Kristian Šafarčík

Evaluation and comparison of therapeutic monitoring of whole-blood levels of cyclosporin A and its metabolites in renal transplantation by HPLC and RIA methods

BACKGROUNDnThe aim of the work was to evaluate the possibility to estimate the level of cyclosporin A (CyA) metabolites as the difference of radioimmunoassay (RIA) non-specific and RIA specific methods.nnnMETHODSnBlood samples of renal transplant patients were analyzed by three different methods: RIA specific method (CYCLO-Trac, DiaSorin, USA) (RIA(SP)), RIA non-specific method (Immunotech, Czech Republic) (RIA(NS)), and high performance liquid chromatography (HPLC) method.nnnRESULTSnAlthough values obtained by RIA(SP) correlated well those obtained by HPLC (RIA(SP)=0.995.HPLC+9.68; r(2)=0.962, n=448), the results of HPLC methods were lower by 8%. The values obtained by RIA(NS) were 2.57 times higher than the values obtained by RIA(SP) (RIA(SP)=0.356RIA(NS); r(2)=0.713, n=448). The ratio (CyA+CyA metabolites)/(CyA) calculated as the ratio RIA(NS)/RIA(SP) values for 42 renal transplant patients was relatively stable for each particular patient. The sum of selected CyA metabolites (M1+M17+M21) measured by HPLC correlated well with that estimated from the difference of RIA(NS)-RIA(SP): HPLC(metab)=0.921.(RIA(NS)-RIA(SP))+21.3; (r(2)=0.746, n=448).nnnCONCLUSIONnThe combination of both the specific and non-specific methods for the determination of CyA presents an improved means for the TDM of CyA and CyA metabolites in renal transplant patients. Moreover, a combination of both methods can help to elucidate some unexpected events, such as the persistence of high cyclosporin blood levels.

High-performance liquid chromatographic method for therapeutic drug monitoring of cyclosporine A and its two metabolites in renal transplant patients

A novel fast HPLC method was developed for the determination of cyclosporine A (CyA) and its two metabolites M17 (AM1) and M21 (AM4N) in blood. Whole blood was precipitated with zinc sulphate, extracted with diethyl ether, evaporated, dissolved in aqueous methanol and partitioned twice with n-hexane. Chromatography was carried out using a microbore RP-column under isocratic elution with acetonitrile-methanol-water (200:80:140, v/v/v) at 70 degrees C and a detector set at 205 nm. Linearity for all three compounds was tested in the range of 1-1000 ng/ml. Recovery was 97-109%, and a coefficient of variation was 1.6-8.8% depending on the particular compound and its concentration. The method was used for a group of renal transplant patients having an inadequate response to CyA therapy in order to evaluate the possible role of CyA and its metabolites on the occurrence of hypertension and other toxicological events.

Reference intervals of plasma matrix metalloproteinases 2, 3, and 9 and serum asymmetric dimethylarginine levels

Abstract Objective. The present study aimed to verify the reference intervals of plasma matrix metalloproteinases (MMPs) 2, 3, and 9 and serum asymmetric dimethylarginine (ADMA) in a healthy population with an average age corresponding to that of patients with cardiovascular diseases. Methods. The study included 180 healthy volunteers. Plasma MMP-2, MMP-3, MMP-9, and serum ADMA levels were determined using an enzyme-linked immunosorbent assay. These levels were analyzed for association with age and gender. The Cbstat5, R software, and NCSS 2007 programs were used for statistical analysis. Results. The average volunteer age was 47.4 years in the group in which MMP-3 and ADMA were analyzed, 40.3 years in the MMP-9 group, and 47.8 years for the MMP-2 group. Serum ADMA levels were determined to be independent of age and gender. Plasma MMP-2 levels were significantly correlated with age (p = 0.001), with lower levels detected in persons ≤ 49 years of age. Plasma MMP-3 was significantly associated with both age (p < 0.0001) and gender, with lower levels detected in persons of ≤ 47 years of age and among women. Plasma MMP-9 levels were not age dependent, but were associated with gender (p = 0.014), showing lower levels in women. Conclusions. Reference intervals of heparin-plasma MMP-2, MMP-3, and MMP-9 and serum ADMA levels were determined. MMP-2 and MMP-3 levels were found to be age dependent, and MMP-3 and MMP-9 levels were gender dependent.

Significant discrepancy in cyclosporin A post-dose concentrations when analyzed with specific RIA and HPLC method.

C(2) or AUC sparse sampling methods are widely recommended for therapeutic monitoring of cyclosporin A (CsA). One additional reason for promoting the C(2) sampling time in place of commonly used C(0) is that the C(2) level may actually provide more accurate measurement of parent drug concentration by immunoassays, as lower portion of metabolites has been formed 2 hours post-dose than at the steady-state trough time point. HPLC and RIA whole blood levels of CsA and its main metabolites AM1, AM9 and AM4N were compared during 12 hours profile after chronic administration. 40 stable renal transplant male patients (age 49 +/- 6 years, body weight 76 +/- 7 kg) were treated with CsA (Sandimmun Neoral, Novartis s.r.o, Prague, Czech Republic) in doses 198 +/- 56 mg twice daily. Samples were collected in steady state (after 2 weeks of regular treatment regimen) as follows: pre-dose, 0.5, 1, 1.5, 2, 3, 5, 8 and 12 hours after dose. CsA concentrations were determined both specific RIA assay (Cyclo-Trac SP Whole, Dia Sorin) and HPLC method, where concentrations of metabolites AM1, AM9 and AM4N were simultaneously analyzed. The AUC(0-12) was calculated by the linear trapezoidal rule. The percentage prediction error defined as [(RIA value-HPLC value)/HPLC value] x 100 was used for estimation of differences. C(max), t(max), and C(avg) were compared using Students t-test. RIA produced significantly higher CsA levels than HPLC method in the period of 0.5 - 5 hours after application. The greatest differences (43 - 56%) occurred between 1 and 3 hours after dose. AUC(0-12), C(max) a C(avg) calculated from RIA results were consequently significantly higher. Only t(max) remained unchanged. The ratio of metabolites/parent drug after CsA intake is decreasing but their absolute concentrations are significantly increasing. Mean levels at C(0)/C(2) were CsA-RIA 82/612, CsA-HPLC 89/425, AM1 121/179, AM9 4.1/81.4, AM4N 9.5/21.0 ng/ml. TDM using C(2) and AUC sparse sampling may cause misleading interpretation using both methods alternately for the same patient.

Validation of Sparse Sampling Strategies to Estimate Cyclosporine A Area Under the Concentration-Time Curve Using Either a Specific Radioimmunoassay or High-Performance Liquid Chromography Method

Introduction: Area under the concentration-time curve (AUC) has been advocated as a better parameter to monitor cyclosporine A than trough concentrations. Up to now, more than 100 equations to estimate AUC using a limited sampling strategy have been published, but not all have been validated. Material and Methods: Eight equations for AUC0-12h and two for AUC0-8h were validated. Concentrations of cyclosporine A were analyzed by high-performance liquid chromatography (HPLC) and a specific radioimmunoassay (RIA) method. Forty male renal transplant patients were included in the study. Blood samples were taken predose and at 0.5, 1, 1.5, 2, 3, 5, 8, and 12 hours after the morning dose when the patient was in steady state. The percentage prediction error (%pe) was used for an assessment of the performance of the equations. Mean %pe less than ±15% and absolute %pe less than 30% in 95% of predictions were considered to be acceptable. Other possibilities such as %pe less than 25%, 20%, and 15% were also tested. Results: Eight equations for AUC0-12h met the requirements using both assays, six in the HPLC set only and four in the RIA set only. The highest precision was obtained with AUC0-12h = 123.792 + 1.165*C1h + 3.021*C3h + 7.33*C8h proposed by de Mattos et al. The mean %pe was 1% ± 8% (-16 to 19) for HPLC (values given as mean ± standard deviation [range]) and -1 ± 5 (-17 to 10) for RIA. Mean absolute %pe was 7 ± 5 (0.0 to 19) for HPLC and 4 ± 4 (0.0 to 17) for RIA. For clinical use, the most suitable equation was AUC0-12h = 363.078 + 8.77*C1h + 3.07*C3h proposed by Wacke et al, which produced the second lowest %pe and used two sampling points in the period of 1 to 3 hours after dose. The mean %pe was -7 ± 10 (-25 to 25) for HPLC and 2.3 ± 6 (-10 to 17) for RIA. Mean absolute %pe was 10 ± 7 (0.4 to 25) for HPLC and 5 ± 4 (0.0 to 17) for RIA. The equation: AUC0-8h = 55.37 + 2.89*C0h + 1.08*C1h0.9*C2h + 2.23*C3h proposed by Foradori et al met the criteria with 95% of prediction with absolute %pe less than 15% in the HPLC set and 10% in the RIA set. Conclusion: The validation of equations is of major importance for prediction precision, whereas the analytical method for limited sampling strategy proposals had no influence. Because of the wide interassay variability, it is also important to know which analytical method was used for AUC calculation when interpreting the results.

The possibility of using the specific RIA method for the area-under-time-concentration curve sparse sampling calculation of cyclosporin A despite a large post-dose overestimation.

OBJECTIVEnTo find limited sampling strategies (LSS) for prediction of the real AUC using the RIA analytical method.nnnMETHODnBlood samples of 40 male renal transplant patients taken pre-dose and after 0.5, 1, 1.5, 2, 3, 5, 8, and 12 h in the steady-state were analyzed with HPLC and the specific RIA method. I. Eight equations for AUC0-12 and one for AUC0-8 obtained from the literature, that produced the mean percentage prediction error (%PE) < ± 15% and absolute %PE < 30% in 95% of predictions, were analyzed for possibility to predict the real AUC of CsA. II. Multiple regression analysis (MRA) was provided for the AUC equation proposal. Patients were divided into two groups according to the AUC0-12. Group I was used for LSS : s proposals while Group II for validation. The bias and precision were expressed as %PE, r2 and RMSE. The relationship of %PE interassay and with LSS:s was expressed as Pearson correlation r. GraphPad InStatt Software was used for MRA and Pearson r calculation.nnnRESULTSnNone of the equations described in the literature predicts AUC of CsA proprietarily. Seven equations for AUC0-12 and five for AUC0-8 were proposed with MRA for prediction of real AUC from RIA values.nnnCONCLUSIONSnLSS:s can moderate the interassay %PE in AUC of CsA. New patients should be tested with both RIA and HPLC for the level of overestimation. The conversion factors should be calculated for patients with an overestimation higher than 90%. Our equation 251.09 + 0.5195 × C1h + 4.926 × C3h or 196.13 + 4.526 Âx8f× C0h + 2.089 × C1.5h for AUC0-12, and 171.80 + 0.4759 × C1h + 4.132 × C3h for AUC0-8 may be used in patients with medium or low RIA and HPLC differences. Repeated analysis with HPLC is thus suggested in cases with AUC:s results close to the lower or upper margin of the therapeutic window.

The role of adrenomedullin and galanin in recurrent vasovagal syncope: a case control study.

AIMSnOrthostatic stimuli are known to elicit changes in vasoactive peptide levels. The hypothesis of no difference in adrenomedullin and/or galanin levels in patients with recurrent vasovagal syncope and healthy controls was tested in a passive 35-min head-up tilt test (HUTT).nnnMETHODSnTwenty eight persons (14 patients and 14 healthy controls) were tested in a 35-min/60° head-up tilt test with telemetry monitoring. Three blood samples were evaluated for each person during the HUTT. Plasma levels of adrenomedullin and galanin were analysed by the Kruskal-Wallis test for all sampling periods. Vagal influence was indirectly assessed by the break index.nnnRESULTSnThere were no significant differences between groups in median values for either adrenomedullin or galanin plasma levels (all 6 p-values were greater than 0.4). For adrenomedullin, no significant difference between groups was found. For galanin, the rate of change between the 1st and 2nd measurement was significantly greater for patients (P=0.04), regardless of HUTT result but between the 2(nd) and 3(rd) measurement it was insignificant (P=0.36). In the group of positive cases, the break index increased significantly (P=0.02).nnnCONCLUSIONnWe confirmed that there is a different galanin secretion pattern during orthostatic provocation in patients with recurrent vasovagal syncope than healthy individuals. For adrenomedullin, no significant difference was found. Axa0significant increment of the break index confirmed increased vagal influence in the subgroup of positive cases.