Liquid chromatography-tandem mass spectrometry method for quantification of gentamicin and its individual congeners in serum and comparison results with two immunoanalytical methods (fluorescence polarization immunoassay and chemiluminiscent microparticle immunoassay).

BACKGROUND: Total gentamicin is a sum of five congeners C1, C1a, C2, C2a and minor C2b, which differ from each other in their methylation on the purpurosamine ring. Liquid chromatography with mass detection (LC-MS/MS) and specified calibration material enables the concentration of total gentamicin and its individual congeners to be analysed. METHODS: 50 µL serum was precipitated with acetonitrile in the presence of 0.5 mol/L formic acid. A RP BEH C18 1.7 µm 2.1x50 mm column maintained at 30 °C and tobramicin as the internal standard were used. Mass detection was performed in positive electrospray. The gentamicin results were compared with fluorescence polarization immunoassay (FPIA) and chemiluminiscent microparticle immunoassay (CMIA). Passing-Bablock regression analysis and Bland-Altman analysis were used. RESULTS: Calibration curves for individual gentamicin congeners were linear with correlation coefficients between 0.997 and 0.998. Recovery was 91.6-102.0% and the coefficients of variation 1.4-8.4%. The total gentamicin concentration was compared with immunoassay FPIA (LC-MSgen = 0.9798xPFIAgen) and CMIA (LC MSgen = 0.9835xCMIAgen) both with significant correlation (p < 0.001). CONCLUSION: The LC-MS/MS method is fast and precise and can be applied to routine TDM in patients. Comparing it to immunoassays makes it possible to measure concentration of gentamicin congeners, which may be important in the case of their different pharmacokinetics.

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

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.

Squamous cell carcinoma antigen as a marker of sinonasal inverted papilloma.

This prospective study aimed to evaluate the usefulness of squamous cell carcinoma antigen (SCCA) as a clinical marker of sinonasal inverted papilloma (IP). The potential benefit of SCCA in the diagnosis of unilateral nasal pathology and as a marker of hidden recurrence was evaluated as well. Blood samples from patients with sinonasal IP were examined to determine serum SCCA levels before surgery, the day after surgery, and every 6 months during follow-up. Preoperative and postoperative levels of SCCA were compared. Twenty consecutive patients with histologically confirmed IP were included in the study, conducted between 2000 and 2011. The mean age of the patients was 54.2 years (range 35-72). The mean serum SCCA level before surgery was 3.885 µg/l (range 0.7-7.6). A decrease of the SCCA level to 0.885 µg/l (range 0.1-1.9) was observed on the 1 day after a radical surgical procedure. A statistically significant difference between the preoperative and postoperative levels was observed (P < 0.001). Elevated levels of SCCA during long-term follow-up were observed in three patients. All of them had a recurrence of IP. We conclude that the serum level of SCCA is a useful clinical marker of the presence of sinonasal IP. The level of SCC antigen was significantly lower in patients after IP was completely removed. According to our results, SCCA level also appears to be useful for long-term follow-up (hidden recurrence diagnosis).

[Thyroid dysfunction during interferon alpha therapy for chronic hepatitis B and C - twenty years of experience].

OBJECTIVE: To determine the incidence of thyroid dysfunction in patients with chronic hepatitis B and C (HBV, HCV) who were treated with interferon (IFN) alpha. PATIENTS AND METHODS: In the years 1992-2013, parameters of the thyroid gland were evaluated in 304 patients (256 with HCV, 48 with HBV) who were treated with conventional or pegylated IFN at the Department of Infectious Diseases in Ostrava. Prior to, during and after completion of antiviral treatment, levels of thyroid stimulating hormone (TSH), thyroxine (T4), triiodothyronine (T3), including their free fractions fT4 and fT3, and anti-thyroid antibodies (anti-thyroglobulin, anti-microsomal fraction) were determined and clinical manifestations of thyroid dysfunction were evaluated. RESULTS: TSH changes were detected in 75 patients (25 %), of whom 68 had HCV and 7 HBV. Hypothyroidism was detected in 39 patients (34 with HCV), of whom 25 required substitute therapy which was subsequently terminated in 5 patients. Hyperthyroidism with transient suppressive therapy with carbimazole developed in 4 HCV patients. In 32 patients, TSH changes were assessed as subclinical hypothyroidism. Abnormal T3 values were found in 188 (62 %) and T4 in 49 (16 %) patients; these changes practically did not correlate with TSH changes. Autoantibodies were detected in 54 (18 %) patients of whom 30 were also found to have changes in TSH. CONCLUSIONS: In a group of 304 patients treated with IFN alpha for chronic hepatitis, thyroid disease with changes in TSH were observed in a quarter of patients; hypothyroidism clearly prevailed. Thyroid diseases developed in half of the patients with the presence of antithyroid antantibodies.

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

AIMS: Orthostatic 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). METHODS: Twenty 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. RESULTS: There 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). CONCLUSION: We 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. A significant increment of the break index confirmed increased vagal influence in the subgroup of positive cases.

Validation of sparse sampling strategies to estimate cyclosporine A area under the concentration-time curve using either a specific radioimmunoassay or high-performance liquid chromatography 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 influence of estradiole and tibolone administration on leptin levels in women with surgically induced menopause.

BACKGROUND: Several studies suggest that changes in estrogens and androgens during menopause play a role in the regulation of leptin production. Some authors present hypothesis that sex hormone replacement therapy can modulate leptin levels but up to date evidence shows that the influence of endogenous estrogens, androgens levels and sex hormone therapy on leptin concentration remains uncertain. AIM: To evaluate the influence of surgically induced menopause on serum leptin levels and the influence of different types of hormonal therapy on serum leptin concentrations. METHODS: 58 women with surgically induced menopause were divided into three groups. Women who did not receive any hormonal substitution (group 1), women who received Estradiol l mg per day (group 2) and women who received Tibolone 2,5 mg per day (group 3). The levels of leptin, estradiol, testosterone, testosterone, dehydroepiandrosterone sulfate, FSH, LH and progesterone were measured in all subjects on the 5th day and after 3 months following the surgical procedure. RESULTS: Mean serum leptin concentrations did not differ statistically in any of the studied groups in the beginning and in the end of the study. There was no correlations between serum leptin and estradiol, LH, FSH, progesterone, testosterone, free testosterone and DHEAS concentrations in any of groups before and after treatment. CONCLUSION: Changes in sex hormone concentrations caused by ovariectomy do not influence serum leptin concentrations. Also the short term administration of low dose estrogen therapy or tibolone in postmenopausal subjects does not change serum leptin levels.

Detection of spontaneous cerebrospinal fluid rhinorrhea.

Radionuclide cisternography (RS) is a highly sensitive, reliable and safe technique used for the confirmation of the presence of cerebrospinal fluid rhinorrhea and the localisation of fistula. The authors describe a patient aged 53 years old with spontaneous cerebrospinal fluid rhinorrhea detected by RS. This kind of rhinorrhea is very rare. After measuring the radioactivity of the nasal wads and examining the RS images, no obvious cause of this rhinorrhea was found. The patient was operated and dura mater cerebroplasty in the anterior cranial fossa was performed. No other cause of rhinorrhea was found during the operation. After the operation the rhinorrhea ceased. The authors report this case because spontaneous rhinorrhea is rare and is well treated by dura mater cerebroplasty.

Effect of the preparation method of control samples on external quality assessment results of PSA testing.

BACKGROUND: The control of PSA tests is included in most systems of External Quality Assessment (EQA). As part of the preparation of control samples, seminal fluid is sometimes used to increase the level of PSA. MATERIAL AND METHODS: In two separate studies, the effect of the method of preparation of control samples was evaluated. The first study, carried out under the conditions of EQA of PSA examination, assessed the results of the analysis of samples prepared using seminal fluid and samples prepared using the addition of human serum. The second study analysed two groups of samples (sera and control samples to which seminal fluid was added) using two systems (IRMA and IMMULITE). RESULTS: The fPSA/tPSA ratio in the control samples prepared using the seminal fluid is very high (over 60%) and does not correspond to the usual values. The use of these samples resulted, when compared with the samples prepared using the addition of serum, in a substantial deterioration of inter-laboratory reproducibility of the results in EQA. The differences were most obvious when the results of individual analytical systems were compared, particularly for the examination of fPSA. CONCLUSION: The use of seminal fluid for the preparation of control samples is not appropriate.