Combination of ultracentrifugation and solid-phase extraction with subsequent chromatographic analysis of α-tocopherol in erythrocyte membranes.

A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid-phase extraction for the determination of α-tocopherol in human erythrocyte membranes by high-performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d-mannitol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and calcium chloride. The α-tocopherol was then extracted from the erythrocyte membranes by solid-phase extraction with n-hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α-tocopherol in real samples from patients. The combined ultracentrifugation and solid-phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid-liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes.

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.

New approach for the clinical monitoring of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by ultra high performance liquid chromatography with MS/MS based on the standard reference material 972.

Biomarkers, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , are important indicators of the vitamin D general status and are monitored in several pathophysiological disorders, such as osteoporosis, diabetes, heart disease, etc. A novel ultra-HPLC with MS/MS methodology for the analysis of 25-hydroxyvitamin D derivatives coupled with a very simple and highly rapid sample preparation step was developed. Analytical parameters obtained showed linearity (R(2) ) above 0.999 for both vitamins with accuracies between 95.8 and 102%. The LODs were as low as 0.22 and 0.67 nmol/L for 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , respectively. Intra-assay precision (%RSD) was lower than 4.5%, and inter-assay precision (%RSD) was lower than 6.5%. The feasibility of the developed methodology to be applied in clinical routine analysis has been proved by its application in blood samples from non-agenarian patients, patients with familial hypercholesterolemia and patients suffering from age-related macular degeneration.

Prognostic significance of serum retinol, serum alpha-tocopherol, and urinary neopterin in patients with head and neck carcinoma treated with external beam radiation.

High serum or urinary neopterin concentrations are associated with poor prognosis in patients with tumors of different primary locations, but reports on neopterin in patients with head and neck carcinoma are relatively less numerous. It has been established that decreased circulating concentrations of retinol and alpha-tocopherol are common in this population. We have evaluated the prognostic significance of urinary neopterin, serum retinol, and alpha-tocopherol in 44 patients with head and neck carcinoma. Urinary neopterin, serum retinol, and alpha-tocopherol were determined with high-performance liquid chromatography. High urinary neopterin and low serum retinol were predictive of poor prognosis, while the prognostic significance of low alpha-tocopherol was of borderline significance. Serum retinol significantly decreased during external beam radiation, but a less marked decrease of alpha-tocopherol during therapy did not reach statistical significance. An increase of urinary neopterin was evident late during the course of treatment. In conclusion, high urinary neopterin and low serum retinol are predictive of poor prognosis in patients with head and neck carcinoma.

Blood levels of antioxidants during age-related macular degeneration treatment by rheohaemapheresis.

AIMS: Rheohaemapheresis treatment influences rheological markers and most likely improves metabolism in affected retinal areas, resulting not only in absorption of soft drusen but also reduction or complete disappearance of drusenoid retinal pigment epithelium detachments. However, the character of the treatment process has raised suspicion that there is a decrease not only in cholesterol but also in antioxidants, such as vitamin E and vitamin A. METHODS: Twenty-three patients with the progressive dry form of age-related macular degeneration were each treated with 8 procedures of rheohaemapheresis. We measured levels of vitamin E (α-tocopherol), the vitamin E/cholesterol ratio in serum and lipoproteins (VLDL, LDL, HDL). Vitamin E in erythrocyte membrane and serum vitamin A (retinol) were also measured. These parameters were determined before and after rheohaemapheresis. Erythrocyte superoxide dismutase, erythrocyte glutathione peroxidase and serum malondialdehyde were analysed as markers of antioxidant activity and lipid peroxidation, respectively. RESULTS: In serum, the VLDL and LDL fraction ratios of vitamin E/cholesterol increased significantly. Additionally, the HDL fraction ratio showed an increase but this was not statistically significant. The patients showed no clinical signs of vitamin E deficiency, and their serum concentrations of vitamin E did not differ from normal values. The results show that rheohaemapheresis in addition to causing a significant reduction in atherogenic LDL cholesterol, may have favourable additive anti-atherogenic effects due to a relative increase in the content of vitamin E in the lipoprotein fractions.

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.

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.

Urinary neopterin concentration and toxicity of radiotherapy in patients with head and neck carcinoma during external beam radiation.

AIM: Increased serum or urinary concentrations of neopterin are predictive of poor prognosis in patients with tumors across a spectrum of primary locations. Less information is available about the significance of changes of urinary neopterin concentrations during therapy. The aim of the present study was to examine the association between urinary neopterin and toxicity of radiotherapy. PATIENTS AND METHODS: We analyzed changes of urinary neopterin and toxicity of therapy in 12 patients with head and neck carcinoma during external-beam radiation. Urinary neopterin was determined daily by high-performance liquid chromatography. RESULTS: In addition to a trend for increased neopterin concentrations during radiation therapy, a significant association between changes of neopterin and toxicity and vice versa was observed with a rise of neopterin predicting a later manifestation of toxicity as well as manifestion of toxicity predicting a later rise of neopterin. CONCLUSION: Urinary neopterin is predictive of toxicity in patients with head and neck carcinoma. An association between toxicity and subsequent rise of urinary neopterin concentrations was also observed.

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.

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.

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 phase's 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.

Intestinal permeability, vitamin A absorption and serum alpha-tocopherol in gastrointestinal stromal tumor patients treated with imatinib.

Administration of imatinib is the therapy of choice in patients with advanced (inoperable) or metastatic gastrointestinal stromal tumors (GIST). Gastrointestinal toxicity is one of the most common side effects of anticancer therapy, including imatinib. Measurement of intestinal permeability represents a method of noninvasive laboratory assessment of gastrointestinal toxicity. We have measured intestinal permeability (by determining absorption of lactulose, mannitol and xylose), vitamin A absorption and serum alpha-tocopherol in 16 patients with advanced/metastatic GIST treated with imatinib. Lactulose/mannitol and lactulose/xylose ratios as well as parameters of vitamin A absorption did not change significantly during the treatment, but a significant decrease of alpha-tocopherol was observed. We conclude that, in contrast to most other anticancer agents studied so far, imatinib does not have an effect on intestinal permeability. No effect on vitamin A absorption was observed, but serum alpha-tocopherol decreased significantly during the treatment.

Erythrocyte alpha-tocopherol in breast cancer patients treated with letrozol.

In an earlier study, we have observed an increase of alpha-tocopherol in breast cancer patients treated with third-generation aromatase inhibitors that was related to tamoxifen withdrawal. We report here the results of measurement of alpha-tocopherol in erythrocytes and alpha-tocopherol/cholesterol ratios in patients treated with letrozol. Alpha-tocopherol in lipoprotein fractions and erythrocytes was determined by high-performance liquid chromatography in 124 post-menopausal women with breast cancer treated with letrozol immediately before the start of treatment as well as 2 and 4 mo later. After a transient decrease after 2 mo of letrozol therapy, erythrocyte alpha-tocopherol concentrations returned to pre-treatment levels 4 mo after the start of treatment. Apart from lower cholesterol in patients pre-treated with tamoxifen, no significant differences were observed at baseline between patients previously treated with tamoxifen and patients who had no prior tamoxifen in any of the other parameters investigated, but the transient decrease of erythrocyte alpha-tocopherol was observed only in patients previously treated with tamoxifen. Alpha-tocopherol content of lipoprotein fractions was significantly increased 4 mo after the start of therapy, but this increase was evident mostly in patients not treated earlier with tamoxifen. In conclusion, only minor changes of alpha-tocopherol, including a transient decrease of alpha-tocopherol in erythrocyte membranes, and an increase of alpha-tocopherol in lipoprotein fractions were observed during the first 4 mo of letrozol therapy.

Serum retinol, alpha-tocopherol and systemic inflammatory response in metastatic colorectal carcinoma patients treated with combination chemotherapy and cetuximab.

Cetuximab is a chimeric antibody registered for the therapy of advanced colorectal carcinoma. Cancer and anticancer therapy are associated with oxidative stress, and disorders of antioxidant balance may be involved in the toxicity associated with anticancer treatment. The aim of the present study was to investigate the changes of serum retinol, alpha-tocopherol and C-reactive protein during the first month of treatment with cetuximab and chemotherapy. Twenty-five consecutive patients with metastatic colorectal carcinoma treated with a combination of chemotherapy and cetuximab were included in the present study. Serum retinol and alpha-tocopherol were determined by high-performance liquid chromatography and serum C-reactive protein was determined using commercial kits. Significant correlation was observed between baseline concentrations of retinol and C-reactive protein (r(s)=-0.54, p<0.01). Median survival of patients who had baseline serum retinol below 1.25 µmol/L was 10 mo compared to 18 mo for patients who had serum retinol equal or above 1.25 µmol/L (p<0.05); median survival of patients who had serum C-reactive protein below 24 mg/L was significantly longer compared to patients with C-reactive protein levels equal or above 24 mg/L (18 vs. 7 mo, p<0.05), but no difference in survival was observed based on alpha-tocopherol levels. Twenty-two patients had evaluation of retinol, alpha-tocopherol and C-reactive protein at least once during the follow up. Serum concentration of alpha-tocopherol decreased significantly during the therapy, but retinol and C-reactive protein concentrations remained unchanged. In conclusion, a significant correlation was observed between serum retinol and C-reactive protein. Serum alpha-tocopherol decreased significantly during the first month of combination therapy with cetuximab. Low retinol and high C-reactive protein concentrations were predictive of poor prognosis in this patient population.