References - Full-text (PDF file)
Last updated: October 17, 2017

Přehled publikací a citací v databázi Google Scholar.
Kompletní přehled 500 publikací od roku 1975 do 2017 je v PDF souboru Reference 2017
Abstrakta publikací zveřejněná v databázi NLM Medline - Abstrakta NLM Medline
Diplomové a bakalářské práce studentů ÚKBLD - Diplomky

45. Doporučení České společnosti klinické biochemie ke správnému používání metody stanovení okultního krvácení ve stolici. (Article in Czech)
Tempus medicorum 2015, 24/10: 30-31
Kocna P., Zima T.
Ústav lékařské biochemie a laboratorní diagnostiky 1.LF UK a VFN Praha
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Abstrakt
Kvantitativní FIT testy pro okultní krvácení jsou v současné době optimálním primárním testem pro screening KRCA. Česká společnost klinické biochemie doporučuje z hlediska kvality a vyhodnocování péče o pacienty a epidemiologické srovnatelnosti výsledků, prováděných v České republice, jednotný postup. Stanovení okultního krvácení ve stolici provádět výhradně kvantitativními FIT testy a výsledky vyjadřovat koncentrací Hb ve stolici, v jednotkách mg/g stolice. Podrobnější text „Stanovisko ke stanovení hemoglobinu ve stolici kvantitativní analýzou“ bylo zveřejněno v časopise Klinická Biochemie Metabolismus (2015,23/2: 78-81) a je na nternetu dostupné on-line.

44. Stanovisko ke stanovení hemoglobinu ve stolici kvantitativní analýzou (Article in Czech)
Klin.Biochem.Metab. 2015, 23/2: 78-81
Kocna P., Zima T.
Ústav lékařské biochemie a laboratorní diagnostiky 1.LF UK a VFN Praha
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Abstrakt
Stanovení hemoglobinu ve stolici kvantitativní imunochemickou technologií (FIT) je v současné době nejpřesnější metodou stanovení okultního krvácení, vhodnou pro celopopulační screening KRCA. Kvantitativní analýza byla doporučena Radou screeningu KRCA ČGS již v roce 2010, avšak populační screening KRCA od roku 2014 umožňuje použití nejrůznějších FIT testů, kvalitativních i kvantitativních. Rizikem kvalitativních FIT testů je vysoká falešná pozitivita, zvyšující nároky na screeningové kolonoskopie, rizikem heterogenity povolených FIT testů je výrazné zkreslení efektivity screeningu pro jednotlivé oblasti České republiky. ČSKB doporučuje z hlediska kvality a vyhodnocování péče o pacienty a epidemiologické srovnatelnosti výsledků, prováděných v České republice jednotný postup vyjadřován koncentrace Hb ve stolici, v jednotkách mg/g stolice. Spolehlivost kvantitativní analýzy prováděné v laboratořích i pomocí POCT analyzátorů má být pravidelně ověřováno externí kontrolou kvality, v souladu s doporučením Evropské unie.

43. Clinical and epidemiological importance of analyzing laboratory data with the data source I-COP (Article in Czech)
Sborník Medsoft 2014, Praha, 2014, 110-122
Kocna P., Májek O., Blaha M.
Ústav lékařské biochemie a laboratorní diagnostiky 1.LF UK a VFN Praha; Institut biostatistiky a analýz Masarykovy universisty Brno
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Summary:
The Czech Republic is, with the value of colorectal cancer (CRC) incidence 80 in 100 thousand inhabitants, one of the most burdened countries in Europe. Prediction of the CRC (ICD -10: C18 - C20) for the year 2014 states 8743 new cases CRC, prevalence prediction for year 2014 shows 59,401 patients with CRC. CRC screening becomes population-based starting from January 2014. The screening programme is organized by the Council for CRC screening of the gastroenterological society and CRC screening committee at the Ministry of Health, in collaboration with health insurance companies. The two-stage CRC screening for persons aged over 50 years includes basic screening for occult blood in the stool by immunochemical FOBT test. In the General University Hospital in Prague, we set FOBT test on an automatic analyzer OC -Sensor, Eiken Japanese manufacturer. During the period 2008 to 2013, 14,495 samples were examined by quantitative test OC-Sensor. The aim of this paper is to describe the clinical and epidemiological significance of analysis of laboratory data from the laboratory information system VFN - OpenLIMS Stapro with the data source I-COP and evaluation aspect of the quantitative analysis of hemoglobin in stool for CRCA screening.

42. Creating personal archives of scientific publications in the portal medvik of the national library of medicine - case study (Article in Czech)
Sborník Medsoft 2014, Praha, 2014, 154-164
Maixnerová L., Kříž F., Bouzková H., Kocna P., Lesenková E., Horsák O., Jarolímková A.
Národní lékařská knihovna, Praha; Ústav lékařské biochemie a laboratorní diagnostiky 1.LF UK a VFN Praha
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Summary:
National Medical Library (NML) in 2013 developed a web application allowing storage of full-text scientific works by authors themselves (self-archiving) using the portal Medvik in the Digital Library of NML. Self-archiving was verified on a particular author – a Czech physician and biochemist. In the first phase an analysis of his publications was made, where it was verified, how much publications the author has mentioned in the BMC databases, PubMed , Embase , Scopus, WoS , GoogleScholar and his personal website. A mutual comparison was made between the databases and the author's page. It was examined whether the works which are not part of the BMC can be included. The works were further divided into those, where the author is listed as the first author, whether the works were published in the Czech Republic or abroad, according to the document type to articles, articles in books, abstracts, monographs, final reports, chapters in monographs, etc. For each category possibilities of self-archiving and access to the public were tested. For works published in the Czech Republic we used the Copyright Act and the actual Civil Code as the main resources. For foreign works the possibility of archiving was verified using the SHERPA/Romeo service. During the process of self-archiving itself, the author is able to select the copyright license (Creative Commons) that determines how the document can be treated by other users. During the study it has proved to be important to add the choice to archive the document, but the document will not be available to the public as it is not authorized by the publisher, or this information is not available, or if the author is not sure whether it is allowed by the co-authors. Currently it is only possible to archive documents that have bibliographical record in BMC - in the future the ability to archive works not included in the BMC will be added. Pilot operation services of self-archiving demonstrated the applicability for the basic types of published works, mainly journal articles. For the wider use of professional public there is a need to incorporate other types of documents. Self-archiving in the institutional or disciplinary repositories is an emerging global trend in the world of publishing scientific literature. In the process of access to the public is self-archiving considered the green way of open access, supported by the NML.

41. Specificities of the diagnostics and therapy of exocrine pancreatic insufficiency (Article in Czech)
Vnitřní Lékařství, 2013, 59(1): 65 - 70
Dítě P., Novotný I., Kocna P., Bojková M., Kupka T., Nechutová H., Kianička B.
Akademické centrum gastroenterologie a gastroonkologie OU Ostrava, Gastroenterologické oddělení Masarykova onkologického ústavu Brno, Ústav lékařské biochemie a laboratorní diagnostiky 1. lékařské fakulty UK a VFN Praha, Interní klinika Lékařské fakulty OU a FN Ostrava, Gastroenterologické oddělení II. interní kliniky Lékařské fakulty MU a FN u sv. Anny Brno
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Summary:
Exocrine pancreatic insufficiency develops steadily; however, the initial reduction in secretion is practically not diagnosable. More advanced stages, which usually replicate morphological changes, can be determined with tests which asses the exocrine pancreatic capacity. Substantial damage of the pancreas and replacement of viable parenchyma with connective tissue is accompanied by the occurrence of steatorrhoea. This corresponds to a reduction in exocrine pancreatic secretion below 10% of physiological secretion. Exocrine pancreatic secretion tests are still not sufficiently sensitive for diagnosing early stages of pancreas defects and thus are not suitable for diagnostics. Furthermore, detecting reduced exocrine secretion does not provide any information about the aetiology of the disease, e.g. inflammation/tumor. The most precise test is a costly examination, including a stimulation of the gland with enterohormones; however, breath tests are usually recommended for the assessment of exocrine insufficiency therapy. Exocrine pancreatic insufficiency therapy consists of administering drugs containing pancreatin (amylase, lipase, and peptidase) to patients diagnosed with steatorrhoea, manifest pancreatic insufficiency. As standard, capsules containing microparticles of 1–2mm are recommended. They have a protective coating that prevents inactivation in the microparticles of the contained enzymes by gastric hydrochloric acid. The drug should be administered during each meal, i.e. several times a day. The most common mistake during pancreatic enzyme therapy is underdosage. The following rule applies to patients with digestive insufficiency: 40,000–50,000 UNT of lipase are to be administered during “main meals” and 25,000 UNT of lipase during morning or afternoon snacks. The drug should be taken during the meal; insufficient treatment and dosage are associated with insufficient digestion and absorption of a number of substances and also with pancreatic malabsorption.

40. Laboratory screening markers in gastroenterology - state of the art
Biomed Pap 2013, 157:(2): 91-97
Kocna P, Vanickova Z, Zima T.
Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Czech Republic.
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Abstract:
INTRODUCTION: Screening tests for gastrointestinal diseases acceptable for population with a high sensitivity and high specificity can now be offered by clinical laboratories. This paper summarizes major recent advances in this area of laboratory medicine. METHODS: Relevant articles published within the last 5 years in the NLM (National Library of Medicine) PubMed - Medline database covering the three gastrointestinal diseases - colorectal cancer, coeliac disease, and atrophic gastritis were included for this overview. RESULTS: In Europe, colorectal cancer (CRCA) is the second most frequent malignant disease. Quantitative immunochemical analysis of the stool for haemoglobin provides the best screening test to date, with both sensitivity and specificity approaching 95%. Even though coeliac disease (CD) affects approximately 1% of the general population, it remains largely unrecognised. Recommended methods for screening currently involve the detection of IgA and IgG antibodies against tissue transglutaminase and deamidated gliadin peptide. Evaluations of screening are now discussed for other diseases of the gastrointestinal tract - such as chronic atrophic gastritis (CAG), and inflammatory bowel disease (IBD). Detection of infection by Helicobacter pylori and stomach-specific plasmatic biomarkers, especially pepsinogen I/II ratio, could help with the prevention of gastric carcinomas. The use of faecal calprotectin as a screening test could substantially reduce the number of invasive methods necessary for the diagnostic work-up of patients with IBD. CONCLUSIONS: Screening tests for CRCA and CD have been used worldwide for many years. Screening strategies for gastrointestinal diseases are suggested in the text, based on recent basic science, clinical papers as well as our own

39. Laboratorní diagnostika v gastroenterologii (Article in Czech)
e-Klinická biochemie - LF Plzeň 2013, kapitola 27, str. 312 - 323,on-line: e-Klinická biochemie
Kocna P.
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Abstrakt
Laboratorní diagnostika v gastroenterologii zahrnuje cílené, diagnostické metody, specifické screeninové programy a neinvazivní funkční vyšetřovací programy využívající moderní metody detekce analytů ve stolici nebo pomocí dechových testů. Screeningové (vyhledávací) programy jsou zaměřeny na časnou diagnostiku onemocnění, které jinak zůstávají, v této časné fázi, nerozpoznány. Screeningové programy se provádějí u asymptomatických osob (bez příznaků onemocnění). U definovaných tzv. vysokorizikových skupin se jedná o programy dispenzární. Gastroenterologický screening v současné době zahrnuje dva základní programy - screening celiakie a screening kolorektálních nádorů.Funkční testy tvoří významnou složku klinicko-diagnostického procesu v gastroenterologii. Doplňují výsledky zobrazovacích technik o podstatnou informaci, kterou je funkce orgánu, resp. schopnost reagovat na stimulaci. Funkční test zahrnuje přesně definovanou stimulaci a výsledek je interpretován jako odpověď orgánu na stimulaci s přihlédnutím k bazálním hodnotám ana-lytu před stimulací. U nepřímých funkčních testů je nutno navíc při interpretaci posuzovat funkci dalších orgánů nebo systémů, které se na procesu podílejí.
Recenzent: MUDr. Milan Dastych; Uvedená práce (dílo) podléhá licenci Creative Commons 3.0

38. The Role and Importance of Screening in Gastrointestinal Disease
in: New Trends in Classification, Monitoring and Management of Gastrointestinal Disease-Handbook, ed. Topic E., Watson I., Homšak E., Leniček Krleza J. EFLM Dubrovnik, 2012, pp. pp. 15-22
Kocna P.
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Abstract
This paper is an overview, and summarizes all of the major recent advances in laboratory methods having been used for screening in gastroenterology, which have been published in the last several decades. In Europe, colorectal cancer is the second most frequent malignant disease, and now with quantitative immunochemical FOBT haemoglobin analysing of the stool we currently have the best screening test to date, with an accuracy of nearly 95%. Celiac disease affects ~1% of most populations, but remains largely unrecognized. The recommended methods for screening are currently by the detection of antibodies for IgG tTGA and IgG DGP. Evaluations of screening are now discussed in other gastroenterology diseases - such as chronic atrophic gastritis (CAG), and inflammatory bowel disease (IBD). Detection of infection by Helicobacter pylori and stomach-specific plasma biomarkers, especially pepsinogen I/II ratio, could help with the prevention of gastric carcinomas, as well. The use of faecal calprotectin as a screening test could substantially reduce the number of invasive measurements necessary in the diagnostic work-up of patients with IBD.

37. Screening and Confirmation of Malabsorption
in: New Trends in Classification, Monitoring and Management of Gastrointestinal Disease-Handbook, ed. Topic E., Watson I., Homšak E., Leniček Krleza J. EFLM Dubrovnik, 2012, pp. pp. 39-47
Vaníčkova Z., Kocna P., Zima T.
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Abstract
Malabsorption is a failure of normal absorption of nutrients and micronutrients regarding transport, digestion or absorption of nutrients in the gut. It differs from malnutrition, which is an inadequate food intake. Malabsorption can be generalized, affecting absorption of a range of nutrients, or specific, where the absorption of only a single nutrient is impaired. Principially three mechanisms are involved in pathophysiology of malabsorption: premucosal (luminal), mucosal and postmucosal (postabsorptive). Premucosal mechanisms lead to maldigestion, mucosal and postmucosal mechanism lead to real malabsorption. There are many diverse causes of malabsorption. Treatment depends on understanding mechanisms of malabsorption involved, identifying the cause by means of laboratory and imaging methods based tests and procedures and giving specific therapy where possible and available.

36. E-learning experiences of national societies of clinical chemistry and laboratory medicine
in Advances in Clinical Chemistry and Laboratory Medicine, edited Renz H., Tauber R., DeGruyter 2012, chapter 2.22, p: 99-101
Kocna P.
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Abstract
E-learning and distance education opportunities in clinical chemistry were reviewed in 2005 by the International Federation of Clinical Chemistry (IFCC) Working Group on Distance Education chaired by D. Juretic, under the auspices of the Committee on Education and Curriculum Development chaired by L.C. Allen. A second survey of national societies was carried out in 2010 by the Committee on Education and Curriculum Development chaired by P. Kocna and the Working Group on Internet-Distance Learning chaired by V.T. Thanh. The aim of this study was to summarize changes and trends in the use of e-learning and distance education by member societies of the IFCC over a 5-year period. Surveys of national societies were performed using printed questionnaires in 2005 and by on-line questionnaire forms in 2010. The response rate from member-societies was ~50% (34 in 2005 and 42 in 2010). National society websites increased from 70.6% in 2005 to 90.5% in 2010. National society websites with educational sections increased from 41.2% in 2005 to 57.1% in 2010. Lectures and presentations published on websites are still the most widely used form of educational resource (79.2% in 2010 and 71.4% in 2005). In 2008 the IFCC Committee on Education and Curriculum Development published an on-line Educational Resource Database recommending 254 resources for education in clinical chemistry and was visited more than 1790 times by visitors from 86 countries. This database is regularly used by 12 national societies and 40% of societies recommended distance education on their websites. The internet educational resource most often recommended by national society websites was the NLM PubMed database (mean mark 2.3) followed by Google (mean mark 2.38). A majority of national societies (76.2%) preferred a unifi ed IFCC educational strategy and many responses promoted the concept of IFCC education credits (59.5% of responding national societies).

35. Kvantitativní imunochemické testy detekce krve ve stolici pro screening kolorektálních tumorů (Article in Czech)
FONS Bulletin, 2012, 3: 4-7
Kocna P., Zima T.
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Abstrakt
Riziko karcinomu tlustého střeva v populaci v České republice trvale narůstá a v roce 2010 dosáhlo hodnoty 80 na 100 tisíc obyvatel, v evropském srovnání je jednou z nejvyšších hodnot. Screeningové programy zahrnují především laboratorní metody detekce okultního krvácení - FOBT, detekci genetických markerů a ze zobrazovacích metod se ve screeningu uplatňují sigmoidoskopie, kolonoskopie, irigografie, virtuální počítačová kolografie s použitím výpočetní tomografie (CT) či magnetické rezonance (MR). Stanovení hemoglobinu ve stolici kvantitativní imunochemickou technologií (qi-FOBT) je v současné době nejpřesnější metodou stanovení okultního krvácení, metodou vhodnou pro screening kolorektálních nádorů - KRCA. Kvantitativní stanovení hemoglobinu koreluje s mírou krvácení prekanceróz (adenomů) a nádorů tlustého střeva a kvantitativní analýza umožňuje, oproti kvalitativním - rapid testům - definovat optimální cut-off hodnotu. Informaci, jak nastavit qi-FOBT v české populaci, přinesla studie provedená v předchozích letech ve spolupráci IV. interní kliniky VFN, II. interní kliniky FTN a Ústavu klinické biochemie a laboratorní diagnostiky VFN a 1. LF UK Praha. Problematika screeningu KRCA a stanovení okultního krvácení ve stolici se tak výrazně stává problematikou oboru klinické biochemie a spoluprací s praktickými lékaři.

34. Improvements in colorectal cancer screening programmes – quantitative immunochemical faecal occult blood testing – how to set the cut-off for a particular population
Biomed Pap 2012, 156(2): 143 - 150
Kovářová J.T., Zavoral M., Zima T., Žák A., Kocna P., Kohout P., Granátová J., Vaníčková Z., Vránová J., Suchánek S., Beneš Z., Čelko M.A., Povýšil C.
4th Department of Internal Medicine, General Teaching Hospital, First Faculty of Medicine, Charles University Prague,; Department of Internal Medicine, Central Military Hospital, First Faculty of Medicine, Charles University; Department of Clinical Chemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University; 2nd Department of Internal Medicine, Thomayer Teaching Hospital; Department of Clinical Chemistry, Thomayer Teaching Hospital; Department of Medical Biophysics and Informatics, Third Faculty of Medicine, Charles University; Department of Epidemiology, Third Faculty of Medicine, Charles University; Department of Pathology, First Faculty of Medicine, Charles University, Prague, Czech Republic
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Abstract:
Objective: The aim of the study was to determine the optimum cut-off value of the quantitative immunochemical test (q-FIT) OC-Sensor® for colorectal cancer and advanced adenomatous polyps in a particular population. Methods: 815 patients were referred for colonoscopy and were offered two q-FIT examinations at two different colonoscopy centers. The patients were classified according to the colonoscopic findings. Test sensitivity, specificity, and accuracy were statistically evaluated using one test and two tests at the levels of 50, 75, 100, 125, and 150 ng/mL of faecal hemoglobin in those patients with advanced polyps and colorectal cancer. The optimum cut-off test level for clinically significant neoplasia was determined using one test. Results: The optimum cut-off value of q-FIT OC-Sensor® for the detection of clinically significant neoplasia in our particular population was determined as 75 ng/mL using one test. This value provides an optimum proportion of 73% sensitivity (±95% CI 60.3% – 83.4%) and 90% specificity (±95% CI 86.8% – 92.8%), PPV and NPV were determined as 54.76% and 95.43% respectively. Conclusions: The first step in the implementation of q-FIT test in the screening program in our country is to determine the optimum cut-off level for a population, and to estimate the number of tests performed with respect to the optimum cost effectiveness and economical climate. Using one test, the optimum level of q-FIT OC-Sensor® in the Czech Republic was determined as 75 ng/mL. This study could serve as a model for further studies in other countries, where screening does not yet exist.

33. Monitoring of Daily Gliadin Intake in Patients on Gluten-free Diets
Prague Medical Report 2011, 112 (1): 5 – 17
Gabrovská D., Kocna P., Rysová J., Borovská D.,Tlaskalová-Hogenová H.
Food Research Institute Prague; Charles University in Prague, First Faculty of Medicine and General University Hospital, Institute of Clinical Biochemistry and Laboratory Diagnostics; Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, and Charles University in Prague, First Faculty of Medicine and General University Hospital; Czech Republic
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Abstract:
The aim of the study was to show patients suffering from the coeliac disease, their real gliadin daily intake, offer them very useful information concerning their diet and to find random possible mistakes. The monitoring was carried out within the context of their routine everyday diet regimen. The daily intake of gliadin in the diet was quantified on the basis of gliadin determination in their current daily food. The gluten-free diet was followed for 30 days. The patients were taking regular daily meals, drinks, and sometimes medicines or food supplements. The patients were provided with instructions, survey forms, digital scales, polyethylen bottles and sacks. The patients took out the stipulated amount, which served as a sample of each of their daily meals. The samples included both homemade meals as well as commercial products. The content of gliadin in daily meal was determined by the sandwich ELISA method. The daily gliadin intake was calculated on the base of the reported amount of meals ingested. 1,900 food samples were analyzed within the framework of this study. Several contaminated commercial foods were found; nevertheless this fact did not influence the otherwise satisfactory overall picture of the daily gliadin intake by the patients followed. The results in 14 patients revealed a satisfactory adherence to the gluten-free diet. It was proved that conscientiousness and awareness on the part of coeliac patients, or those taking care of them, is of paramount importance in determining the choice of foods comprising a gluten-free diet.

32. Doporučené diagnostické a terapeutické postupy pro všeobecné praktické lékaře - Laboratorní metody – Část 1. Biochemické metody (Article in Czech)
Klin. biochemie a metabolismus, 2008, 17, 1: 56-68
Zima T., Racek J., Dastych M., Kreidlová M., Springer D., Kocna P., Dražďáková M., Štěpán J., Marečková H. Seifert B., Laňková J., Mucha C., Vojtíšková J.
Doporučení České společnosti klinické biochemie ČLS JEP a Společnosti všeobecného lékařství ČLS JEP
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Úvod
Praktický lékař může v různých fázích diagnostického a diferenciálnědiagnostického procesu využívat širokou paletu laboratorních metod. Pod tlakem regulace nákladů je motivován k jejich efektivnímu využívání, racionální indikaci a správné interpretaci. Na pomoc praktickým lékařům v oblasti racionálního využívání laboratorních metod byl vypracován tento doporučený postup. Předkládáme jeho první část věnovanou biochemickým a vybraným imunologickým metodám. Zahrnuje obecné zásady laboratorních vyšetření a jejich 3 fází: preanalytické, analytické a postanalytické. Ve své speciální části popisuje portfolio současných biochemických a základních imunologických metod, pravidla jejich indikace a správné interpretace. Popisuje metody použitelné přímo v ordinaci (Point Of Care Testing, POCT) a definuje pravidla zajištění jejich kvality. Přináší algoritmy pro využití v nejvýznamnějších klinických situacích. Záměrně neobsahuje algoritmy biochemické diagnostiky a monitorování zpracované podrobně v jiných doporučených postupech pro praktické lékaře.

31. Testy exokrinní funkce pankreatu - elastáza 1 ve stolici a dechový test s 13C-MTG. (Article in Czech)
HPB Bulletin 2006, 14; 3: 97 - 99
Kocna, P.; Vaníčková, Z.; Krechler, T.; Lukáš, M.; Doseděl, J.; Kohout, P.
Ústav klinické biochemie a laboratorní diagnostiky 1.LF UK a VFN; 4. interní klinika 1.LF UK a VFN; Interní oddělení Nemocnice milosrdných sester sv. Karla Boromejského, 2.interní klinika Fakultní Thomayerova nemocnice, Praha
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Abstract
Problematika funkční diagnostiky exokrinního pankreatu zůstává stále diskutovaným tématem - jak z pohledu laboratorních metodik, tak z pohledu klinických aplikací. Klinický význam laboratorních testů pankreatické funkce zahrnuje široké spektrum od primární diagnostiky především chronické pankreatitidy a cystické fibrózy, stanovení stupně postižení pankreatu, dlouhodobého sledování nemocných s chronickou pankreatitidou, včetně monitorování suplementační terapie pankreatickými enzymy, až po diferenciální diagnostiku malabsorpčního syndromu.
Naše čtyřleté zkušenosti potvrzují význam obou dvou testů exokrinní funkce pankreatu. Stanovení elastázy-1 ve stolici je základním screeningovým, jednoduchým testem, který lze doporučit pro dlouhodobé sledování nemocných a progresi onemocnění. Test lépe koreluje s morfologickým aspektem postižení pankreatu, než dechový test 13C-MTG. 13C-MTG dechový test je vhodný pro kvantitativní posouzení funkční insuficience, dobře koreluje se steatorhoeou, umožňuje posouzení dynamiky trávicího procesu, jeho kinetiku a optimalizaci substituční terapie pankreatickými enzymy. Diagnostický význam jednotlivých ukazatelů 13C-MTG dechového testu - cPDR, DOBmax, DOBtime, charakter křivky - je předmětem další studie. Závěry hodnocení obou testů v souboru 184 susp. CHP prokazují rozdílnost výsledků dvou testů exokrinní funkce pankreatu ve 40,1%, a to ve skupině CHP-B a CHP-D. Funkční testy FELA a 13C-MTG proto mají odlišnou indikaci v diagnostickém algoritmu CHP.

30. Breath Tests - The Modern, Non-invasive Diagnostics (Article in Czech)
Interní Med. 2006; 7 - 8: 336 - 341
Kocna P.
Ústav klinické biochemie a laboratorní diagnostiky 1. LF UK, Praha
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Abstract
Breath tests present moderne, non-invasive diagnostic tool in gastroenterology and hepatology. Both the hydrogen breath tests (H2) and tests with stable isotope of carbon (13C) are clinically helpful in the wide range of diagnostic indications. In this review there are discussed prinicples of hydrogen and 13C-breath tests, methods and test procedures, analytical instruments and selected clinical applications. The range of diseases that can be identified include Helicobacter pylori infection, lactose and fructose intolerance, bacterial overgrowth, abnormal small bowel transit, gastric emptying, pancreatic insufficiency, liver dysfunction. Five years experiences with 13C-breath tests used for Helicobacter pylori (13C-urea), exocrine pancreatic function (13C-mixed triglycerides) and small bowell bacterial ove rgrowth (13C-xylose) are presented. These tests were analysed by infrared analysers Isomax-4000 and HeliFAN plus in the Laboratory of Gastroenterology, Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Medical Faculty of Charles University in Prague.

29. Hyperamylasemia, Laboratory and Clinical Aspects (Article in Czech)
Cas. Lek. Ces., 145 (6), 2006, p. 449 - 452.
Kocna P., Zima T.
Ústav klinické biochemie a laboratorní diagnostiky 1. LF UK, Praha
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Biochemical estimation of the total a-amylase represents in the Czech Republic almost two millions of assessments per year. Estimation of the total a-amylase for the diagnostics of pancreatic diseases has a very low specificity and it is therefore recommended to analyze specific pancreatic enzymes – pancreatic isoenzyme a-amylase and pancreatic lipase. Paper summarizes laboratory and clinical aspects aimed namely on hyperamylasamia.

28. Faecal Elastase I. Assessment – Its Use in Diagnosis of Chronic Pancreatitis (Article in Czech)
Cas. Lek. Ces., 145 (6), 2006, p. 480 - 483.
Krechler T., Kocna P., Vaníčková Z., Švestka T., Lukáš M., Doseděl J., Kohout P., Žák A.
IV. interní klinika 1. LF UK a VFN, Ústav klinické biochemie a laboratorní diagnostiky 1. LF UK, Nemocnice U Milosrdných sester sv. Karla Boromejského, Fakultní Thomayerova nemocnice 1. LF UK, Praha
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Background. The diagnosis of chronic pancreatitis is based on the imaging methods. These imaging methods show the main morphological changes in the pancreatic ducts and its parenchyma, but they do not define the function of the pancreas. The aim of our study was Faecal Elastase I. determination in patients with chronic pancreatitis. The test is a simple, non-invasive method of the investigation of the pancreatic exocrine insufficiency. The Faecal Elastase I occurring in the stool was correlated with the level of the damage of pancreatic tissue together with the control group of the patients with different diagnoses. Methods and Results. Faecal Elastase I (mean values in ug/g of stool) detection is a simple, non-invasive method which correlates well with the damage of pancreatic tissue, stemming from chronic pancreatitis. This test is routinely used especially in the diagnosis of chronic pancreatitis. The classification of chronic pancreatitis currently depends on the morphological changes of the pancreatic duct system (the patho-morphological changes). We are currently missing the classification describing simultaneously the morphological changes of the gland and the function of the pancreas. In our studies we have used a newly proposed classification system, which was put together in Bern, 2000 (1). This new system encompasses morphological and functional changes. Faecal Elastase I was determined by a microplate ELISA method using monoclonal antibody to human pancreatic protein. The Faecal Elastase I. was tested in the stool of the 196 patients with chronic pancreatitis stemming from alcoholism. The occurrence of Faecal Elastase I. was classified according to the levels assigned by the classification system. The control group used in this study included 144 patients with different diagnoses. The results demonstrate a very good correlation of Faecal Elastase I. with the grading of the newly proposed classification system of chronic pancreatitis. Patients with the highest levels of the damage of the pancreas had a significantly lower occurrence of Faecal Elastase I. in comparison with the non-pancreatic control group and in patients with chronic pancreatitis who had no clinical complications or damage of endocrine and exocrine functions of the pancreas.

27. Laboratorní diagnostika malabsorpčního syndromu.
in Gastroenterologie 2006 - Collectio novissima, ed. Bureš J., Triton-Praha 2006, 181 - 198
Kocna P.
Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Prague, Czech Republic
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Abstract
Maldigesce, malabsorpce a malasimilace popisují poruchy (vrozené nebo získané) jedné nebo více základních funkcí trávicího traktu, trávení, vstřebávání, sekrece amotility. Malabsorpční syndrom může být způsoben řadou různých onemocnění a je provázen širokým spektrem klinických projevů, symptomů i biochemických nálezů. Laboratorní testy zaměřené na malabsorpční syndrom jsou orientovány na základní diagnostiku nozologických jednotek primárního i sekundárního procesu, na posouzení funkčního aspektu trávení a absorpce cukrů, tuků a bílkovin, a konečně i na posouzení a monitorování terapie.

26. Isotope Selective Nondispersive Infrared Spectrometry Can Compete with Isotope Ratio Mass Spectrometry in Cumulative 13CO2 Breath Tests: Assessment of Accuracy
Klin. Biochem. Metab., 13 (34), 2005, No. 2, p. 92–97.
Chleboun J., Kocna P.
Mathematical Institute, Academy of Sciences and Faculty of Mechanical Engineering, Czech Technical University; Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Prague, Czech Republic
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To measure a patient’s metabolic response to an administered 13C-enriched substrate, isotope selective nondispersive infrared spectrometry is used. Isotope abundance levels are relative, i. e., reported as differences between a tested sample and a reference sample. The reference 13C abundance is not known exactly. This uncertainty, uncertainty in CO2 production, and the inaccuracy of the measuring instrument contribute to the uncertainty in the results of breath tests. In this study, the particular impacts of uncertainty are estimated and expressed in a mathematical way by an uncomplicated formula illustrated by an example dealing with real-life data. It is shown that the uncertainty in the reference 13C abundance does not have severe consequences, so that the method can compete with the spectrometry methods that are able to deliver an absolute value of the 13C abundance. The inaccuracy of the measuring instrument is also manageable, though its influence is greater than in the previous case. The analysis reveals that the uncertainty in CO2 production deserves great attention because it is difficult to estimate and its influence is rather strong. The problem of determination of a proper cut-off level is outlined.

25. Effect of eradicating H. pylori on the appearance of esophageal reflux disease: Randomized double blind study (Article in Czech)
Prakt. Lék., 2005, 85, No. 3, p. 133–138.
Martínek J., Špičák J., Beneš M., Zavoral M., Lukáš M., Mandys V., Kocna P., Kykal J.
Klinika hepatogastroenterologie, IKEM, Praha, II. interní oddělení ÚVN a Subkatedra gastroenterologie IPVZ, Praha, Gastrocentrum, IV. interní klinika VFN, Praha, Ústav patologie III. LF UK a FN KV, Praha, Ústav klinické biochemie VFN a I. LF UK, Praha, Interní oddělení Nemocnice Říčany
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Background and aims: Eradication of H. pylori may provoke gastroesophageal reflux disease (GERD) but many studies don’t support this hypothesis. The aim of our randomized, double blind study was to find out whether the eradication of the infection is a risk factor for the development of GERD. Methods: One hundred and twenty-six patients with functional dyspepsia participated at screening and 40 patients were randomized. All were infected by H. pylori. Twenty patients received the eradication treatment (omeprazol, amoxicillin and clarithromycin for 7 days) and 20 patients received placebo instead of antibiotics. The follow-up was 12 months. The main endpoint was the development of GERD defined as reflux esophagitis and/or typical reflux symptoms. Results: The follow-up was completed in 38 patients (19 in each group). De novo GERD was observed in 1 patient (in the placebo group). Dyspeptic symptoms significantly improved in 79% of patients in the eradication group and in 58% patients in the placebo group (p<0.05). Successful eradication was achieved in 89.5% of patients. We confirmed the reliability of stool antigen test for the detection of H. pylori. The RUT was false negative in 18% of patients. Conclusions: 1. Keeping in mind the small number of randomized patients, we did not observe a higher risk of development of GERD following eradication of H. pylori infection. 2. The efficacy of eradication regimen with omeprazole + 2 antibiotics was very high. 3. Both the eradication and short – term course of acid suppressive therapy are effective for the treatment of functional dyspepsia. 4. RUT alone is not appropriate to make a diagnosis of H. pylori infection.

24. On cumulative 13C breath tests: An effort to improve the accuracy of test results revealed a substantial uncertainty in input data.
in Programs and Algorithms of Numerical Mathematics, ed.: Chleboun J., Přikryl P. and Segeth K. Institute of Mathematics AS CR, Prague, 2004. pp. 82–87
Chleboun J., Kocna P
Institute of Mathematics AS CR, Prague; Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Prague.
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A cumulative 13C breath test can be used to detect a pancreatic disorder, for example. The test is burdened by uncertain input data. Among them, the estimation of CO2 production rate is a key issue. An established estimate based on the body surface area could be replaced by an estimate using the basal metabolic rate. Although the latter estimate might be considered more appropriate than the former, the differences between them are large in some sex and age groups. Such disagreements pose a danger to the reliability of the cumulative breath tests and ask for further research.

23. Distribution of molecular markers in sporadic colorectal cancer, adjacent and distant mucosa
Folia Gastroenterol Hepatol 2004; 2 (2): 62 - 71
Frič P, Sovová V, Roth Z, Šloncová E, Kocna P, Jirásek A, Čermák J.
Second Department of Medicine, Central Military Hospital, Postgraduate Institute of Medicine and Department of Gastroenterology and Hepatology, Institute of Clinical Biochemistry and Laboratory Diagnostics, Institute of Pathology, Department of Surgery, First Faculty of Medicine, Charles University; Institute of Molecular Genetics, Czech Academy of Sciences; Department of Biostatistics and Informatics, State Health Institute; Praha, Czech Republic
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Purpose: Molecular markers of carcinomatous transformation of the colonic mucosa have been predominantly determined in the tumour tissue. Their behaviour in the neighbouring mucosa has been only seldom followed and data on comparison of their distribution in individual locations are rare. Methods: In 53 consecutive subjects with sporadic colorectal cancer the frequency and intensity of expression of p53, c-Src protein kinase activity, c-erbB2, EGF-r, PCNA and arginase activity were followed in the tumour tissue, the adjacent (less than 2 cm) and distant (more than 5 cm from the tumour margin) mucosa. Results: Frequency and intensity of the expression of molecular markers displayed two different distribution patterns. A decreasing pattern (the highest value in the tumour and the lowest in the distant mucosa) was observed in p53, arginase and to some extent also in c-Src protein kinase activity. An increasing pattern was found in c-erbB2 and EGF-r. Logistic regression analysis of frequency expression gave the following data: 1. p53, c-erbB2 and arginase displayed significant differences among tumour and mucosal locations. 2. In the case of arginase the frequency of expression between both mucosal locations was also significantly different. 3. Individual subjects differed in their ability to express EGF-r and arginase. Intensity of expression (paired t-test: 2-tailed) was significantly different among tumour and adjacent as well as distant mucosa in p53, c-erbB2 and arginase. In c-Src protein kinase activity the intensity of expression differed significantly in both mucosal locations. In relation to Dukes staging (stages AB versus CD) the intensity of expression was significantly different (non-paired t-test: 2-tailed) only in PCNA (distant mucosa) and arginase (adjacent mucosa). Conclusions: Different distribution patterns of frequency and intensity of the expression of individual molecular markers in colorectal cancer and neighbouring mucosa reflect the complex character of carcinomatous transformation of the epithelial cells. The results may be exploited in prospective prognostic studies with the use of molecular markers.

22. Test exokrinní funkce pankreatu - 13C-MTG dechový test. (Article in Czech)
HPB Bulletin 2004, 12; 3: 76 - 77
Kocna, P.; Vaníčková, Z.; Krechler, T.; Lukáš, M.; Doseděl, J.
Ústav klinické biochemie a laboratorní diagnostiky 1.LF UK a VFN; 4. interní klinika 1.LF UK a VFN; Interní oddělení Nemocnice milosrdných sester sv. Karla Boromejského, Praha
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Abstract
13C-MTG substrát je přednostně hydrolyzován pankreatickou lipázou, která odštěpuje stearyl v poloze 1 a 3 a vzniklý 13C-octanoát je pak metabolizován jaterní b-oxidací. Stanovení nerozštěpeného substrátu ve stolici, resp. 13C markeru, metodou GC-MS prokázalo zbytkovou frakci 13C-substrátu ve stolici v rozmezí 1 - 5 %. Kumulativní exkrece cPDR významně koreluje s výdejem pankreatické lipázy (r = 0.89) a rovněž s kvantitativním ukazatelem steatorhey. Naše dvouleté zkušenosti potvrzují význam 13C-MTG dechového testu pro hodnocení exokrinní funkce pankreatu. Ve srovnání s kvantitativním ukazatelem koncentrace pankreatické elastázy ve stolici (FELA), která velmi dobře koreluje s gradingem chronické pankreatitidy, umožňuje 13C-MTG dechový test posouzení dynamiky trávicího procesu, jeho kinetiku a optimalizaci substituční terapie pankreatickými enzymy. Diagnostický význam jednotlivých ukazatelů 13C-MTG dechového testu - cPDR, DOBmax, DOBtime, charakter křivky - je předmětem další studie, která vyžaduje především rozšíření jednotlivých skupin CHP a jejich detailní analýzu. Závěry hodnocení 108 provedených testů prokazují rozdílnost výsledků dvou testů exokrinní funkce pankreatu ve 24 %, ve skupině CHP-B je frekvence nejvyšší - 41,2b %. Funkční testy FELA a 13C-MTG proto mají odlišnou indikaci v diagnostickém algoritmu CHP.

21. Přínos stanovení elastázy I ve stolici u chronické pankreatitidy. (Article in Czech)
HPB Bulletin 2004, 12; 3: 84-85
Krechler, T.; Kocna, P.; Vaníčková, Z.; Švestka, T.; Doseděl, J.; Kohout, P.; Bortlík, M.; Lukáš, M.
Ústav klinické biochemie a laboratorní diagnostiky 1.LF UK a VFN; 4. interní klinika 1.LF UK a VFN; Interní oddělení Nemocnice milosrdných sester sv. Karla Boromejského, Praha
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Chronická pankreatitida (CHP) je onemocnění, jehož diagnostika v poslední době zaznamenala prudký rozvoj, a to především díky rozvoji moderních zobrazovacích metod. V první fázi diagnostického procesu pacient s chronickou pankreatitidou (CHP) přichází k lékaři s nejrůznější symptomatologií. M ezi nejčastější příznaky patří : chronická bolest břicha (5 %), recidivující bolesti břicha (10 %), recidivující ataky akutních pankreatitid (50 %), steatorhea (75 %), malnutrice (10 %) a diabetes mellitus (2 %). Při stanovení diagnózy CHP bychom měli k pacientovi přistupovat z několika aspektů: 1) Z pohledu morfologického potvrzení stanovení diagnózy CHP. Tento krok je v současné době při stanovení diagnózy CHP nejdůležitější a zahrnuje celou škálu zobrazovacích metod: ERCP (endoskopická retrográdní cholangiopankreatografie), která je stále považována za zlatý standard v diagnostice CHP a M RCP ( magnetická rezonance). Tyto metody hodnotí změny především ve vývodném systému žlázy. Změny v parenchymu žlázy hodnotíme při vyšetření abdominální ultrasonografie, endoskopické ultrasonografie, M RCP a výpočetní tomografie (CT). Zobrazovací techniky však prakticky nic nevypovídají o funkci slinivky břišní. 2) Z pohledu funkčního postižení žlázy se využívá celá škála funkčních testů. Pro naše klinické pracovníky jsou prakticky k disposici kromě rutinních stanovení sérových hladin pankreatických enzymů 2 testy: stanovení elastázy I ve stolici a dechové testy.

20. IgA and IgG Antigliadin, IgA Anti-tissue Transglutaminase and Antiendomysial Antibodies in Patients with Autoimmune Thyroid Diseases and Their Relationship to Thyroidal Replacement Therapy
Physiol. Res.; 2003, 52: 79-88
J.Jiskra, Z. Límanová, Z. Vaníčková, P. Kocna
Third Medical Department and 1Department of Clinical Biochemistry, First Medical Faculty, Charles University, Prague, Czech Republic
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Abstract
Celiac disease is a chronic illness of the small bowel caused by gliadin intolerance in genetically predisposed subjects. The aim of this study was to investigate serum levels of IgA and IgG antigliadin antibodies, IgA antiendomysial antibodies, and IgA anti-tissue transglutaminase antibodies in 169 patients with autoimmune thyroid diseases, i.e. chronic thyroiditis and Graves´ disease. Antiendomysial antibodies were positive in 2 out of 169 persons (1.18 %), IgA antigliadin antibodies in 15.98 %, IgG antigliadin antibodies in 51.48 %, and IgA anti-tissue transglutaminase in 14.79 %. The prevalence of positivity was higher compared to the 1312 control blood donors described in our previous study (Vančíková et al. 2002) (p<0.05). Patients with chronic thyroiditis treated with a high replacement dosage of levothyroxin (125-200 µg daily) had higher serum levels of IgA antigliadin antibodies in comparison with patients treated with a lower dosage (50-100 µg daily) (medians: 13.00 vs. 19.69, p=0.033). We found a negative correlation of IgA anti-tissue transglutaminase antibodies and total calcium serum levels (r = –0.480, p=0.0236, n=22). We can conclude that in persons with autoimmune thyropathy there is a high prevalence of positive antigliadin, anti-tissue transglutaminase and antiendomysial antibodies. Latent celiac disease may lead to impaired resorption of therapeutically administered levothyroxine, calcium, or other substances.

19. Tissue Transglutaminase-Serology Markers for Coeliac Disease
Clin Chem Lab Med 2002; 40 (5): 485 – 492
Petr Kocna, Zdislava Vaníčková, Jindřiška Perušičová and Miloš Dvořák
Institute of Clinical Biochemistry, 3rd Clinic of Internal Medicine, 4th Clinic of Internal Medicine, 1st Faculty of Medicine & General Faculty Hospital, Charles University, Prague, Czech Republic
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Abstract
Serology markers of coeliac disease (CD) – antigliadin IgA/IgG antibodies (AGA/AGG) with purified a-gliadin, antiendomysium IgA antibodies (EmA) and anti-tissue transglutaminase (atTG) IgA/IgG antibodies – determined in 1451 serum samples, were analysed with respect to different screening algorithms. Determination of atTG using five ELISA methods was compared taking into account the impact of human recombinant antigen and IgG class of atTG. A subgroup of 119 patients undergoing small intestinal biopsy was used to calculate sensitivity and specificity of CD markers. The highest sensitivity (94%) was obtained for AGG, and the highest specificity (93.5%) was obtained for EmA. All coeliac disease patients were detected using the combination of all four CD markers, resulting in 100% sensitivity. CD and type 1 diabetes mellitus autoantigens were determined in 139 diabetic patients. The atTG IgA mean value (16.7 IU/ml) was higher in the antiglutamate dehydrogenase antibody (GAD)-positive subgroup, where at least one CD marker was positive in 83.6% subjects. In the GAD-negative subgroup atTG IgA was 8.73 IU/ml and at least one CD marker was positive in 57.4% subjects. atTG in IgA and IgG classes could be recommended as valuable serological markers of CD in the differential diagnosis of malabsorption as well as in various screening algorithms. ELISA determination of atTG with human antigen could increase the specificity, especially in patients with other autoimmune diseases.

18. The Serologic Screening for Celiac Disease in the General Population (Blood Donors) and in Some High-Risk Groups of Adults (Patients with Autoimmune Diseases, Osteoporosis and Infertility) in the Czech Republic
Folia Microbiol. 2002, 47 (6), 753 - 758
Z. Vančíková A, V. Chlumecký B, D.Sokol, D.Horáková, E.Hamšíková, T.Fučíková, I. Janatková , Z. Ulčová-Gallová, J. Štěpán, Z. Límanová , M. Dvořák , P. Kocna, D. Sanchéz , L. Tučková, H. Tlaskalová-Hogenová
Department of Pediatrics, University Hospital Motol, Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences, lnstitute of Hematology and Blood Transfilsion, lnstitute of Clinical lmmunology, 3rd Departmellt of Internal Medicine 1st Medical School, Charles University, Department of Gynecology and Obstetrics, Medical School, Charles University, and Faculty Hospital Pilsen, Czech Republic
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Abstract
The prevalence of celiac disease (CD) was determined in healthy blood donors and in highrisk groups of adults (a total of 1835 adults - randomly selected 1312 healthy blood donors, 102 patients with primary osteoporosis, 58 patients with autoimmune diseases and 365 infertile women). It was calculated on the basis of a two-step serologic screening method - in the first step IgA and lgG antigliadin antibodies (AGA) and lgA anti-7-glutamyltransferase ('transglutaminase') antibodies.(ATG) were estimated, in the second step sera positive for lgA AGA and/or lgA ATG were exainined for antiendomysial IgA (AEA) antibodies. Immunoenzymic assay (ELISA) was used for determining of AGA and ATG antibodies; immunoflurescence method, performed on human umbilical cord tissue, was used for assaying of AEA antibodies. Total serum IgA level in only lgG AGA positive subjects was measured by routine turbidimetric method. 0.45 % of healthy blood donors, 0.98 % of osteoporotic patients, 2.7 % of patients suffering from autoimmune disease and 1.13 % of women with infertility considered as immunologically mediated were found to be positive in both steps of serologic screening (AGA and/or ATG and antiendomysium positive). The presumed high prevalence of seropositivity for CD in apparently healthy Czech adult population was confirmed. In the highrisk groups, the prevalence of seropositivity for CD was approximately 2-4 times higher than in healthy blood donors. The real prevalence of CD in the tested groups, however, can be estimated after performing small intestinal biopsy in the seropositive patients.

17. Arginase Activity Determination - A Marker of Large Bowel Mucosa Proliferation.
Eur J Clin Chem Clin Biochem 1996; 34: 619 - 623
Kocna P., Fric P., Zavoral M. and Pelech T.
Laboratory of Gastroenterology and Department of Internal Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic.
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Abstract
Arginase activity of the intestinal mucosa was tested as a proliferative marker in the adenoma-carcinoma sequence. The enzyme activity was determined by an end-point colorimetric method with L-arginine as substrate. Arginase activity was evaluated in 430 biopsy samples of large bowel mucosa, polyps and cancer tissue. The activities (U/g protein, mean ± SE; n) were: normal mucosa 83.2 ± 7.3; 25, adenomas 199.4 ± 19.1; 40, carcinomas 1269.7 ± 174.9; 40, inflammatory bowel disease 1210.7 ± 247.1; 34. The arginase activity differs significantly in the adenoma-carcinoma sequence according to the Duncan's test (p < 0.05).

16. Interaction of natural and synthetic peptides derived from gliadin with different cell lines.
Advances in Mucosal Immunology, 1995, 37, 2, 1371-1373
Farré, M.A.; Tlaskalová, H.; Huan, N.H.; Kocna, P.; Tučková, L.; Krchňák, V.; Větvička, V.; Baudišová, M.; Pospíšil, M.; Frič, P.
Department of Immunology and Gnotobiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
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Abstract
The mechanism by which cereal protein gluten affecks the small intestinal mucosain coeliac disease is still unknown. Weiser and Douglas proposed that gluten contains a toxic lectin which binds to altered membrane glycoproteins of coeliac enterocytes. This hypothesis was supported by Kottgen who confirmed that gluten has lectin-like properties and that gliadin peptides can bind to glycoproteins present on immature crypt cells of rat intestine. Auricchio reported that peptic-tryptic-digest of bread wheat gliadin and A-gliadin agglutinated the undifferentiated K562 human cell line. In our work different cell lines of lymphoblastoid and epithelial origin were compared in order to study the interaction between gliadin peptides and cell membranes.

15. Binding of gliadin to lymphoblastoid, myeloid and epithelial cell lines.
Folia Microbiol (Praha). 1995; 40 (4): 431 - 435
Farre Castany MA, Kocna P, Tlaskalova-Hogenova H.
Department of Immunology and Gnotobiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
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Abstract
The aim of our work was to investigate the in vitro reactivity of gliadin peptides of natural and synthetic origin with various cell lines. We have found that all tested cell lines of human, mouse and rat origin were agglutinated by enzymically digested gliadin (peptic-tryptic- and peptic-tryptic pancreatic digest of alpha-gliadin) in a concentration dependent manner. In order to test the specificity of binding, inhibition studies were performed using a panel of sugars as well as natural and synthetic peptides derived from gliadin. We have found that among twelve tested sugars only fetuin and phosphomannan were able to inhibit the agglutination of K562 cells with peptic-tryptic- but not with peptic-tryptic pancreatic digest of alpha-gliadin. The lack of inhibition by gliadin peptides and most of the saccharides suggests that agglutinating activity of gliadin is the result of a nonspecific binding of gliadin to the cell membrane.

14. Characterization of human, mouse and rabbit anti-gliadin antibodies by ELISA and western blotting.
Folia Microbiol (Praha). 1995; 40 (6): 659 - 664
Vancikova Z, Kocna P, Tuckova L, Fric P, Dvorak M, Stoyanov S, Tlaskalova-Hogenova H.
Department of Clinical Immunology, Faculty Hospital Bulovka, Prague, Czech Republic.
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Abstract
Monoclonal, hyperimmune rabbit and human serum anti-gliadin antibodies were analyzed by ELISA and immunoblotting techniques. In Western blotting the difference in reactivity between monoclonal and human antibodies was quantitative rather than qualitative. Rabbit antisera differed in reactivity according to the protein used for immunization. The rabbits immunized by the peptic-tryptic pancreatic digest of gliadin reacted similarly to the patients. In ELISA, significantly higher reactivity with crude, A-, glyc-gli, alpha-, beta- and omega-gliadins was found in the patients' sera than in controls.

13. Molecular mimicry as a possible cause of autoimmune reactions in celiac disease? Antibodies to gliadin cross-react with epitopes on enterocytes.
Clin Immunol Immunopathol. 1995 Feb; 74 (2): 170 - 176
Tucková L, Tlaskalová-Hogenová H, Farré MA, Karská K, Rossmann P, Kolínská J, Kocna P.
Department of Immunology and Gnotobiology, First Faculty of Medicine, Prague, Czech Republic.
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Abstract
Structural similarities between external antigen and self components are believed to be one of the possible causes of autoimmunity. This study describes the presence of similar structures shared by gliadin and enterocyte surface molecules recognized by antigliadin mAbs. The reactivity of mAbs to gliadin was followed by ELISA using fixed enterocytes, their brush-border membranes, or purified enterocyte antigen. The specificity of reaction was confirmed by ELISA inhibition studies and by immunohistochemical staining of rat tissue sections using biotin-avidin-peroxidase technique. Immunoprecipitation analysis of 125I-labeled intestinal epithelial cells using antigliadin mAb revealed the presence of two main cross-reactive molecules of 28 and 62 kDa. The 62-kDa and an associated 66-kDa protein were isolated by affinity chromatography. Immunoblotting analysis showed that a 28-kDa protein detected by immunoprecipitation also reacted with IgA of celiac disease patient sera.

12. Effective peritoneal therapy of acute pancreatitis in the rat with glutaryl-trialanin-ethylamide: a novel inhibitor of pancreatic elastase.
Gut. 1992 May; 33 (5): 701 - 706
Fric P, Slabý J, Kasafírek E, Kocna P, Marek J.
Department of Medicine, Charles University, Prague, Czechoslovakia.
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Abstract
The six hour peritoneal lavage with glutaryl-trialanin-ethylamide, a low molecular competitive inhibitor of pancreatic elastase (IC50-8 mumol/l), effectively suppresses the evolution of taurocholate induced acute pancreatitis in the rat. The lavage alone is followed by a marked decrease of fat necrosis and amylase and lipase activity in serum. The area of pancreatic haemorrhage was significantly reduced only after the lavage solution was supplemented with Glt-Ala3-NHEt. The effect was not enhanced by a bolus injection of the inhibitor before starting the lavage. The combination of Glt-Ala3-NHEt with aprotinin or nafamstate mesilate produced only marginal greater benefit. The effect of Glt-Ala3-NHEt on pancreatic haemorrhage is time and dose related even with delayed onset of the lavage. Animals treated with peritoneal lavage without Get-Ala3-NHEt lived longer than controls (p less than 0.05), but by 60 hours the survival rate of both groups was almost the same (76 v 74%). All animals lavaged with Glt-Ala3-NHEt survived 120 hours and the difference in the survival rate between this and both remaining groups was significant (100% v 76% v 74% - p less than 0.05). The results were considered favourable and preliminary clinical trials of Glt-Ala3-NHEt in subjects with acute pancreatitis justified.

11. Short-term cultivation of duodenal biopsies in coeliacs. Effect of the gluten challenge and gliadin peptides.
Intern.Coeliac Symposium, Dublin 1992, 37
Kocna, P.; Frič, P.; Tlaskalová, H.; Krchňák, V.; Kasafírek, E.
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Summary:
The goal of this study was to confirm a high biological effect of peptide 8-19 on human intestine, to test a relationship between mucosal enzymes and immunological response in the culture medium and an effect of the gluten challenge on coeliac patients in remission. The -gliadin purified on Sulfopropyl-Sephadex, proteolyticaly degraded fragments (PTP-digest) and peptide 8-19 has been added to the culture medium. The short-term (24 hr) cultivation of duodenal biopsies has been carried out by the technique reported by Falchuk, and the medium composition was prepared according to Jos.

10. Relationship between gliadin peptide structure and their effect on the fetal chick duodenum.
Z Lebensm Unters Forsch. 1991 Feb; 192 (2): 116 - 119
Kocna P, Mothes T, Krchnak V, Fric P.
Laboratory of Gastroenterology, Faculty of General Medicine, Charles University, Prague, Czechoslovakia.
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Abstract
The tendency to form a beta-turn in alpha-gliadin was estimated using the B-cell determinant prediction program based on the Chou and Fasman probability of beta-turn formation. Six sequences possessing a high probability of beta-turn formation were found. A statistically high agreement was found between these six sequences and three areas in alpha-gliadin with the occurrence of Pro-Ser-Gln-Gln sequence which has recently been considered responsible for toxicity in coeliac disease. By means of solid-phase synthesis seven peptides were obtained covering the above-mentioned regions. Their toxicity was tested using the fetal chick duodenum. The results support the suggestion that peptides containing the sequences Pro-Ser-Gln-Gln and Gln-Gln-Gln-Pro may be involved in the pathogenesis of coeliac disease.

9. Isolation and properties of lectin-like glycopeptide from wheat gluten.
Lectins-Biology; Biochem.Clinical Biochemistry, 1990, 7, 45-48
Kocna, P.; Frič, P.; Kasafírek, E.; Slabý, J.; Tlaskalová, H.; Vančíková, Z.; Hekkens, W.Th.J.M.
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Summary:
We prepared thc glyc-gli glycopeptide according to Douglas by trichloracetic acid precipitalion of the ethanol-acetic extract of the crude gluten in the amount of 800 mg. By the SDS-PAGE in 10% polyacrylamide gel the main fraction with molecular mass 15 400 and some additional high-molecular-weight fractions were found. A considerable heterogenity of pI across the whole range of pH 3.5-9.5 was demonstrated by isoelectric focusing. Compared with other gliadin preparations, only in glyc-gli were these protein bands in the acidic area (pH 4-6). The analysis of glyc-gli we performed by gel-permeation chromatography (GPC) using the automated HPLC Perkin-Elmer on the TSKG2000-SW column eluted with 0.1% TFA at a flow of 0.4 ml/min. The molecular mass estimated by this method was found to be 10 000 for the main peak.

8. Isolation and analysis of peptidic fragments of alpha-gliadin using reversed-phase high-performance liquid chromatography.
J Chromatogr. 1988 Dec 30; 434 (2): 429 - 438
Kocna P, Fric P, Kocova-Holakova M, Slaby J, Kasafirek E, Hekkens WT.
Laboratory of Gastroenterology, Charles University, Faculty of General Medicine, Prague, Czechoslovakia.
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Abstract
Peptidic fragments of alpha-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the alpha-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa1-PTPa3. The subfraction PTPa2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C18 sorbent. A 100-mg amount of the PTPa2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300-3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.

7. Inhibitors of pancreatic and leukocyte elastase.
Collect. Czech. Chem. Commun. 1987, 52, 3034-3041
Kasafírek E., Frič P., Slabý J., Kocna P.
Research Institute for Pharmacy and Biochemistry, Prague; Laboratory of Gastroenterology, Charles University, Prague; Department of Medicine, Policlinic of Charles University, Prague
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Summary:
The following alkylamides of ?-carboxyalkanoyldi- and tripeptides of the Ala-Pro or Ala-Ala-Pro sequence have been prepared: ethylamide-, propylamide-, and isobutylamide of 3-carboxypropionylalanyl-proline, ethylamide of 3-carboxypropionylalanyl-alanyl-proline and ethylamide-, propylamide, and isobutylamide of 4-carboxybutyrylalanyl-alanyl-proline. The inhibitors were synthesized by fragment condensation in solution or by gradual construction. The inhibition constants Ki were determined by means of pancreatic elastase (substrates - p-nitroanilides of 4-carboxybutyrylalanyl-alanyl-alanyl-alanine and 3-carboxypropionylalanyl-alanyl-alanyl-alanine) and leukocyte elastase (substrate - p-nitroanilide of 4-carboxybutyrylalanyl-alanyl-alanyl-valine). The strongest inhibitions (Ki) were recorded in ethylamide of 4-carboxybutyrylalanyl-alanyl-proline, 2.0 and 1.6 µmol l-1 for pancreatic elastase, and in propylamide of 4-carboxybutyrylalanyl-alanyl-proline, 0.4 mmol l-1 for leukocyte elastase.

6. Modification of a Microprocessor-Controlled HPLC System (Perkin-Elmer).
Journal of Chromatographic Science, 1985, 23 (3), 132-136
Kocna P. Mittermüller B.J.
Laboratory of Gastroenterology, Faculty of Medicine, Charles University, Prague, Czechoslovakia; Perkin-Elmer Service Department, Vienna, Austria
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Summary:
Three modifications of a microprocessor-controlled high performance liquid chromatography system are described: initialization of peripheral devices by the injection valve enabling more precise estimation of retention constants, automatic return of the automatic switching valve to the RUN position after stop-flow spectroscopy, and solvent-saving by program cycling during overnight column stabilization.

5. Simple starch-gel electrophoresis of gliadin proteins using the LKB-multiphor system.
Zeitschrift für Lebensmitteluntersuchung Forschung A, 1983, 177 (6): 454 - 456
Kocna P, Holáková-Kocova M, Sasek A.
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Abstract
A simple modification of the horizontal starch-gel electrophoresis of gliadin proteins with the use of LKB Multiphor System has been developed. The thin-layer starch gel is prepared by using the original gel-moulding cassette and the procedure on the whole is easier. The sensitivity is comparable with that of the original method. Our modification moreover widens the field of application of the LKB Multiphor System.

4. Cleavage of p-nitroanilides of N-acylated tri- and tetrapeptides by alanine endopeptidase from the brush border membranes of rat enterocytes.
Experientia. 1983 Apr 15; 39 (4): 389 - 390
Kocna P, Kasafirek E, Fric P, Slaby J.
2nd Research Division of Gastroenterology and Internal Department of Policlinic, Faculty of General Medicine Charles University, and Research Institute for Pharmacy and Biochemistry, CS-121 11 Prague (Czechoslovakia).
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Abstract
The activity of the alanine endopeptidase from the intestinal brush border was studied using chromogenic substrates of the general formula Sc-Ala2-X-pNA. Sc-Y-Z-Ala-pNA and W-Ala3-pNA respectively. Substrates with C-terminal Leu or Nle are hydrolyzed more readily than Ala-analogues. At least one Ala-residue in one of the positions adjacent to the C-terminus is necessary for the enzyme activity. An Na-substituent has no effect on the activity.

3. Determination of the Protein Concentration in Biological Material by the Coomassie Briliant Blue G-250. (Article in Czech)
Biochm.Clin.Bohemoslov. 1982, 11 (1): 57 - 65
Kocna P, Frič P, Loucký K, Slabý J.
II.Vědecké oddělení gastroenterologické, FVL UK; Ústav termomechaniky ČSAV, Interní oddělení Fakultní polikliniky FVL UK, Praha (Czechoslovakia).
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Summary:
The method of protein estimation is based on photometrical measurement of a colour complex formed by Coomassie Brilliant Blue with protein. For estimation in urine, cerebrospinal fluid, ascites and homogenized tissues are the results achieved with this method comparable with the method according to Lowry. In addition is this method simpler, quicker and more sensitive. This paper showes further mathematical expression of the calibration curve and its linear regresion, which may be used in the range 2.5 - 20 ug protein probation sample. For a wider range up to 60 ug protein a non-linear regresion must be used. The use of a programable calculator Hewlett-Packard 9815A for the calculation both the calibration curve and the protein concentration from the absorbance is advantageous.

2. Endopeptidase of the brush border membrane of rat enterocyte. Separation from aminopeptidase and partial characterization.
Hoppe Seylers Z Physiol Chem. 1980 Sep; 361 (9): 1401 - 1412
Kocna P, Fric P, Slaby J, Kasafirek E.
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Summary:
The brush border of the enterocytes of the rat was isolated by the method of differential centrifugation with CaCl2 according to Schmitz. This material was solubilized with papain, trypsin and Triton X-100. The greatest amount of membrane enzymes was released to the supernatant (105 000 X g) with the use of Triton X-100. The tritonized supernatant was treated in the next step by papain, bromelain, ficin and trypsin (individually or in combinations). After simultaneous proteolysis with papain and bromelain a partial separation of the aminopeptidase from the endopeptidase by Sephadex G-200 chromatography was observed. These two enzyme activities were distinctly separated by isoelectric focusing at pH 4--6. Two enzymatically active bands (RF 0.13 and 0.24) in the aminopeptidase fraction and one single active band (RF 0.16) in the endopeptidase fraction using polyacrylamide gel electrophoresis were found. Co-migrating proteins to all of these activities were detected. Endopeptidase activity splits 3-carboxypropionyltrialanin-4-nitroanilide (SucAla3NAp) in the position P2-P1. Liberated aminoacyl-NAP may be further split to generate chromogenic 4-nitroaniline through aminopeptidase activity. Endopeptidase of the brush border of the rat enterocytes is characterized by the following properties: 1) molecular mass 130000 +/- 15 000 dalton; 2) Km value (substrate: SucAla3NAp) 1.1 X 10(-3) M; 3) pI 5.23; 4) ph optimum 8.5; 5) 50% activity remains after 15 min of preincubation at 50 degrees C; 6) activity is strongly inhibited by EDTA, p-chloromercuribenzoate, Mn2 and Co2.

1. Endopeptidase Isolation in Rat Enterocyte Brush Border. (Article in Czech)
Čas.Lék.Čes. 1978, 117 (40): 1258 - 1261
Kocna P., Frič P., Slabý J., Kasafírek E.
II.Vědecké oddělení gastroenterologické, FVL UK; Interní oddělení Fakultní polikliniky FVL UK; Výzkumný ústav pro farmacii a biochemii, Praha (Czechoslovakia).
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Summary:
Rat enterocyte brush border was isolate by differential centrifugation using calcium chloride according to Schmitz (22). The material was then solubilized with papaine, trypsine and Triton X-100 (1%) which released a maximum of membrane enzymes in the supernatant. Tritonized supernatant (105.000 xg) was treated with papaine, bromelin, ficin and trypsine (individually and in combinations). Following the simulaneous action of papaine and bromeline, Sephadex G-200 chromatography helped to achieve the partial separation of aminopeptidase (substrate: AlaNAp) from endopeptidase activity (substrate: SucAla3NAp Both activities were visibly separated from one another by means of isoelectric focusation (pI aminopeptidase - 4.73; pI endopeptidase - 5.23). Rat enterocyte endopeptidase has so far failed to be either described in detail or localized.


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