Zdeněk Zadák

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.

Urinary neopterin in patients with advanced colorectal carcinoma.

In previous studies, mostly in patients with early stage colorectal carcinoma, neopterin, an indicator of systemic immune activation, has been associated with poor prognosis. The aim of the present study was to evaluate urinary neopterin in patients with advanced or metastatic colorectal carcinoma treated with chemotherapy. A retrospective analysis was performed of urinary neopterin, determined by high-performance liquid chromatography, in 88 patients with advanced or metastatic colorectal carcinoma. Peripheral blood cell count and serum carcinoembryonic antigen (CEA) were determined in 72 patients before the start of chemotherapy. Urinary neopterin in colorectal carcinoma patients was significantly increased compared to controls, but lower than in patients with inflammatory bowel disease. Neopterin correlated significantly with serum CEA, age, peripheral blood leukocyte and platelet counts. The median survival of colorectal carcinoma patients with urinary neopterin below 214 mol/mol creatinine was significantly longer compared to that of patients with higher neopterin concentrations (median 18 vs 5 months, log-rank test p=0.003). CEA and hemoglobin were also associated with survival in univariate analysis, but in multivariate analysis only urinary neopterin and serum CEA were independent predictors of survival. High urinary neopterin during follow-up was also predictive of poor prognosis.

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.

Small bowel permeability in patients with cytostatic therapy

The purpose of this study was to examine the influence of cytostatic therapy on the barrier function of the small bowel. In 16 patients with tumors in the gastrointestinal tract with metastatic involvement of the liver, small bowel permeability was measured using the lactulose/mannitol test. The patients were treated for 5 d with fluorouracil (750-1000 mg/d) and leucovorin (25-50 mg/d). The examination was performed on the first day, at the beginning of the cytostatic therapy, and also 5, 12, and 28 d after the therapy had begun. In comparing the start and the end of this therapy, the index of permeability was significantly increased (as measured 7 d after the end of therapy). These results show the damage of small bowel barrier after cytostatic therapy.

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.

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.

Bioanalysis of PUFA metabolism and lipid peroxidation in coronary atherosclerosis

Twenty eight men (age 34-77 years) who underwent an elective coronary angiography for coronary artery disease (CAD), were studied. They were divided into group A (luminal narrowing < 50%; n = 11) and group B (luminal narrowing > 50%; n = 17). Capillary gas chromatography was used for determination of fatty acids. Retinol and alpha-tocopherol were analyzed by reversed-phase high-performance liquid chromatography (HPLC), other parameters were determined spectrofluorometrically and spectrophotometrically. Severe coronary atherosclerosis in group B was associated with higher serum low density lipoprotein/high density lipoprotein (LDL/HDL) cholesterol ratio, triacylglycerols, and phospholipids (P < 0.05). Erythrocyte membrane fatty acids C14:0, C16:1 and C22:6n3 were significantly higher in group B (P < 0.05). We found significantly higher plasma polyunsaturated fatty acids (PUFA) C18:3n6 in group B, whereas plasma linoleic acid was not changed significantly. There was a significant increase of IDL-C18:0, LDL-C14:0 and HDL-C22:6n3 PUFA in group B. We conclude that disturbances in saturated fatty acids (SUFA) and PUFA metabolism are associated with coronary atherogenesis. Such abnormalities may include enhanced extrahepatic transport of C14:0 SUFA via LDL and its incorporation into cell membranes, and enhanced clearance of anti atherosclerotic C22:6n3 PUFA via serum HDL.

Bioanalysis of age-related changes of lipid metabolism in nonagenarians

The aim of the present study was the bioanalysis of lipid metabolism in the aged patients and to study the relationship between these biochemical markers and longevity. Eleven nonagenarians, nine women and two men, aged 94+/-3 years and ten control patients, six women and four men, aged 84+/-5 years, followed at the Department of Metabolic Care and Gerontology, Charles University, Teaching Hospital entered the study. All subjects were self-sufficient, without major illnesses and free living. At the start of the project the free fatty acids (FFA), thiobarbituric reactive substances (TBARS), retinol, alpha tocopherol, ascorbic acid, cholesterol, triacylglycerols, phospholipids in serum, in lipoprotein fractions and fatty acids (FA) and phospholipids in erythrocyte membrane were determined. We used capillary gas chromatography for determination of fatty acids. Retinol and alpha tocopherol were analysed by reversed-phase high-performance liquid chromatography, other parameters were determined spectrophotometrically or spectrofluorometrically. We found significantly higher LDL polyunsaturated fatty acids (PUFA) 22:4n--3 (P=0.028) and 22:6n--3 (P=0.018) and a significant increase of HDL alpha tocopherol/cholesterol ratio (P=0.034) in nonagenarians. There were not any significant differences in erythrocyte membrane fatty acids and phospholipids. In serum we found significantly higher level of TBARS (3.22+/-1.22 vs 1.98+/-0.71 micromol/l, P=0.012) in nonagenarians, other parameters were not changed significantly. The higher concentration of PUFAs in LDL and alpha tocopherol in HDL might be parameters related to longevity.

The Energetic and Metabolic Effect of Ringerfundin (B. Braun) Infusion and Comparison with Plasma-Lyte (Baxter) in Healthy Volunteers

Measurement of parameters of energy requirement, respiratory quotient (RQ), rate of oxygen consumption (VO2) and rate of carbon dioxide production (VCO2) reveal Ringerfundin as an excellent and metabolically stable-acting balanced ionic solution, which does not increase the consumption of O2 or the total energy requirement. In conclusion, Ringerfundin was very well tolerated and in no case were observed undesirable effects.