Radomír Hyšpler

Determination of isoprene in human expired breath using solid-phase microextraction and gas chromatography–mass spectrometry

An analytical method for determination of isoprene in expired breath as a marker of body cholesterol synthesis was developed with a special emphasis on breath sampling. Patients were breathing controlled air using respiratory masks for 2 min (washout period) and then their expired breath was collected in 8-1 Tedlar bags. The bags were heated to 40 degrees C and the solid-phase microextraction fiber Carboxen-polydimethylsiloxane 75 microm was inserted through the septum. Extraction time was 10 min. Analytes were desorbed in the GC injector for 2 min at 270 degrees C. Analyses were performed on a Q-PLOT column and fragment ions 68, 67 and 53 were quantified. The concentration range was 1-40 nmol/l, limit of detection was 0.25 nmol/l, the calibration curve was linear. Precision, expressed as RSD, was 5.5-12.5%. These tests are non-invasive, feasible and relatively inexpensive.

A simple, optimized method for the determination of sulphide in whole blood by GC–MS as a marker of bowel fermentation processes

Hydrogen sulphide is produced in human large intestine by anaerobic fermentation and may play a pathogenic role. An analytical method for determination of sulphide in whole blood using an extractive alkylation technique was optimised and validated for this purpose. The sample was mixed with organic phase containing pentafluorobenzyl bromide as an alkylating agent. The benzalkonium chloride was used as a phase-transfer catalyst. The quantitative determination was performed using GC-MS technique in selected ion monitoring mode. The blood levels of sulphide of healthy controls were measured (35-80 microM/l). The method is versatile, reproducible (RSD=2.7%) and suitable for research of anaerobic fermentation in vivo.

Intestinal permeability in the assessment of intestinal toxicity of cytotoxic agents.

The diagnosis and assessment of the severity of intestinal mucosal damage in cancer patients treated with cytotoxic drugs still rely on anamnestic data. There is cumulative evidence that measurement of intestinal permeability may represent a sensitive indicator of intestinal damage by cytotoxic agents. The intestinal permeability testing is based on differential permeability of tight junctions along the crypt-villus axis to nonmetabolized sugars. Cytotoxic drugs induce flattening of villi, leading to increased exposure of luminal contents to crypts and increased disaccharide absorption. An increased disaccharide/monosaccharide ratio and decreased xylose absorption have been described in patients treated with different cytotoxic drugs across a spectrum of malignant tumors that correlated with clinical manifestations, and were used to monitor the effect of therapeutic interventions.

Polypharmacy and malnutrition.

Purpose of reviewMalnutrition and polypharmacy increase with age and polymorbidity and their relationship is based on a number of mechanisms. The occurrence of malnutrition in both in-patients and out-patients and its dependence on polymorbidity and age are well known, but the interrelation of polypharmacy and malnutrition has been far less investigated. The countries with the highest occurrence of polypharmacy in Europe include the Czech Republic and Finland, whereas the lowest prevalence of polypharmacy is found in Norway and the Netherlands. Recent findingsThe occurrence, consequences and mutual relationship of malnutrition and polypharmacy are described. Up-to-date knowledge regarding the influence of drugs on nutritional status is summarized. SummaryThe effect of polypharmacy on nutrition is suggested from the observations that problems with nutrition occur mostly in elderly patients, and that such patients are more frequently subject to polypharmacy. It is known that about 65% of hospitalized patients have a worse nutritional status than their healthy contemporaries. A worsened nutritional status may adversely influence the process of treatment.

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.

Cholesterol metabolism in active Crohn's disease

ZusammenfassungErniedrigte Cholesterin-Spiegel gelten als typisches Merkmal einer schweren Erkrankung und sind mit einer schlechten Prognose vergesellschaftet. Der Morbus Crohn ist eine entzündliche Erkrankung, die mit verschiedenen Störungen des Stoffwechsels einhergeht. In den vergangenen Dekaden haben verschiedene klinische Studien einen Zusammenhang zwischen dem Lipidstoffwechsel und der systemischen Entzündung etabliert. In unserer Studie haben wir nicht nur das Cholesterin-Profil (Cholesterin, LDL und HDL Cholesterin) im Plasma bestimmt, sondern auch Veränderungen im Cholesterin-Absorptions- beziehungsweise -Synthese-Prozess durch Bestimmung der Konzentrationen der Nicht-Cholesterin-Sterole im Serum untersucht. Die Plasmakonzentrationen des Gesamtcholesterins, des LDL- und HDL-Cholesterins sowie der Nicht-Cholesterin-Sterole (Squalene, Lathosterol, Campesterol, Sitosterol) wurden bei 24 Patienten mit aktivem Morbus Crohn in einer Zeitspanne von 28 Tagen erhoben. Die Plasmakonzentrationen des Gesamtcholesterins (p < 0,001) und des LDL-, und HDL-Cholesterins (p < 0,05) waren signifikant niedriger als die bei Kontrollpersonen erhobenen Werte. Im Vergleich zur Kontrolle hatten die Patienten mit aktivem Morbus Crohn signifikant niedrigere Plasmaspiegel von Lathosterol (p < 0,001) mit gleichzeitig nicht signifikant erhöhten Konzentrationen von Squalene. Die Campesterol-Plasmakonzentrationen waren signifikant (p < 0,001) erniedrigt. Sitosterol auch – aber nicht signifikant. Die aktive Phase des Morbus Crohn ist durch Veränderungen des Lipidstoffwechsels – vor allem von Cholesterin – gekennzeichnet. Unsere Ergebnisse zeigen Plasmakonzentrationen der Nicht-Cholesterin-Sterole außerhalb der Norm und sprechen dafür, dass die Cholesterin-Synthese und -Absorption beim aktiven Morbus Crohn verändert ist.SummaryHypocholesterolemia has been investigated as a typical feature of critical illness and is connected with poor prognosis. Crohns disease is an inflammatory process and is associated with several metabolic disturbances. In recent decades clinical studies have established a link between lipid metabolism and systemic inflammation. In our study we examined the serum profile of cholesterol (total cholesterol, LDL- and HDL-cholesterol) and changes in the cholesterol absorption/synthesis process by determination of plasma non-cholesterol sterol (squalene, lathosterol, campesterol, sitosterol) concentrations. Serum concentrations of total cholesterol, LDL- and HDL-cholesterol and non-cholesterol sterols were evaluated in 24 patients with active Crohns disease during a period of 28 days. We detected lower serum levels of total cholesterol (P < 0.001), LDL- and HDL-cholesterol (P < 0.05) in the patients with active Crohns disease than in the control group. In addition, the patients had significantly lower plasma levels of lathosterol (P < 0.001) and higher concentrations of squalene, although without significant differences. A significant decrease of campesterol plasma levels (P < 0.001) was detected, but lower plasma concentrations of sitosterol were without statistical significance. The active phase of Crohns disease is characterized by altered metabolism of lipids, mainly of cholesterol. Our results show abnormalities in plasma concentrations of non-cholesterol sterols and provide evidence that the process of cholesterol synthesis and absorption is altered in active Crohns disease.

Epigallocatechin gallate enhances biliary cholesterol secretion in healthy rats and lowers plasma and liver cholesterol in ethinylestradiol-treated rats

The beneficial effect of the major green tea catechin, epigallocatechin gallate (EGCG), on cholesterol homeostasis has been studied mainly in relation to the intestinal absorption of cholesterol; however, how EGCG affects cholesterol metabolism in the liver is not entirely known. The present study investigated the effect of EGCG on liver cholesterol metabolism in healthy and ethinylestradiol-treated rats. EGCG treatment reduced plasma total cholesterol in ethinylestradiol-treated animals and very low density lipoprotein cholesterol in both groups receiving EGCG. In healthy rats, despite the decrease in bile flow, EGCG markedly enhanced biliary secretion of cholesterol and phospholipids. These changes were correlated with increased expression of ATP-binding cassette transporter G5 and G8 and scavenger receptor class B type 1, and decreased expression of acyl-CoA:cholesterol acyltransferase. Ethinylestradiol treatment caused marked hepatic cholesterol accumulation with a concomitant liver weight increase and plasma cholesterol reduction. In ethinylestradiol-treated rats, EGCG co-administration attenuated the increase in liver cholesterol and liver weight. Furthermore, EGCG blunted induction of acyl-CoA:cholesterol acyltransferase and raised reduced levels of ATP-binding cassette transporter G5 and G8 and 3-hydroxy-3-methyl-glutaryl-CoA reductase in ethinylestradiol-treated rats. In conclusion, this study has demonstrated for the first time the ability of EGCG to enhance biliary cholesterol secretion and to attenuate ethinylestradiol-induced liver cholesterol accumulation. Changes in the expression of relevant enzymes and transporters suggest evidence of another mechanism that may contribute to the overall effect of EGCG on cholesterol metabolism and imply new physiological consequences of this widely used compound.

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.

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.