Development and validation of a reversed‐phase HPLC method for CYP1A2 phenotyping by use of a caffeine metabolite ratio in saliva

CYP1A2 is important for metabolizing various clinically used drugs. Phenotyping of CYP1A2 may prove helpful for drug individualization therapy. Several HPLC methods have been developed for quantification of caffeine metabolites in plasma and urine. Aim of the present study was to develop a valid and simple HPLC method for evaluating CYP1A2 activity during exposure in xenobiotics by the use of human saliva. Caffeine and paraxanthine were isolated from saliva by liquid‐liquid extraction (chlorophorm/isopropanol 85/15v/v). Extracts were analyzed by reversed‐phase HPLC on a C₁₈ column with mobile phase 0.1% acetic acid/methanol/acetonitrile (80/20/2 v/v) and detected at 273nm. Caffeine and paraxanthine elution times were <13min with no interferences from impurities or caffeine metabolites. Detector response was linear (0.10–8.00µg/ml, R²>0.99), recovery was >93% and bias <4.47%. Intra‐ and inter‐day precision was <5.14% (n=6). The limit of quantitation was 0.10µg/ml and the limit of detection was 0.018±0.002µg/mL for paraxanthine and 0.032±0.002µg/ml for caffeine. Paraxanthine/caffeine ratio of 34 healthy volunteers was significantly higher in smokers (p<0.001). Saliva paraxanthine/caffeine ratios and urine metabolite ratios were highly correlated (r=0.85, p<0.001). The method can be used for the monitoring of CYP1A2 activity in clinical practice and in studies relevant to exposure to environmental and pharmacological xenobiotics. Copyright © 2015 John Wiley & Sons, Ltd.     

High spin limits and non-abelian T-duality

The action of the non-abelian T-dual of the WZW model is related to an appropriate gauged WZW action via a limiting procedure. We extend this type of equivalence to other σ-models with non-abelian isometries and their non-abelian T-duals, focusing on Principal Chiral models. We reinforce and refine this equivalence by arguing that the non-abelian T-duals are the effective backgrounds describing states of an appropriate parent theory corresponding to divergently large highest weight representations. The proof involves carrying out a subtle limiting procedure in the group representations and relating them to appropriate limits in the corresponding backgrounds. We illustrate the general method by providing several non-trivial examples.     

One-dimensional unsteady inertial flow in phreatic aquifers induced by a sudden change of the boundary head

We are examining the classical problem of unsteady flow in a phreatic semi-infinite aquifer, induced by sudden rise or drawdown of the boundary head, by taking into account the influence of the inertial effects. We demonstrate that for short times the inertial effects are dominant and the equation system describing the flow behavior can be reduced to a single ordinary differential equation. This equation is solved both numerically by the Runge-Kutta method and analytically by the Adomian’s decomposition approach and an adequate polynomial-exponential approximation as well. The influence of the viscous term, occurring for longer times, is also taken into account by solving the full Forchheimer equation by a finite difference approach. It is also demonstrated that as for the Darcian flow, for the case of small fluctuations of the water table, the computation procedure can be simplified by using a linearized form of the mass balance equation. Compact analytical expressions for the computation of the water stored or extracted from an aquifer, including viscous corrections are also developed.     

Exit Times,Overshoot and Undershoot for a Surplus Process in the Presence of an Upper Barrier

We study the movement of a surplus process with initial capital u in the presence of two barriers: a lower barrier at zero and an upper barrier at b (b > u). More specifically, we consider the behaviour of the surplus: (a) in continuous time; and (b) only at claim arrival times. For each of these cases, we find the expected time until the process exits the interval [0,b]. We also obtain results related to the undershoot and overshoot of the surplus which, in particular for case (b) above, are derived under the assumption that the distribution of claim sizes and/or claim interarrival times belongs to the mixed Erlang class. In the final section we discuss the implementation of the methods in a number of examples using computer algebra software. These examples illustrate the efficiency of the methods even in fairly complicated cases.     

The black hole and FRW geometries of non-relativistic gravity

We consider the recently proposed non-relativistic Ho?ava–Lifshitz four-dimensional theory of gravity. We study a particular limit of the theory which admits flat Minkowski vacuum and we discuss thoroughly the quadratic fluctuations around it. We find that there are two propagating polarizations of the metric. We then explicitly construct a spherically symmetric, asymptotically flat, black hole solution that represents the analog of the Schwarzschild solution of GR. We show that this theory has the same Newtonian and post-Newtonian limits as GR and thus, it passes the classical tests. We also consider homogeneous and isotropic cosmological solutions and we show that although the equations are identical with GR cosmology, the couplings are constrained by the observed primordial abundance of ⁴He.     

Tail bounds for the joint distribution of the surplus prior to and at ruin

For the classical risk model with Poisson arrivals, we study the (bivariate) tail of the joint distribution of the surplus prior to and at ruin. We obtain some exact expressions and new bounds for this tail, and we suggest three numerical methods that may yield upper and lower bounds for it. As a by-product of the analysis, we obtain new upper and lower bounds for the probability and severity of ruin. Many of the bounds in the present paper improve and generalise corresponding bounds that have appeared earlier. For the numerical bounds, their performance is also compared against bounds available in the literature.     

Asymptotics for the Moments of the Time to Ruin for the Compound Poisson Model Perturbed by Diffusion

We obtain the asymptotic behaviour of the k-th moment of the time to ruin in the classical risk model perturbed by diffusion for the case where the claim size distribution has a heavy tail.     

In vivo evaluation of CYP1A2, CYP2A6, NAT-2 and xanthine oxidase activities in a Greek population sample by the RP-HPLC monitoring of caffeine metabolic ratios

A RP-HPLC method was developed for the assessment of caffeine and its metabolites in urine and was used for the evaluation of the CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyl-transferase-2 (NAT-2) in vivo activities in 44 Greek volunteers (21 men, 23 women). Spot urine samples were analyzed 6 h after 200 mg caffeine consumption, following a 30 h methylxantine-free diet. The major urinary caffeine metabolites are 1-methyluric acid (1U), 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (1X), 1,7-dimethyluric acid (17U) and 1,7-dimethylxanthine (17X). CYP1A2, CYP2A6, XO and NAT-2 activities were estimated from the metabolic ratios (AFMU + 1U + 1X)/17U, 17U/17X, 1U/(1X + 1U) and AFMU/(AFMU + 1U + 1X), respectively. Metabolites and internal standard were extracted with chloroform/isopropanol (85:15, v/v) and separated on a C18 column by an isocratic HPLC system using a two-step elution with manual switch from solvent A (0.1% acetic acid-methanol-acetonitrile, 92:4:5 v/v) to solvent B (0.1% acetic acid-methanol, 60:40, v/v), and detected at 280 nm. The method exhibited adequate metabolite separation (resolution factors >1.48), accuracy (94.1-106.3%) and intraday and interday precision <8.02 and <8.78%, respectively (n = 6). Smoking affected only CYP1A2, whereas gender had no effect in any enzyme activity. NAT-2 exhibited bimodal distribution, 63.6% of volunteers being slow acetylators. The developed RP-HPLC method was fully validated and successfully applied for the evaluation of CYP1A2, CYP2A6, XO and NAT-2 activities.