Miloslav Hronek

In Vitro Antiplatelet Activity of Flavonoids from Leuzea Carthamoides

Plants and their secondary metabolites, including flavonoids, exhibit a wide range of biological effects. Consequently, natural substances are receiving an increased attention in medicinal research. Owing to these facts, in vitro antiplatelet activity of ethanol summary extract and four flavonoids from Leuzea carthamoides was determined in human platelet-rich plasma. Arachidonic acid (AA), adenosine diphosphate (ADP), collagen (COL), and thrombin were used as agonists of platelet aggregation. The summary extract showed a significant inhibition of the aggregation induced by COL and ADP. Of the tested flavonoids, eriodictyol (1) and patuletin (2) influenced COL- and AA-induced aggregation. Their IC50 values are presented. Flavonoid glycosides eriodictyol-7-β-glucopyranoside (3) and 6-hydroxykaempferol-7-O-(6″-O-acetyl-β-D[small cap]-glucopyranoside) (4) were found to be weak antiplatelet agents. These results confirmed the fact that glucosylation decreases the antiplatelet activity. Quantitative composition of tested flavonoids in L. carthamoides extract was also determined. Though two of the tested flavonoids inhibited platelet aggregation, further evaluation of L. carthamoides, in order to discover other antiplatelet active compounds and possible adverse health effects, is needed.

Clinically relevant determinants of body composition, function and nutritional status as mortality predictors in lung cancer patients

Lung cancer belongs to the type of tumors with a relatively high frequency of malnutrition, sarcopenia and cachexia, severe metabolic syndromes related to impairment of physical function and quality of life, resistance to therapy and short survival. Inexpensive and accessible methods of evaluating changes in body composition, physical function and nutrition status are for this reason of great importance for clinical practice to enable the early identification, monitoring, preventing and treatment of these nutritional deficiencies. This could lead to improved outcomes in the quality of life, physical performance and survival of patients with lung cancer. The aim of this article is to summarize the recent knowledge for the use of such methods, their predictability for patient outcomes and an association with other clinically relevant parameters, specifically with lung cancer patients, because such an article collectively describing their practical application in clinical practice is lacking. The interest of this article is in the use of anthropometry, handgrip dynamometry, bioelectrical impedance analysis derived phase angle and nutritional screening questionnaires in lung cancer patients.

The association between specific nutritional antioxidants and manifestation of colorectal cancer

Determination of specific antioxidants for examination of oxidative balance and immune responses may be of value in the early diagnosis of colorectal cancer. In the present report, we investigated urinary excretion of zinc, copper, and neopterin and serum levels of vitamins A, C, and E in 30 patients (age = 64 +/- 12 y) with colorectal cancer at the time of diagnosis and in 30 control subjects (age = 61 +/- 11 y) with benign disorders not associated with a systemic inflammatory response. Urinary excretion of zinc, copper, and neopterin was significantly elevated, and serum concentration of vitamin A was decreased in patients with colorectal cancer; these changes are characteristics of systemic immune activation. These phenomena may be of use for the detection of tumor progression and immune response to neoplasm.

The serum levels of calcium, magnesium, iron and zinc in patients with recurrent vulvovaginal candidosis during attack, remission and in healthy controls.

The real cause of recurrent vulvovaginal candidosis (RVVC) is concealed and the etiopathogenesis of this disease remains to be determined. In a cohort study, concentrations of metals in 44 patients with RVVC and 30 healthy age‐matched women were measured and compared. The concentrations of serum calcium (Ca), magnesium (Mg) and iron (Fe) were measured photometrically, the zinc (Zn) levels were determined using flame atomic absorption spectrometry. For statistical analysis were used the Students t‐tests (paired analysis for attack vs. remission; non‐paired analysis for patient vs. control). Although all measured metals were within normal ranges the patients with RVVC had in contrast to the healthy controls significantly lower levels of serum Ca, Mg and Zn and insignificantly higher levels of Fe. These relative changes may contribute to the development of attacks in patients with RVVC.

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.

New equation for the prediction of resting energy expenditure during pregnancy.

OBJECTIVE The equation for the prediction of resting energy expenditure (REE) during pregnancy is unknown. The aim of this prospective longitudinal study was to determine a new equation for prediction of REE in pregnancy. METHODS A total of 152 randomly recruited healthy pregnant Czech women (nonsmokers, not users of chronic medications or abusers of alcohol or drugs, normoglycemic, euthyroid, and not anemic) were divided into two cohorts: group 1 (n=31) was used for determination of the equation for calculation of pregnant REE and group 2 (n=121) for cross-validation of this formula. The REE of the pregnant women in both study groups was examined by indirect calorimetry (REE-IC) along with anthropometry after 12h of fasting in four periods of pregnancy. A statistical comparison of three basic equations (Harris Benedict, Schofield, and Kleiber) was used for the prediction of REE. RESULTS Through correlation analysis and linear regression, a new predictive equation of REE during pregnancy (P REE) was derived from the Harris Benedict equation. We observed high concordance between values from P REE and REE-IC in group 2. Analysis of alternative predictive equations of REE with the addition of kilocalories and a corrected multiplication factor for each stage of pregnancy expressed low concordance. CONCLUSIONS The equation for REE in kilocalories during pregnancy, P REE=346.43943+13.962564 x W + 2.700416 x H - 6.826376 x A (W, weight; H, height; A, age), with SD 116 kcal/d, corresponds closely to REE-IC and maternal changes in each phase of pregnancy. P REE can be applied for prediction of REE during gestation.

Detection of DNA crosslinks in peripheral lymphocytes isolated from patients treated with platinum derivates using modified comet assay.

Platinum-based chemotherapeutic agents induce the formation of crosslinks in DNA, which are accepted as being responsible for the cytotoxicity of these agents. In this study, we used a modification of the alkaline comet assay for detection of the presence of DNA crosslinks in vitro caused by cisplatin, and in peripheral lymphocytes of patients with non-small cell lung carcinoma undergoing chemotherapy with platinum derivatives. The comet technique modified for the detection of DNA crosslinks was calibrated in vitro by treating HeLa cells and human lymphocytes from healthy donors with different concentrations of cisplatin. A cisplatin dose-dependent formation of DNA crosslinks was observed in in vitro measurements using 10-200 µM concentrations of cisplatin. Lymphocytes from cancer patients were also assayed for the formation and repair of DNA crosslinks. Evidence of crosslink formation and repair was observed in peripheral blood lymphocytes of all cancer patients in this study, although some inter-individual differences were observed in the response to chemotherapy and in repair of DNA crosslinks. We propose that monitoring the number of DNA crosslinks in peripheral blood lymphocytes might be a quick and sensitive method for monitoring a patients sensitivity to this agent. Modification of the method by incubation of analysed cells with styrene oxide before crosslink analysis by comet assay extends the use of the method also to laboratories which have no facilities to use ionizing irradiation for introducing DNA breaks into the cells.

Skinfold Anthropometry –The Accurate Method for Fat Free Mass Measurement in COPD

Abstract Purpose: Fat free mass index (FFMI) is an independent predictor of metabolic and functional consequences in COPD. For its measurement dual energy X-ray absorptiometry (DEXA), skin-fold anthropometry (SFA), bioelectrical impedance analysis (BIA) and bioimpedance spectroscopy (BIS) are used in clinical practice. The aim of our pilot study was to analyse precisely and critically which method is most accurate and available for common use in clinical practice for measurement of FFM by assessment against relevant DEXA in patients with COPD. Methods: This was an observational cross-sectional study of consecutive COPD subjects. FFM by methods of SFA, two versions of BIA, and BIS was compared with that from clinically relevant DEXA in 41 outpatients (mean age 66.5 ± 7.7 yrs) with stable COPD, 34 men and 7 women, with mean BMI 28.2 ± 6.1 kg.m−2. Results: All methods underestimate FFM in comparison with DEXA. In the general evaluation non-significant differences with the smallest mean bias were demonstrated for SFA (1.2 kg) and BIA (3.8 kg), but there was a difference of more than 9 kg using BIS and BIA COPD methods (p < 0.0001). The best agreement between DEXA and SFA was demonstrated via Lins concordance coefficient and Bland–Altman test. Conclusions: SFA has been demonstrated as an accurate, available and cheap method for determination of FFM and FM with application of the Durnin Womersley equation for body density and with the Siri equation for FM in patients with COPD. SFA can be easily applied in routine clinical practice.

Anthropometric measured fat-free mass as essential determinant of resting energy expenditure for pregnant and non-pregnant women

OBJECTIVE There is conflicting evidence as to whether anthropometric parameters are related to resting energy expenditure (REE) during pregnancy. The aim of this prospective longitudinal study was to precisely assess a major anthropometric determinant of REE for pregnant and non-pregnant women with verification of its use as a possible predictor. METHODS One hundred fifty-two randomly recruited, healthy, pregnant Czech women were divided into groups G1 and G2. G1 (n = 31) was used for determination of the association between anthropometric parameters and REE. G2 (n = 121) and a group of non-pregnant women (G0; n = 24) were used for verification that observed relations were suitable for the prediction of REE during pregnancy. The women in the study groups were measured during four periods of pregnancy for REE by indirect calorimetry and anthropometric parameters after 12 h of fasting. RESULTS Associations were found in all groups between measured REE by indirect calorimetry and anthropometric parameters such as weight, fat mass, fat-free mass (FFM), body surface area, and body mass index (P < 0.0001). The best derived predictor, REE/FFM (29.5 kcal/kg, r = 0.70, P < 0.0001), in group G1 was statistically verified in group G2 and compared with G0. CONCLUSION Anthropometrically measured FFM with its metabolically active components is an essential determinant of REE in pregnancy. REE/FFM can be used for the prediction of REE in pregnant and non-pregnant woman.

DNA crosslinks, DNA damage and repair in peripheral blood lymphocytes of non-small cell lung cancer patients treated with platinum derivatives

Lung cancer is the leading cause of cancer-related mortality in the world. Chemotherapy has been the mainstay of treatment for advanced non-small cell lung cancer (NSCLC) and platinum-based derivatives have been shown to improve overall survival. The aim of the present study was to investigate the DNA damage [single strand breaks (SSBs) and DNA crosslinks] and DNA repair in peripheral blood lymphocytes in patients with NSCLC treated with platinum derivatives using modified comet assay. Twenty patients in the final (4th) stage of NSCLC and 10 age-corresponding healthy controls participated in the study. Alkaline comet assay was performed according to the appropriate protocol. The DNA base excision repair (BER) activity of the controls was significantly higher compared to that of cancer patients, and the activity of DNA nucleotide excision repair (NER) was almost at the same level both in controls and patients. We observed changes in the amount of SSBs and DNA crosslinks during the course of chemotherapy. We found a significantly higher level of SSBs immediately after administration of chemotherapy. Similarly, we found the highest incidence of DNA crosslinks immediately or 1 day after chemotherapy (compared to measurement before chemotherapy). Moreover, we compared the levels of DNA repair in patients who survived chemotherapy with those in patients who died in the course of chemotherapy: the activity of BER was higher in the case of surviving patients, while the levels of NER were essentially the same. The data arising from the present study confirm the findings of other studies dealing with DNA damage and repair in cancer patients treated with chemotherapy. Moreover, our results indicated that despite the fact that cisplatin-DNA adducts are removed by the NER pathway, BER may also play a role in the clinical status of patients and their survival.