIVE: To develop a predictive model to identify individuals with an increased risk for undiagnosed diabetes, allowing for the availability of information within the health care system. RESEARCH DESIGN AND METHODS: A sample of participants from the Rotterdam Study (n = 1,016), aged 55-75 years, not known to have diabetes completed a questionnaire on diabetes-related symptoms and risk factors and underwent a glucose tolerance test. Predictive models were developed using stepwise logistic regression analyses with the absence or presence of newly diagnosed diabetes as the dependent variable and various items with a plausible connection to diabetes as the independent variables. The models were evaluated in another Dutch population-based study, the Hoorn Study (n = 2,364), in which the participants were aged 50-74 years. Performances of the predictive models were compared by using receiver-operator characteristics (ROC) curves. RESULTS: We developed three predictive models (PMs), PM1 contained information routinely collected by the general practitioner, while PM2 also contained variables obtainable by additional questions. The third predictive model, PM3, included variables that had to be obtained from a physical examination. These latter variables did not have additive predictive value, resulting in a PM3 similar to PM2. The area under the ROC curve was higher for PM2 than for PM1, but the 95% Cls overlapped (0.74 [0.70-0.78] and 0.68 [0.64-0.72], respectively). CONCLUSIONS: Using only information normally present in the files of a general practitioner, a predictive model was developed that performed similarly to one supplemented by information obtained from additional questions. The simplicity of PM1 makes it easy to implement in the current health care setting.     

Angiographic distribution of lower extremity atherosclerosis in patients with and without diabetes.

AIMS: To determine differences in the anatomic site of atherosclerosis in the lower extremity between patients with and patients without diabetes. DESIGN: Cross-sectional study of patients who underwent angiography of both legs because of symptoms of intermittent claudication, rest and/or night pain, ulceration or gangrene. METHODS: The angiographies of 37 patients with diabetes and 37 patients without diabetes, matched for age, sex and smoking behaviour, were evaluated using the Bollinger scoring system. RESULTS: The mean (sd) Bollinger score in the upper leg (from the abdominal aorta to and including the superficial femoral artery) was higher (P = 0.01) for patients without diabetes (35.3 (22.8)) than for patients with diabetes (23.3 (16.1)). In the lower leg (from the popliteal artery to the posterior tibial artery) patients with diabetes tended to have a higher score than patients without diabetes: 47.4 (34.2) and 37.6 (32.9), respectively (P = 0.22). CONCLUSION: This angiographic study confirms the clinical notion that lower limb atherosclerosis in diabetes is more severe in distal segments of the lower extremity, while the proximal segments remain less attenuated compared with patients without diabetes.     

In vitro effect of a mucolytic thiol agent on the activity of polymorphonuclear leucocyte elastase and antileucoprotease.

We have studied the effects of the mucolytic thiol agent mercapto-ethanesulphonate (mesna) on the activity of both polymorphonuclear leucocyte (PMN) elastase and antileucoprotease in vitro. In all tests a specific synthetic substrate was used to measure elastase activity, which was then related to enzyme activity in the absence of mesna. The relative elastase activity decreased to 67.5% of control values after the enzyme had been incubated in a 120 mmol/l mesna solution. In the sol phase of purulent sputum, elastase activity decreased to 45% after the sol phase had been incubated in a 600 mmol/l mesna solution. The inability to reverse the inhibition of mesna by increasing the substrate concentration indicated that mesna acts as a non-competitive inhibitor of PMN elastase. Incubation of elastase with antileucoprotease reduced the relative elastase activity to 21%. When antileucoprotease was preincubated in a 60 mmol/l mesna solution under identical assay conditions, a relative elastase activity of 39% was observed. Inhibition experiments with mesna treated antileucoprotease, in which sulphydryl groups were blocked with iodoacetamide, strongly suggested that the dissociation constant (Ki) of the fraction of antileucoprotease that retains activity after the incubation with mesna was not changed. Elastase inhibitory activity in mucoid sol phase, which can be ascribed mainly to antileucoprotease, decreased to 53% after incubation with mesna at a concentration of 960 mmol/l. Incubation of PMN elastase/antileucoprotease complex with mesna did not result in any release of active PMN elastase from the antileucoprotease. It is concluded that mesna and other thiol compounds, when locally administered, may influence the proteinase-antiproteinase balance in the airways by their effect on both PMN elastase and antileucoprotease.