Lenka Kujovská Krčmová

Urinary Neopterin in Patients with Ovarian Cancer

Abstract Urinary neopterin, an indicator of systemic immune activation, is increased in most patients with epithelial ovarian carcinoma (EOC) and is an independent prognostic indicator. The data on prognostic significance of neopterin in EOC have been collected before the advent of paclitaxel that has changed the management and natural history of the disease. In the present study, we have evaluated the prognostic significance of urinary neopterin in 49 patients with primary and secondary ovarian neoplasms treated in the late 1990s and in 2000s. Urinary neopterin was measured by high-performance liquid chromatography. Compared to controls, urinary neopterin was significantly increased in patients with both primary ovarian cancer and ovarian metastases of other tumors (341 ± 343, and 328 ± 277 vs. 133 ± 40 μmol/mol creatinine; p <0.001 ). Serious toxicity of chemotherapy was observed in 8 out of 12 (67%) patients with urinary neopterin equal or above 338 μmol/mol creatinine (mean of all patients) compared to 2 of 19 ( 11%) of patients with urinary neopterin below 338 μmol/mol creatinine (Fisher exact test, p - 0.001). No significant changes were observed in urinary neopterin concentrations during the treatment with paclitaxel/platinum. A significant correlation was observed between urinary neopterin and percentage of xylose absorbed (rs = -0.58, p = 0.03), and positive correlations were observed between urinary neopterin and lactulose/ mannitol (rs = 0.63, p = 0.02), lactulose/xylose (rs = 0.79, p = 0.0007) and sucrose/xylose (rs = 0.60, p = 0.02) ratios. Survival was significantly longer in patients with urinary neopterin below 338 μιηοΐ/ιτιοί creatinine in the whole group of 49 patients with ovarian cancer, in 36 patients with primary ovarian cancer as well as in 13 patients in ovarian metastases of other primary tumors. A significant difference in survival was also observed when 37 pretreated patients or 24 pretreated EOC patients were evaluated (p = 0.05). In conclusion, neopterin remains a significant prognostic indicator in patients with recurrent ovarian cancer in the era of newer chemotherapeutic agents. Increased urinary neopterin was associated with chemotherapy toxicity.

Intestinal permeability and vitamin A absorption in patients with chemotherapy-induced diarrhea.

Objective:Gastrointestinal toxicity is one of the most common side effects of anticancer therapy. Measurement of intestinal permeability represents one of the potential methods of noninvasive laboratory assessment of gastrointestinal toxicity. The aim of the present study was to investigate intestinal permeability and vitamin A absorption in patients with chemotherapy-induced diarrhea (CID). Methods:We have assessed intestinal permeability, by measuring absorption of lactulose, mannitol, xylose, and vitamin A absorption, in 11 patients with CID, 10 healthy controls, and 24 untreated patients with gastrointestinal tumors. Urinary lactulose, mannitol and xylose were measured by capillary gas chromatography and serum retinol and retinyl esters were determined by high performance liquid chromatography. The results obtained in patients and controls were compared by Mann-Whitney U test. Results:Lactulose/mannitol and lactulose/xylose ratios were increased and retinol esters (retinyl palmitate and retinyl stearate) were decreased significantly in patients with CID. Conclusions:Measurements of intestinal permeability and vitamin A absorption may represent sensitive tools in the assessment of CID.

Intima-media thickness, myocardial perfusion and laboratory risk factors of atherosclerosis in patients with breast cancer treated with anthracycline-based chemotherapy

An increased incidence of complications of atherosclerosis has been noted in cancer survivors. The aim of the present study was to evaluate, in patients with breast carcinoma, the effect of antracycline-based chemotherapy on carotid intima-media thickness (IMT), myocardial perfusion, assessed by single-photon emission tomography (SPECT) and laboratory parameters associated with the risk of atherosclerosis. Thirty-six patients with breast cancer were evaluated before and after anthracycline-based chemotherapy. Retinol, alpha-tocopherol, glycosylated hemoglobin and urinary neopterin were measured by high-performance liquid chromatography. Peripheral blood cell count, D-dimers, fibrinogen, antithrombin, glucose, magnesium, creatinine, uric acid, albumin, C-reactive protein, lipoprotein (a), cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, homocysteine, urinary albumin and N-acetyl-beta-d-glucosaminidase (NAG) were determined with routine methods. No significant differences were observed between patients and 16 controls. Compared to the measurement before the start of therapy, peripheral blood leukocyte and platelet count, hemoglobin, creatinine, HDL cholesterol, retinol, albumin, urinary albumin and NAG decreased, and total cholesterol, LDL cholesterol, triglycerides, neopterin and mean IMT increased significantly after the treatment. Of the 36 patients who had SPECT after treatment, perfusion defects were noted only in two cases, including the patient who had perfusion defects at baseline examination and a patient who did not have a baseline SPECT. In conclusion, a significant increase in carotid IMT, total cholesterol, LDL cholesterol, triglycerides and urinary neopterin and a decrease of peripheral blood leukocyte and platelet counts, hemoglobin, creatinine, HDL cholesterol, retinol, albumin and NAG were observed after the treatment.

HPLC method for simultaneous determination of retinoids and tocopherols in human serum for monitoring of anticancer therapy

A simple and rapid HPLC method requiring small volumes (250 microL) of human serum after C18 SPE sample preparation was developed using monolithic technology for simultaneous determination of all-trans-retinoic acid, 13-cis-retinoic acid, retinol, gamma- and alpha-tocopherol. The monolithic column, Chromolith Performance RP-18e (100x4.6 mm), was operated at ambient temperature. The mobile phase consisted of a mixture of acetonitrile (ACN) and 1% ammonium acetate in water (AMC) at pH 7.0. The mobile phase started at 98:2 (v/v) ACN/AMC (column pre-treatment) at a flow rate of 2 mL/min, then changed to 95:5 (v/v) ACN/AMC for 4 min at a flow rate of 1.5 mL/min and a further 3 min at a flow rate of 3.2 mL/min. Detection and identification were performed using a photodiode array detector. Retinol, 13-cis- and all-trans-retinoic acid were monitored at 325 nm. Both alpha- and gamma-tocopherol were detected at 295 nm. The total analysis time was 7.2 min. Tocol (synthesized tocopherol, not occurring in humans) was used as internal standard. The method was linear in the range of 0.125-10.00 micromol/L for all-trans-retinoic acid, 0.125-5.00 micromol/L for 13-cis-retinoic acid, 0.25-10.00 micromol/L for retinol, 0.5-50.00 micromol/L for gamma-tocopherol, and 0.5-50.00 micromol/L for alpha-tocopherol. The present method may be useful for monitoring of retinoids and tocopherols in clinical studies.

Rapid sample preparation procedure for determination of retinol and α-tocopherol in human breast milk.

The liposoluble vitamins (retinol and α-tocopherol) concentration in human breast milk is of a cardinal knowledge especially for nutrition of prematurely born. It enables the feeding optimization of these important micronutrients for preterm infants. The novel rapid liquid-liquid extraction procedure for human breast milk investigation was developed and validated according to FDA guidelines. The recovery of retinol was 82-90% measured at three concentration levels 1.0, 2.5 and 5.0 μmol/L, for α-tocopherol 92-109% at concentration levels 2.5, 5.0 and 10.0 μmol/L. The repeatability of extraction procedure expressed as relative standard deviation was 3.26% for retinol and 4.79% for α-tocopherol. Developed extraction procedure was applied on 120 human breast milk samples. The separation of vitamins was completed using advantages of a monolithic column which accomplished demands of acceleration made by modern bio-analytical HPLC methodology. The analytes of interest were detected by diode-array detector at wavelengths 325 nm for retinol and 290 nm for α-tocopherol.

Comparison of a new high‐resolution monolithic column with core‐shell and fully porous columns for the analysis of retinol and α‐tocopherol in human serum and breast milk by ultra‐high‐performance liquid chromatography

The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high-throughput assay methods in bioanalysis. It can be achieved either by ultra-high-performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction--monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second-generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core-shell and fully porous sub-2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice.

Recent trends in the analysis of vitamin D and its metabolites in milk--a review.

Vitamin D plays an important role in calcium metabolism and affects other metabolic pathways. Despite the intense interest in vitamin D, no comprehensive overview addressing the analysis of vitamin D in milk has been published. Historically, immunoassay techniques have been mainly used for the routine quantification of vitamin D and its metabolites. However, the greater accuracy and precision of chromatography makes it one of the most important methods in the analysis of vitamin D. The determination of vitamin D and its metabolites by LC-MS is the gold standard for its assessment. LC-MS has unique advantages for vitamin D determination and quantification due to its high sensitivity and specificity. In this review, the current status of vitamin D and its metabolites analysis in milk, human and bovine, including sample pre-treatment and chromatography analysis, are critically discussed and summarised.

Determination of neopterin, kynurenine, tryptophan and creatinine in human serum by high throuput HPLC.

A new HPLC method for simultaneous determination of neopterin, creatinine, kynurenine and tryptophan in human serum was developed and validated. Monolithic stationary phases technology (two monolithic columns RP-18e were connected with guard monolithic cartridge 4.6 mm × 50 mm+3.0 mm × 100 mm and 4.6 × 10 mm) and special auto sampler for micro titration plates (samples are storage in dark cooled place protected against evaporation) were combined with easy sample preparation step. As mobile phase 15 mmol/L phosphate buffer at pH 4.50 was used. Neopterin and tryptophan were detected using fluorescent detection and kynurenine and creatinine were detected by diode-array detection. This method may be suitable for large sequences of samples in clinical research and routine practice.

Citrulline as a biomarker of gastrointestinal toxicity in patients with rectal carcinoma treated with chemoradiation.

Abstract Background: Gastrointestinal toxicity is the principal toxicity of chemoradiation in the treatment of rectal carcinoma. The assessment of this toxicity still relies mostly on the symptoms reported by the patient. Methods: Plasma citrulline, serum neopterin and urinary neopterin were followed weekly in 49 patients with rectal carcinoma during chemoradiation. Results: Citrulline significantly (p<0.05) decreased while serum and urinary neopterin concentrations increased during therapy. Irradiated gut volume correlated significantly inversely with citrulline and positively with urinary neopterin. Statistically significant inverse correlations were also observed between urinary neopterin and plasma citrulline concentrations during the treatment. Urinary neopterin concentrations were significantly higher and citrulline concentrations were lower in patients who experienced grade ≥3 gastrointestinal toxicity. Conclusions: Citrulline represents a promising biomarker of gastrointestinal toxicity. Moreover, the volume of irradiated gut correlated with urinary neopterin concentrations and an association was observed between gastrointestinal toxicity evidenced by lower citrulline concentrations and systemic immune activation reflected in increased concentrations of urinary neopterin.

Application of core–shell technology for determination of retinol and alpha-tocopherol in breast milk

Breast milk is a main source of fat-soluble vitamins for newborns and it is needful to monitor the nutritional status prior to its application. In this work a novel, high-throughput and low-cost method for monitoring of retinol and alpha-tocopherol in breast milk was developed, validated and compared with reference method using monolithic column. For this purpose five various porous shell and monolithic columns were tested on the basis of relationship between HETP and linear mobile phase velocity, analysis time and consumption of solvents. Finally the core-shell analytical column Kinetex C18 (2.6 μm, 100 Å, 100×4.6 mm) was chosen as the best and optimal values of flow rate, injection volume and temperature of analysis were established. The detection of retinol and alpha-tocopherol was carried out at 325 and 295 nm, respectively by diode array detector. The LOD 0.004 μmol/L and 0.078 μmol/L, the LOQ 0.012 μmol/L and 0.182 μmol/L for retinol and alpha-tocopherol, respectively were calculated. The validation data showed good linearity, repeatability of retention time with RSD 0.22% and 0.12%, repeatability of peak area with RSD 6.94% and 1.75%, recovery 114.1-116.3% and 99.0-108.6% for retinol and alpha-tocopherol, respectively. Moreover, the newly developed method substantially decreased the solvent consumption by about 263 mL per 100 samples with the total time of analysis 1.75 min in comparison with analysis time 1.80 of the reference method.