Is It Useful to Monitor Thiopurine Metabolites in Pediatric Patients with Crohn's Disease on Combination Therapy? A Multicenter Prospective Observational Study.

BACKGROUND: The additional value of azathioprine concomitant treatment on infliximab pharmacokinetics in children is not well described yet. AIMS: In the present study, we aimed to describe the relationship between thiopurine metabolite levels, infliximab trough levels, anti-IFX antibody formation, and clinical and laboratory markers of disease activity in pediatric patients with Crohn's disease, and to assess non-adherence. METHODS: Data were collected prospectively during repeated visits from pediatric patients followed for Crohn's disease in two Czech pediatric inflammatory bowel disease centers between January 2016 and June 2017. Thiopurine metabolites (6-thioguanine and 6-methylmercaptopurine) were measured by high-performance liquid chromatography. Infliximab trough levels and anti-IFX antibody serum levels were measured routinely by ELISA. The risk of loss of response to infliximab therapy was also assessed. RESULTS: A significant association between infliximab serum levels and 6-thioguanine erythrocyte levels was observed when tested as categorical variables (63 patients, 321 observations). To predict infliximab levels > 5 µg/mL, we propose a 6-thioguanine cutoff of 278 pmol/8 × 108 erythrocytes (sensitivity, 0.799; specificity, 0.347). A higher loss-of-response-to-infliximab rate (tested in a subgroup of 51 patients) was observed in patients with undetectable 6-thioguanine levels than in those with detectable levels (p = 0.026). Non-adherence to azathioprine therapy was suspected in 20% of patients. CONCLUSION: Thiopurine metabolite monitoring in pediatric patients with Crohn's disease is useful when optimizing combination therapy. Pediatric patients with undetectable 6-thioguanine levels are more likely to lose response to infliximab therapy. When targeting optimal infliximab levels, the 6-thioguanine cutoff levels in children appear to be higher than in adults.

Prediction of Thiopurine Metabolite Levels Based on Haematological and Biochemical Parameters.

OBJECTIVES: Therapeutic drug monitoring of thiopurine erythrocyte levels is not available in all centers and it usually requires quite a long time to obtain the results. The aims of this study were to build a model predicting low levels of 6-thioguanine and 6-methylmercaptopurine in pediatric inflammatory bowel disease (IBD) patients and to build a model to predict nonadherence in patients treated with azathioprine (AZA). METHODS: The study consisted of 332 observations in 88 pediatric IBD patients. Low AZA dosing was defined as 6-thioguanine levels <125 pmol/8 × 10 erythrocytes and 6-methylmercaptopurine levels <5700 pmol/8 × 10 erythrocytes. Nonadherence was defined as undetectable levels of 6-thioguanine and 6-methylmercaptopurine <240 pmol/8 × 10 erythrocytes. Data were divided into training and testing part. To construct the model predicting low 6-thioguanine levels, nonadherence, and the level of 6-thioguanine, the modification of random forest method with cross-validation and resampling was used. RESULTS: The final models predicting low 6-thioguanine levels and nonadherence had area under the curve, 0.87 and 0.94; sensitivity, 0.81 and 0.82; specificity, 0.80 and 86; and distance, 0.31 and 0.21, respectively, when applied on the testing part of the dataset. When the final model for prediction of 6-thioguanine values was applied on testing dataset, a root-mean-square error of 110 was obtained. CONCLUSIONS: Using easily obtained laboratory parameters, we constructed a model with sufficient accuracy to predict patients with low 6-thioguanine levels and a model for prediction of AZA treatment nonadherence (web applications: https://hradskyo.shinyapps.io/6TG_prediction/ and https://hradskyo.shinyapps.io/Non_adherence/).

3,5,7-Substituted Pyrazolo[4,3- d]pyrimidine Inhibitors of Cyclin-Dependent Kinases and Their Evaluation in Lymphoma Models.

Cyclin-dependent kinases are therapeutic targets frequently deregulated in various cancers. By convenient alkylation of the 5-sulfanyl group, we synthesized 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2) H-pyrazolo[4,3- d]pyrimidines with various substitutions at position 5 with potent antiproliferative activity in non-Hodgkin lymphoma cell lines. The most potent derivative 4.35 also displayed activities across more than 60 cancer cell lines. The kinase profiling confirmed high selectivity of 4.35 toward cyclin-dependent kinases (CDKs) 2, 5, and 9, and the cocrystal with CDK2/cyclin A2 revealed its binding in the active site. Cultured lymphoma cell lines treated with 4.35 showed dephosphorylation of CDK substrates, cleavage of PARP-1, downregulation of XIAP and MCL-1, and activation of caspases, which collectively confirmed ongoing apoptosis. Moreover, 4.35 demonstrated significant activity in various cell line xenograft and patient-derived xenograft mouse models in vivo both as a monotherapy and as a combination therapy with the BCL2-targeting venetoclax. These findings support further studies of combinatorial treatment based on CDK inhibitors.

LC-MS/MS method for determination of cyclin-dependent kinase inhibitors, BP-14 and BP-20, and its application in pharmacokinetic study in rat.

N2-(4-Amino-cyclohexyl)-9-cyclopentyl-N6-(6-furan-2-yl-pyridine-3-ylmethyl)-9H-purine-2,6-diamine (BP-14) and 2-(5-{[2-(4-amino-cyclohexylamino)-9-cyclopentyl-9H-purine-6-ylamino]-methyl}-pyridine-2-yl)-phenol (BP-20) are novel cyclin-dependent kinase inhibitors, structurally related to roscovitine, with significant biological activity. A simple, selective and sensitive liquid chromatography - tandem mass spectrometry method for determining them in rat plasma, using roscovitine as an internal standard, was developed and validated. Chromatographic separation was performed in reversed phase mode on Acquity BEH C18 column (100 × 2.1 mm, 1.7 µm) by gradient elution with mobile phases composed of 15 mM ammonium formate pH 4.0 and methanol at flow rate 0.25 mL/min at 40 °C. The analytes were detected based on their characteristic multiple reaction monitoring transitions in positive electrospray ionization mode m/z 473.07 > 157.93 for BP-14, m/z 499.62 > 184.2 for BP-20 and m/z 355.5 > 90.86 for internal standard. In plasma the method provided good linearity within the entire concentration range: 1-10,000 nmol/L (r2 = 0.9989) for BP-14 and 10-25,000 nmol/L (r2 = 0.9994) for BP-20; the limit of detection was 0.6 nmol/L for BP-14 and 6.1 nmol/L for BP-20. Validation was also performed in bile and urine. The results of validation fit within the acceptance limits following European Medicines Agency guidelines. The method was applied in a pharmacokinetic study of BP-14 and BP-20 in vivo in rats following intravenous and intraduodenal administration including plasma pharmacokinetics, tissue distribution and excretion (renal and biliary). Both compounds showed low bioavailability after intraduodenal administration (0.630 and 1.58% for BP-14 and BP-20, respectively). Distribution into all the analyzed tissues (brain, lungs, liver, kidney, spleen, muscle, adipose tissue) was observed 3 h after single dose administration, the highest and lowest concentrations being reached in the adipose tissue and brain, respectively. The biliary excretion of the parent BP-14 and BP-20 compounds accounted for 4.81% and 10.6% of the doses, respectively, and renal excretion for <0.5% in both cases. The obtained results represent pilot knowledge for further development of a new generation of compounds with strong anticancer activities.

Stable isotope dilution ultra-high performance liquid chromatography-tandem mass spectrometry quantitative profiling of tryptophan-related neuroactive substances in human serum and cerebrospinal fluid.

Many compounds related to L-tryptophan (L-TRP) have interesting biological or pharmacological activity, and their abnormal neurotransmission seems to be linked to a wide range of neurodegenerative and psychiatric diseases. A high-throughput method based on ultra-high performance liquid chromatography connected to electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) was developed for the quantitative analysis of L-TRP and 16 of its metabolites in human serum and cerebrospinal fluid (CSF), representing both major and minor routes of L-TRP catabolism. The combination of a fast LC gradient with selective tandem mass spectrometry enabled accurate analysis of almost 100 samples in 24h. The standard isotope dilution method was used for quantitative determination. The method's lower limits of quantification for serum and cerebrospinal fluid ranged from 0.05 to 15nmol/L and 0.3 to 45nmol/L, respectively. Analytical recoveries ranged from 10.4 to 218.1% for serum and 22.1 to 370.0% for CSF. The method's accuracy ranged from 82.4 to 128.5% for serum matrix and 90.7 to 127.7% for CSF matrix. All intra- and inter-day coefficients of variation were below 15%. These results demonstrate that the new method is capable of quantifying endogenous serum and CSF levels of a heterogeneous group of compounds spanning a wide range of concentrations. The method was used to determine the physiological levels of target analytes in serum and CSF samples from 18 individuals, demonstrating its reliability and potential usefulness in large-scale epidemiological studies.

In Vitro and In Vivo Comparison of Selected Ga-68 and Zr-89 Labelled Siderophores.

PURPOSE: Some [(68)Ga]siderophores show promise in specific and sensitive imaging of infection. Here, we compare the in vitro and in vivo behaviour of selected Ga-68 and Zr-89 labelled siderophores. PROCEDURES: Radiolabelling was performed in HEPES or sodium acetate buffer systems. Radiochemical purity of labelled siderophores was determined using chromatography. Partition coefficients, in vitro stability and protein binding affinities were determined. Ex vivo biodistribution and animal imaging was studied in mice. RESULTS: Certain differences among studied siderophores were observed in labelling efficiency. Protein binding and stability tests showed highest stabilities and lowest protein binding affinities for Ga-68 and [(89)Zr]triacetylfusarinine C (TAFC). All studied Ga-68 and [(89)Zr]siderophores exhibited a similar biodistribution and pharmacokinetics in mice with the exception of [(89)Zr]ferrioxamine E (FOXE). CONCLUSIONS: Zr-89 and [(68)Ga]siderophores showed analogous in vitro and in vivo behaviour. Tested [(89)Zr]siderophores could be applied for longitudinal positron emission tomography (PET) studies of fungal infections and especially TAFC for the development of novel bioconjugates.

The Effect of LDL-Apheresis and Rheohaemapheresis Treatment on Vitamin E.

Lipid apheresis (extracorporeal lipoprotein elimination) is administered to patients with familial hypercholesterolemia who fail to respond to standard therapy. The nature of the treatment process raises the suspicion that it decreases not only cholesterol but also antioxidants. A group of 12 patients (average age 47±17 y, 4 homozygous and 8 heterozygous individuals) with familial hypercholesterolemia treated by LDL-apheresis or rheohaemapheresis for 3-12 y was included in the study. In addition to cholesterol and triacylglycerol levels, vitamin E and vitamin A and also other markers of antioxidant activity were investigated. Nevertheless, the most important determined parameter was the vitamin E/cholesterol ratio in serum and lipoproteins. The results indicate that both extracorporeal elimination methods are effective and suitable ways to treat severe familial hypercholesterolemia, as the LDL fraction of cholesterol decreased by approximately 77% and 66% following LDL-apheresis and rheohaemapheresis, respectively. In addition, the serum vitamin E decreased by 54% and 57% and the decrease of the serum vitamin A was approximately 20%. However, the main marker of antioxidant capacity, vitamin E/cholesterol ratio, in the serum, VLDL and LDL significantly increased. The increase of vitamin E levels in the erythrocyte membranes of 2% following LDL-apheresis and a significant increase of 4% following rheohaemapheresis were confirmed. The presented results indicate that LDL-apheresis and rheohaemapheresis can be considered to be safe procedures according to the antioxidant capacity of the serum, VLDL and LDL lipoprotein fractions and the erythrocyte membrane.

Selected 68Ga-siderophores versus 68Ga-colloid and 68Ga-citrate: biodistribution and small animal imaging in mice.

BACKGROUND: (68)Ga-triacetylfusarinine C (TAFC) and (68)Ga-ferrioxamine E (FOXE) show great potential to be used as highly sensitive and selective tracers for Aspergillus infection imaging. Here we report on a comparison of the ex vivo biodistribution and small animal imaging of (68)Ga-TAFC and (68)Ga-FOXE versus (68)Ga-colloid and (68)Ga-citrate as unspecific control in mice. METHODS: The radiochemical purity of tested (68)Ga labelled tracers was determined by RP-HPLC or ITLC-SG. Ex vivo biodistribution was studied in normal DBA/2 mice 30 min and 90 min p.i. Static and dynamic imaging were performed using µPET/CT. RESULTS: (68)Ga-TAFC and (68)Ga-FOXE showed rapid renal excretion and low blood values even 90 min p.i. (68)Ga-TAFC showed almost no retention in other organs while (68)Ga-FOXE displayed some uptake in gastrointestinal tract. (68)Ga-colloid and (68)Ga-citrate revealed significantly different ex vivo biodistribution. (68)Ga-colloid showed pronounced radioactivity retention in the liver, while (68)Ga-citrate displayed high blood values and significant retention of radioactivity in highly perfused organs. CONCLUSIONS: From the results, both (68)Ga-TAFC and (68)Ga-FOXE have excellent and significantly different in vivo behaviour compared to (68)Ga-colloid and (68)Ga-citrate. (68)Ga-TAFC in particular confirmed its great potential use as a specific tracer for Aspergillus infection imaging.

Intestinal permeability, vitamin A absorption and serum alpha-tocopherol during therapy with gefitinib.

Measurement of intestinal permeability represents one of the potential methods of noninvasive laboratory assessment of gastrointestinal toxicity of anticancer therapy. We have assessed intestinal permeability (by measuring absorption of lactulose, mannitol, and xylose), vitamin A absorption and serum alpha-tocopherol in patients with non-small cell lung carcinoma or head and neck carcinomas treated with gefitinib. Lactulose, mannitol and xylose were determined by capillary gas chromatography, and retinol, alpha-tocopherol, retinyl stearate and retinyl palmitate were determined by high-performance liquid chromatography. Compared to healthy controls, patients had significantly increased lactulose/mannitol ratio and lower postprandial retinyl palmitate and retinyl stearate concentrations. Compared with pre-treatment values, xylose absorption was decreased and lactulose/mannitol and lactulose/xylose ratios were increased during the therapy. A significant decrease of serum alpha-tocopherol was evident throughout the course of therapy. In contrast, only minor alterations of vitamin A absorption were observed. In conclusion, an alteration in intestinal permeability reflected in increased lactulose/mannitol and lactulose/xylose ratios was observed during gefitinib therapy. Potential association between decreased serum alpha-tocopherol concentrations and the toxicity of gefitinib therapy should be further investigated.

Intestinal permeability, vitamin A absorption, alpha-tocopherol, and neopterin in patients with rectal carcinoma treated with chemoradiation.

Although gastrointestinal toxicity is one of the most common side effects of anticancer therapy, the diagnosis and assessment of this toxicity still depend mostly on anamnestic data. Measurement of intestinal permeability is one of potential methods of non-invasive laboratory evaluation of gastrointestinal toxicity. The aim of the present study was to investigate intestinal permeability, vitamin A absorption, serum alpha-tocopherol, and urinary neopterin in patients with rectal carcinoma treated with chemoradiation. We have studied intestinal permeability, vitamin A absorption, serum alpha-tocopherol, and urinary neopterin in 17 patients with rectal carcinoma treated with chemoradiation. Urinary lactulose, mannitol, and xylose were measured by capillary gas chromatography, and serum alpha-tocopherol, retinol, retinyl esters, and urinary neopterin were determined by high-performance liquid chromatography. Lactulose/mannitol ratio was increased 5 and 6 weeks after the start of the treatment. Serum alpha-tocopherol was decreased significantly throughout the course of treatment, but no significant changes were observed in postprandial serum concentrations of retinyl esters or in the concentrations of urinary neopterin. A correlation was observed between baseline parameters of intestinal permeability and urinary neopterin. The measurement of intestinal permeability using the lactulose/mannitol test may represent a sensitive tool in the detection of changes associated with chemoradiation in patients with rectal carcinoma. The therapy is also associated with a decrease of alpha-tocopherol.

Effect of aromatase inhibitors on lipid metabolism, inflammatory response and antioxidant balance in patients with breast carcinoma.

BACKGROUND: Aromatase inhibitors may affect lipid metabolism, inflammatory response and antioxidant balance. PATIENTS AND METHODS: One hundred and eighty-six post-menopausal patients with breast carcinoma underwent evaluation of parameters of lipid metabolism, inflammatory response and antioxidant balance immediately before as well as 2 and 4 months after the start of therapy with aromatase inhibitors. RESULTS: A significant increase in total, very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol, lipoprotein (a), retinol, C-reactive protein and fibrinogen was observed. The changes of serum lipid concentrations were restricted mostly to the patients pre-treated with tamoxifen who had significantly lower baseline levels of these parameters. CONCLUSION: An increase of serum cholesterol, lipoprotein (a), C-reactive protein and fibrinogen in patients treated with aromatase inhibitors is the result of tamoxifen withdrawal rather than a direct effect of therapy. No significant changes in serum lipids were observed in patients treated with aromatase inhibitors in the first-line setting.

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.

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.

Urinary neopterin, hemoglobin and peripheral blood cell counts in breast carcinoma patients treated with dose-dense chemotherapy.

BACKGROUND: Among other actions, chemotherapy may induce an activation of systemic inflammatory and immune response. PATIENTS AND METHODS: Urinary neopterin was evaluated, using high-performance liquid chromatography, before and during dose-dense combination chemotherapy with doxorubicin, cyclophosphamide and sequential paclitaxel (neoadjuvant or adjuvant) in 194 patients with breast carcinoma. Hemoglobin, peripheral blood cell count and, in a subgroup of patients, iron metabolism were also evaluated. RESULTS: Urinary neopterin increased significantly during the chemotherapy. The increase in urinary neopterin was accompanied by a gradual decrease of hemoglobin. A marked increase in serum ferritin concentration was observed during the chemotherapy, along with fluctuations of iron concentrations. Among 161 patients treated with primary chemotherapy, the pathological response was evaluable in 150. Pathological complete response was observed in 37 cases (25%). In patients with pathological complete response, significantly lower serum ferritin concentrations were observed. CONCLUSION: Present data demonstrate the presence of systemic immune activation, reflected in increased urinary neopterin concentrations, in breast carcinoma patients treated with dose-dense chemotherapy. Lower ferritin concentrations were predictive of pathological complete response.

Serum alpha-tocopherol, retinol and neopterin during paclitaxel/carboplatin chemotherapy.

BACKGROUND: Disorders of antioxidant balance are considered to be involved in the toxicity associated with radiotherapy or chemotherapy. PATIENTS AND METHODS: Serum alpha-tocopherol and retinol were determined, by high performance liquid chromatography, before and during therapy with a combination of paclitaxel and carboplatin in 28 patients with breast and ovarian cancer. Serum neopterin and cholesterol were measured using a radioimmunoassay and enzymatic colorimetric method, respectively. RESULTS: Compared to pretreatment concentrations, a significant increase was observed in serum alpha-tocopherol and retinol concentrations during therapy that was associated with decreased serum neopterin concentrations. Serum alpha-tocopherol concentrations were significantly higher during therapy in patients who did not experience serious toxicity. CONCLUSION: An increase in alpha-tocopherol and retinol during therapy with combination paclitaxel/carboplatin may be explained by inhibition of systemic immune activation secondary to control of the tumor with effective chemotherapy. Lower alpha-tocopherol concentrations were associated with the toxicity of therapy.

Development and validation of a liquid chromatography method for the simultaneous determination of alpha-tocopherol, retinol and retinyl esters in human serum using a monolithic column for the monitoring of anticancer therapy side effects.

Among other side effects, administration of anticancer agents is accompanied by manifestations of gastrointestinal toxicity and disturbances of antioxidant balance. The monitoring of these toxic effects in clinical practice is impeded by a dearth of reliable laboratory methods. Therefore, a simple and rapid reversed-phase high-performance liquid chromatography procedure for selective and sensitive determination of retinol, a-tocopherol, and retinyl esters (retinyl-palmitate and retinyl-stearate) in blood serum has been developed and presented in this study. A Series 200 LC HPLC instrument from Perkin Elmer (Norwalk, USA) with diode-array detector (DAD) was used for the analysis. Separations of retinol, alpha-tocopherol, retinyl-palmitate, and retinyl-stearate were performed using a Chromolith Performance RP-18e, 100 x 4.6 mm monolithic column from Merck (Darmstadt, Germany). Gradient elution was used at a flow rate of 3 mL/min; the mobile phase was methanol-water (95:5, v/v) for 0-2.1 min and methanol-2-propanol (60:40, v/v) for 2.1-4.9 min. The total time of analysis was 6 min. The injection volume was 20 microL and the analysis was performed at ambient temperature. Detection of retinol, alpha-tocopherol, and retinyl esters was carried out at 325, 295, and 330 nm, respectively. For practical assessment of the method, the vitamin A absorption test was performed on seven healthy controls as well as on six patients with non-small cell lung carcinoma or head and neck carcinoma previously treated by chemotherapy and/or radiotherapy, six patients with rectal carcinoma before chemoradiotherapy, four patients with gastrointestinal stromal tumor (GIST) before treatment with imatinib, and a breast cancer patient with chemotherapy-induced diarrhea. Present data demonstrate the feasibility of large scale HPLC determination of vitamin E, vitamin A, and retinyl esters in human serum using a silica monolithic column, and this method may represent a valuable aid in the laboratory monitoring of the toxicity of anticancer therapy.

Urinary neopterin in patients with liver tumors.

AIMS AND BACKGROUND: Primary and secondary liver tumors are associated with poor prognosis. Neopterin is an indicator of systemic immune activation, and increased neopterin concentrations have been associated with poor prognosis in a wide range of malignant tumors. METHODS: Urinary neopterin was determined by high-performance liquid chromatography in 154 patients with primary and secondary liver tumors. The survival of different groups of patients was compared by log-rank test, and Cox regression was used for multivariate analysis. RESULTS: Urinary neopterin was significantly increased in patients compared to controls. A statistically significant correlation was observed between urinary neopterin and age of the patients, hemoglobin concentration, mean erythrocyte volume and peripheral blood leukocyte or platelet count. In univariate analysis, urinary neopterin below 214 micromol/mol creatinine, peripheral blood leukocytes below 8 x 10(9)/L, hemoglobin equal to or above 125 g/L, no extrahepatic tumor, stage of liver involvement, and colorectal, breast or ovarian primary were significant prognostic factors for survival. In multivariate analysis, Bengtsson stage, presence of extrahepatic involvement, primary other than colorectal, breast or ovarian carcinoma, peripheral blood leukocyte count and urinary neopterin were independent prognostic factors. Increased urinary neopterin during and at the end of follow-up was also associated with poor prognosis. CONCLUSIONS: Urinary neopterin is increased in patients with liver tumors. Neopterin is an independent prognostic indicator in patients with liver tumors along with Bengtsson stage, presence of extrahepatic disease, primary site and peripheral blood leukocyte count.

Optimization and validation of a high performance liquid chromatography method for the simultaneous determination of vitamins A and E in human serum using monolithic column and diode-array detection.

In this study a novel, simple and rapid reversed-phase high performance liquid chromatography (HPLC) procedure for simultaneous determination of vitamins A and E (retinol and alpha-tocopherol) in blood serum has been developed and validated using monolithic column and diode-array detection (DAD). The monolithic column Chromolith Performance RP-18e (100 mm x 4.6 mm) was operated at ambient temperature. One hundred percent methanol at flow rate 2.5 ml min(-1) was used as a mobile phase. Detection of both compounds was performed with diode-array detector, retinol was monitored at 325 nm and alpha-tocopherol at 295 nm. The linear dependence between peak area and concentration ranged from 0.25 to 10.00 micromol l(-1) for retinol and 0.5-50.0 micromol l(-1) for alpha-tocopherol. The limit of detection (LOD) for retinol was 0.02 micromol l(-1) and limit of quantification (LOQ) was 0.07 micromol l(-1). The limit of detection (LOD) for alpha-tocopherol was 0.1 micromol l(-1) and limit of quantification (LOQ) was 0.3 micromol l(-1). Retinol was eluted in 0.8 min and alpha-tocopherol in 1.4 min. The simultaneous analysis of vitamin A and E can be achieved in less than 2 min. The implementation of monolithic column Chromolith Performance shortens the time of analysis of both vitamins four times in comparison with using traditional particulate column Pecosphere C18 (150 mm x 4.6 mm), 5 microm. This fact may play an important role for routine clinical analysis of biological samples.