Thesis (Ph.D.)--Flinders University, School of Medicine, Dept. of Medical Biochemistry. / Typescript bound. Includes bibliographical references: (leaves 211-230) Also available online.

Thesis (M.Sc. (Biochemistry)--Mahidol University, 1983.

As principais causas de morbidade e mortalidade em cirurgia biliar relacionam-se a complicações sépticas. Estas associam-se à presença de bife colonizada por bactérias. Pacientes portadores de patologias biliares apresentam colonização bacteriana da bile em uma prevalência que varia de 11 a 1 00°/o. A maior parte dos estudos identifica uma prevalência de colonização da bile em torno de 1/3 dos casos. As espécies bacterianas mais freqüentemente isoladas são as da flora entérica, destacando-se a Escherichia co/i, Klebsiella sp e Streptococcus faeca/is. As bactérias anaeróbicas são encontradas em zero a 40°/o dos pacientes, sendo as mais prevalentes dos gêneros Bacteroides e Clostridium. No presente estudo, foram coletadas biópsias hepáticas intra-operatórlas com agulha de Vinn Silvermann e material denominado de aspirado hepático, constituído do sangue aspirado pela agulha após a retirada do fragmento da biópsia. O estudo visa identificar a acurácia da cultura bacteriana destes materiais no diagnóstico da colonização bacteriana da bile. A cultura da bile do colédoco, coletada por cateterização transcística da via biliar, foi escolhida como padrão-ouro. Alem destas, foram realizadas culturas de bile da vesícula biliar e sangue periférico. Foram, também, coletados dados de idade, sexo, diagnóstico pré-operatório, fatores de risco para colonização biliar e uso de antibióticos profiláticos. A população amestrada foi de pacientes submetidos a cirurgia biliar no Hospital de Clínicas de Porto Alegre. Foram excluídos pacientes em uso de antibióticos em regime terapêutico, uso de corticóides ou imunossupressores, presença de patologias parenquimatosas hepáticas e patologias infecciosas abdominais fora das vias biliares. As culturas aeróbicas foram feitas usando meio de agar-tripnase .. soja enriquecido com sangue humano desfibrinado e agar-MacConkey, incubados por 24 horas. As cultura anaeróbicas foram semeadas em caldo de tioglicolato enriquecido com vitamina K-hemina 1 o/o, agar-Brucella e agar-fenil-etanol enriquecidos com sangue de carneiro desfibrinado e vitamina K-hemina. o tempo de incubação foi de 48 horas. Entraram no estudo 57 pacientes, sete foram excluídos, permanecendo 50. A média de idade foi de 45,1 anos (:!:_15,3). Quarenta e oito pacientes tinham o diagnóstico pré-operatório de colecistite crônica, sendo 13 deles associada à coledocolitíase. Havia um caso de colecistite aguda e um de estenose pós-cirúrgica de via biliar. O fator de risco para colonização da bile de maior prevalência foi a coledocolitíase, com 13 pacientes. Vinte e nove pacientes não apresentavam nenhum fator de risco. Foram isoladas bactérias na bile do colédoco de nove pacientes. As espécies encontradas foram: Klebsiella pneumoniae, Escherichia co/i, Streptococcus faecalis, Enterobacter c/oacae, Klebsiella oxytoca, Streptococcus a-haemolitico. Na bile da vesícula biliar, foram isoladas bactérias em 7 pacientes: Klebsiella pneumoniae, Escherichia co/i, Streptococcus faeca/is, Enterobacter cloacae. Houve concordância entre os resultados da bile do colédoco e da vesícula biliar em 97,7% dos casos. A cultura da biópsia hepática foi positiva em dois casos. Em um, foi isolado Staphilococcus epidermidis, onde provavelmente houve contaminação externa. No outro, foi isolada Klebsiella pneumoniae que também havia sido encontrada na bile do colédoco do mesmo paciente. Neste paciente foi isolada Klebsiella no aspirado hepático. Foi o único caso de cultura positiva no aspirado hepático. Foi o único caso de cultura positiva no aspirado hepático. A cultura da biópsia hepática teve uma sensibilidade de 0,11, especificidade de 0,98, valor preditivo positivo de 0,50 e valor preditivo negativo de 0,83. A cultura do aspirado hepático apresentou uma sensibilidade de O, 11, especificidade de 1 ,O , valor preditivo positivo de 1 ,O e valor preditivo negativo de 0,84. Conclui-se que a cultura da biópsia e aspirado hepáticos com a técnica bacteriólogica empregada no estudo demonstrou uma acurácia diagnóstica baixa. A sua utilidade clínica na identificação de pacientes portadores de bactérias na bile é limitada. / The main causes of morbidity and mortality in biliary tract surgery are related to septic complications. Those relate to the presence of bile colonized by bacteria. Patients with biliary pathology show bacteria colonization of bile in a prevalence which varies from 11 to 100°/o. The majority of the studies identify a prevalence of colonization of bile about 1/3 of the cases. lhe bacterial species most frequently isolated are those of enteric flora, distinguishing the Escherichia co/i, Klebsiella sp and Streptococcus faecallis. The anaerobic bacteria are found in zero to 40o/o of the cases, being the most prevailing of the species Bactoides and Clostridium. In the present study there were collected hepatic intra-surgical biopsies with a needle of Vinn Silvermann and material named hepatical aspirated, made of aspirated blood through the needle after the removal of the pieces of the biopsy. lhe study aims to identify the accuracy of the bacterial culture from those substances in the diagnostic of the bacterial colonization of the bile. The culture of the bife from the common duct, collected by cystic duct probing, was chosen as the gold-standard. Besides those, there were made cultures o f bile o f the gallbladder and peripheral blood. lt was also collected data of age, sex, prcoperativo diagnosis, risk factors to biliary tract colonization and the use of prophylactic antibiotics. The sampled population was of patients undergoing on biliary tact surgery at the "Hospital de Clínicas de Porto Alegre". Patients in therapeutical use of antibiotics, using corticoesteroids or immunossupressive drugs, with hepato-cellular pathologies and abdominal infectious pathologies out · of the biliary tract were excluded. The aerobic cultures were made by the use of agar-tripnase-soy improved by desfibrinated human blood and agar-McConkey, incubated for 24 hours. The anaerobic cultures were sowed in a broth of tioglicolatus improved by vitamins K-hemine 1o/o, agar-Brucella and agar-fenil-ethanol improved with desfibrinated sheep blood and vitamin K-hemine. The time of incubation was of 48 hours. From 57 patients that begun the study, 7 were excluded, remaining 50. The age average was of 45,7 years old (± 15,3). Forty-eight patients had the preoperative diagnosis of chronic cholecystitis, 13 of them being related to choledocholithiasis. There was a case of acute cholecystitis and one of postoperative biliary tract stenosis. The risk factor to bile colonization of major prevalence was the choledocholithiasis (13 patients). Twenty-nine patients did not show any risk factor. They were isolated bacteria in the common duct bile of nine patients. The species found were Klebsiella penumoniae, Eschericha coli, Streptococcus faecalis, Enterobacter cloacae, Klebsiella oxytoca, Streptococcus a-haemolitico. In the gallbladder bile bacteria were isolated in 7 patients: Klebsie/la pneumoniae, Escherichia co/i, Streptococcus faecalis, Enterobacter cloacae. There was agreement between the results of the common duct and gallbladder bile in 97,7% o f the cases. The culture o f the hepatic biopsy was positiva in two cases. In one, it was isolated Staphi/ococcus epidermidis , were probably there was externai contamination. In the other one, it was isolated Klebsiella penumoniae, which had also been found in the common duct bile of the same patient. In that patient it was isolated Klebsiella in the hepatic aspirated. lt was the only case of positive culture in the hepatic aspirated. The hepatic biopsy culture had a sensibility of O, 11, a specificity of 0,98, a predictive positiva value of 0,50 and a predictive negative value of 0,83. The hepatic aspirated culture showed a sensibility of O, 11, a specificity of 1 ,O, a predictive positive value of 1 ,O and a predictive negative value of 0,84. We concluded that the culture of the biopsy and hepatic aspirated, with the bacteriological technique used in the study, showed a low diagnostic accuracy. lt has a low clinicai use in the identification of patients with biliary tract colonization.

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Obesity is considered to be a worldwide epidemic disease characterized by an accumulation of AT. Increased adiposity can perturb normal metabolic functions and lead to the development of diseases like insulin resistance and other metabolic disorders. A large amount of clinical studies have been shown that changes in inflammatory signaling in adipose tissue cells, increased infiltration of immune cells into AT as well as stress of endoplasmic reticulum belong to the key molecular steps leading to the development of metabolic disturbances associated with this disease. Adverse metabolic effects of AT accumulation can be diminished by calorie restriction resulting in weight loss. In addition, stress of endoplasmic reticulum could be alleviated by chemical chaperones including bile acids. These two approaches for the treatment of obesity or the obesity-associated disturbances were basis for this PhD thesis. In the first part of this work, we studied inflammation status of gluteal in comparison with abdominal AT and differentiation and secretory capacity of adipocytes after weight loss in obese patients. We revealed that inflammatory profile of gluteal AT, estimated by mRNA level of macrophages and cytokines as markers of inflammatory status of the body, did not explain the different clinical impact of...

As principais causas de morbidade e mortalidade em cirurgia biliar relacionam-se a complicações sépticas. Estas associam-se à presença de bife colonizada por bactérias. Pacientes portadores de patologias biliares apresentam colonização bacteriana da bile em uma prevalência que varia de 11 a 1 00°/o. A maior parte dos estudos identifica uma prevalência de colonização da bile em torno de 1/3 dos casos. As espécies bacterianas mais freqüentemente isoladas são as da flora entérica, destacando-se a Escherichia co/i, Klebsiella sp e Streptococcus faeca/is. As bactérias anaeróbicas são encontradas em zero a 40°/o dos pacientes, sendo as mais prevalentes dos gêneros Bacteroides e Clostridium. No presente estudo, foram coletadas biópsias hepáticas intra-operatórlas com agulha de Vinn Silvermann e material denominado de aspirado hepático, constituído do sangue aspirado pela agulha após a retirada do fragmento da biópsia. O estudo visa identificar a acurácia da cultura bacteriana destes materiais no diagnóstico da colonização bacteriana da bile. A cultura da bile do colédoco, coletada por cateterização transcística da via biliar, foi escolhida como padrão-ouro. Alem destas, foram realizadas culturas de bile da vesícula biliar e sangue periférico. Foram, também, coletados dados de idade, sexo, diagnóstico pré-operatório, fatores de risco para colonização biliar e uso de antibióticos profiláticos. A população amestrada foi de pacientes submetidos a cirurgia biliar no Hospital de Clínicas de Porto Alegre. Foram excluídos pacientes em uso de antibióticos em regime terapêutico, uso de corticóides ou imunossupressores, presença de patologias parenquimatosas hepáticas e patologias infecciosas abdominais fora das vias biliares. As culturas aeróbicas foram feitas usando meio de agar-tripnase .. soja enriquecido com sangue humano desfibrinado e agar-MacConkey, incubados por 24 horas. As cultura anaeróbicas foram semeadas em caldo de tioglicolato enriquecido com vitamina K-hemina 1 o/o, agar-Brucella e agar-fenil-etanol enriquecidos com sangue de carneiro desfibrinado e vitamina K-hemina. o tempo de incubação foi de 48 horas. Entraram no estudo 57 pacientes, sete foram excluídos, permanecendo 50. A média de idade foi de 45,1 anos (:!:_15,3). Quarenta e oito pacientes tinham o diagnóstico pré-operatório de colecistite crônica, sendo 13 deles associada à coledocolitíase. Havia um caso de colecistite aguda e um de estenose pós-cirúrgica de via biliar. O fator de risco para colonização da bile de maior prevalência foi a coledocolitíase, com 13 pacientes. Vinte e nove pacientes não apresentavam nenhum fator de risco. Foram isoladas bactérias na bile do colédoco de nove pacientes. As espécies encontradas foram: Klebsiella pneumoniae, Escherichia co/i, Streptococcus faecalis, Enterobacter c/oacae, Klebsiella oxytoca, Streptococcus a-haemolitico. Na bile da vesícula biliar, foram isoladas bactérias em 7 pacientes: Klebsiella pneumoniae, Escherichia co/i, Streptococcus faeca/is, Enterobacter cloacae. Houve concordância entre os resultados da bile do colédoco e da vesícula biliar em 97,7% dos casos. A cultura da biópsia hepática foi positiva em dois casos. Em um, foi isolado Staphilococcus epidermidis, onde provavelmente houve contaminação externa. No outro, foi isolada Klebsiella pneumoniae que também havia sido encontrada na bile do colédoco do mesmo paciente. Neste paciente foi isolada Klebsiella no aspirado hepático. Foi o único caso de cultura positiva no aspirado hepático. Foi o único caso de cultura positiva no aspirado hepático. A cultura da biópsia hepática teve uma sensibilidade de 0,11, especificidade de 0,98, valor preditivo positivo de 0,50 e valor preditivo negativo de 0,83. A cultura do aspirado hepático apresentou uma sensibilidade de O, 11, especificidade de 1 ,O , valor preditivo positivo de 1 ,O e valor preditivo negativo de 0,84. Conclui-se que a cultura da biópsia e aspirado hepáticos com a técnica bacteriólogica empregada no estudo demonstrou uma acurácia diagnóstica baixa. A sua utilidade clínica na identificação de pacientes portadores de bactérias na bile é limitada. / The main causes of morbidity and mortality in biliary tract surgery are related to septic complications. Those relate to the presence of bile colonized by bacteria. Patients with biliary pathology show bacteria colonization of bile in a prevalence which varies from 11 to 100°/o. The majority of the studies identify a prevalence of colonization of bile about 1/3 of the cases. lhe bacterial species most frequently isolated are those of enteric flora, distinguishing the Escherichia co/i, Klebsiella sp and Streptococcus faecallis. The anaerobic bacteria are found in zero to 40o/o of the cases, being the most prevailing of the species Bactoides and Clostridium. In the present study there were collected hepatic intra-surgical biopsies with a needle of Vinn Silvermann and material named hepatical aspirated, made of aspirated blood through the needle after the removal of the pieces of the biopsy. lhe study aims to identify the accuracy of the bacterial culture from those substances in the diagnostic of the bacterial colonization of the bile. The culture of the bife from the common duct, collected by cystic duct probing, was chosen as the gold-standard. Besides those, there were made cultures o f bile o f the gallbladder and peripheral blood. lt was also collected data of age, sex, prcoperativo diagnosis, risk factors to biliary tract colonization and the use of prophylactic antibiotics. The sampled population was of patients undergoing on biliary tact surgery at the "Hospital de Clínicas de Porto Alegre". Patients in therapeutical use of antibiotics, using corticoesteroids or immunossupressive drugs, with hepato-cellular pathologies and abdominal infectious pathologies out · of the biliary tract were excluded. The aerobic cultures were made by the use of agar-tripnase-soy improved by desfibrinated human blood and agar-McConkey, incubated for 24 hours. The anaerobic cultures were sowed in a broth of tioglicolatus improved by vitamins K-hemine 1o/o, agar-Brucella and agar-fenil-ethanol improved with desfibrinated sheep blood and vitamin K-hemine. The time of incubation was of 48 hours. From 57 patients that begun the study, 7 were excluded, remaining 50. The age average was of 45,7 years old (± 15,3). Forty-eight patients had the preoperative diagnosis of chronic cholecystitis, 13 of them being related to choledocholithiasis. There was a case of acute cholecystitis and one of postoperative biliary tract stenosis. The risk factor to bile colonization of major prevalence was the choledocholithiasis (13 patients). Twenty-nine patients did not show any risk factor. They were isolated bacteria in the common duct bile of nine patients. The species found were Klebsiella penumoniae, Eschericha coli, Streptococcus faecalis, Enterobacter cloacae, Klebsiella oxytoca, Streptococcus a-haemolitico. In the gallbladder bile bacteria were isolated in 7 patients: Klebsie/la pneumoniae, Escherichia co/i, Streptococcus faecalis, Enterobacter cloacae. There was agreement between the results of the common duct and gallbladder bile in 97,7% o f the cases. The culture o f the hepatic biopsy was positiva in two cases. In one, it was isolated Staphi/ococcus epidermidis , were probably there was externai contamination. In the other one, it was isolated Klebsiella penumoniae, which had also been found in the common duct bile of the same patient. In that patient it was isolated Klebsiella in the hepatic aspirated. lt was the only case of positive culture in the hepatic aspirated. The hepatic biopsy culture had a sensibility of O, 11, a specificity of 0,98, a predictive positiva value of 0,50 and a predictive negative value of 0,83. The hepatic aspirated culture showed a sensibility of O, 11, a specificity of 1 ,O, a predictive positive value of 1 ,O and a predictive negative value of 0,84. We concluded that the culture of the biopsy and hepatic aspirated, with the bacteriological technique used in the study, showed a low diagnostic accuracy. lt has a low clinicai use in the identification of patients with biliary tract colonization.

Bile acids are essential for the efficient digestion and absorption of lipids, and are re-absorbed by aNa+ /bile acid co-transport protein within the ileum. Investigation into the transport protein, using BBMV and Xenopus laevis oocytes, revealed that pig ileal Na +/bile acid co-transport protein was not strictly dependent of the presence of an inwardly directed Na + gradient, as previously determined. Cations which had ionic radii between 0.8 - 1.33A, most notably Na+, K+ and Ca2+, stimulated secondary active transport of taurocholate. Taurocholate uptake in the presence of inwardly directed cation gradients of Na + and K+, demonstrated Michealis Menten Kinetics, concentrative accumulation, competitive inhibition and was temperature sensitive. Preventing the translation of the gene encoding the Na+/bile acid co-transport protein, abolished secondary active transport in the presence ofboth Na+ and K+. Isolation and computer modelling of the gene which encoded the pig ileal Na +/bile acid co-transport protein, revealed that this protein was composed of 8 transmembrane domains and lead to the identification of proposed cation and bile acid binding sites. The ability of K+ to stimulate the Na +/bile acid co-transport protein could be of physiological importance in vivo, because of the depleted Na + concentration present within the ileum. Though, the rate of taurocholate transport in the presence of K+ is reduced when compared to Na+, the transport protein has very similar affinities for taurocholate in the presence of both cations and therefore could use K+ for efficient re-absorption of taurocholate. Therefore, for this study it was proposed that the Na+/bile acid co-transport protein had a preference for Na+ rather than a strict dependence as previously concluded.

As principais causas de morbidade e mortalidade em cirurgia biliar relacionam-se a complicações sépticas. Estas associam-se à presença de bife colonizada por bactérias. Pacientes portadores de patologias biliares apresentam colonização bacteriana da bile em uma prevalência que varia de 11 a 1 00°/o. A maior parte dos estudos identifica uma prevalência de colonização da bile em torno de 1/3 dos casos. As espécies bacterianas mais freqüentemente isoladas são as da flora entérica, destacando-se a Escherichia co/i, Klebsiella sp e Streptococcus faeca/is. As bactérias anaeróbicas são encontradas em zero a 40°/o dos pacientes, sendo as mais prevalentes dos gêneros Bacteroides e Clostridium. No presente estudo, foram coletadas biópsias hepáticas intra-operatórlas com agulha de Vinn Silvermann e material denominado de aspirado hepático, constituído do sangue aspirado pela agulha após a retirada do fragmento da biópsia. O estudo visa identificar a acurácia da cultura bacteriana destes materiais no diagnóstico da colonização bacteriana da bile. A cultura da bile do colédoco, coletada por cateterização transcística da via biliar, foi escolhida como padrão-ouro. Alem destas, foram realizadas culturas de bile da vesícula biliar e sangue periférico. Foram, também, coletados dados de idade, sexo, diagnóstico pré-operatório, fatores de risco para colonização biliar e uso de antibióticos profiláticos. A população amestrada foi de pacientes submetidos a cirurgia biliar no Hospital de Clínicas de Porto Alegre. Foram excluídos pacientes em uso de antibióticos em regime terapêutico, uso de corticóides ou imunossupressores, presença de patologias parenquimatosas hepáticas e patologias infecciosas abdominais fora das vias biliares. As culturas aeróbicas foram feitas usando meio de agar-tripnase .. soja enriquecido com sangue humano desfibrinado e agar-MacConkey, incubados por 24 horas. As cultura anaeróbicas foram semeadas em caldo de tioglicolato enriquecido com vitamina K-hemina 1 o/o, agar-Brucella e agar-fenil-etanol enriquecidos com sangue de carneiro desfibrinado e vitamina K-hemina. o tempo de incubação foi de 48 horas. Entraram no estudo 57 pacientes, sete foram excluídos, permanecendo 50. A média de idade foi de 45,1 anos (:!:_15,3). Quarenta e oito pacientes tinham o diagnóstico pré-operatório de colecistite crônica, sendo 13 deles associada à coledocolitíase. Havia um caso de colecistite aguda e um de estenose pós-cirúrgica de via biliar. O fator de risco para colonização da bile de maior prevalência foi a coledocolitíase, com 13 pacientes. Vinte e nove pacientes não apresentavam nenhum fator de risco. Foram isoladas bactérias na bile do colédoco de nove pacientes. As espécies encontradas foram: Klebsiella pneumoniae, Escherichia co/i, Streptococcus faecalis, Enterobacter c/oacae, Klebsiella oxytoca, Streptococcus a-haemolitico. Na bile da vesícula biliar, foram isoladas bactérias em 7 pacientes: Klebsiella pneumoniae, Escherichia co/i, Streptococcus faeca/is, Enterobacter cloacae. Houve concordância entre os resultados da bile do colédoco e da vesícula biliar em 97,7% dos casos. A cultura da biópsia hepática foi positiva em dois casos. Em um, foi isolado Staphilococcus epidermidis, onde provavelmente houve contaminação externa. No outro, foi isolada Klebsiella pneumoniae que também havia sido encontrada na bile do colédoco do mesmo paciente. Neste paciente foi isolada Klebsiella no aspirado hepático. Foi o único caso de cultura positiva no aspirado hepático. Foi o único caso de cultura positiva no aspirado hepático. A cultura da biópsia hepática teve uma sensibilidade de 0,11, especificidade de 0,98, valor preditivo positivo de 0,50 e valor preditivo negativo de 0,83. A cultura do aspirado hepático apresentou uma sensibilidade de O, 11, especificidade de 1 ,O , valor preditivo positivo de 1 ,O e valor preditivo negativo de 0,84. Conclui-se que a cultura da biópsia e aspirado hepáticos com a técnica bacteriólogica empregada no estudo demonstrou uma acurácia diagnóstica baixa. A sua utilidade clínica na identificação de pacientes portadores de bactérias na bile é limitada. / The main causes of morbidity and mortality in biliary tract surgery are related to septic complications. Those relate to the presence of bile colonized by bacteria. Patients with biliary pathology show bacteria colonization of bile in a prevalence which varies from 11 to 100°/o. The majority of the studies identify a prevalence of colonization of bile about 1/3 of the cases. lhe bacterial species most frequently isolated are those of enteric flora, distinguishing the Escherichia co/i, Klebsiella sp and Streptococcus faecallis. The anaerobic bacteria are found in zero to 40o/o of the cases, being the most prevailing of the species Bactoides and Clostridium. In the present study there were collected hepatic intra-surgical biopsies with a needle of Vinn Silvermann and material named hepatical aspirated, made of aspirated blood through the needle after the removal of the pieces of the biopsy. lhe study aims to identify the accuracy of the bacterial culture from those substances in the diagnostic of the bacterial colonization of the bile. The culture of the bife from the common duct, collected by cystic duct probing, was chosen as the gold-standard. Besides those, there were made cultures o f bile o f the gallbladder and peripheral blood. lt was also collected data of age, sex, prcoperativo diagnosis, risk factors to biliary tract colonization and the use of prophylactic antibiotics. The sampled population was of patients undergoing on biliary tact surgery at the "Hospital de Clínicas de Porto Alegre". Patients in therapeutical use of antibiotics, using corticoesteroids or immunossupressive drugs, with hepato-cellular pathologies and abdominal infectious pathologies out · of the biliary tract were excluded. The aerobic cultures were made by the use of agar-tripnase-soy improved by desfibrinated human blood and agar-McConkey, incubated for 24 hours. The anaerobic cultures were sowed in a broth of tioglicolatus improved by vitamins K-hemine 1o/o, agar-Brucella and agar-fenil-ethanol improved with desfibrinated sheep blood and vitamin K-hemine. The time of incubation was of 48 hours. From 57 patients that begun the study, 7 were excluded, remaining 50. The age average was of 45,7 years old (± 15,3). Forty-eight patients had the preoperative diagnosis of chronic cholecystitis, 13 of them being related to choledocholithiasis. There was a case of acute cholecystitis and one of postoperative biliary tract stenosis. The risk factor to bile colonization of major prevalence was the choledocholithiasis (13 patients). Twenty-nine patients did not show any risk factor. They were isolated bacteria in the common duct bile of nine patients. The species found were Klebsiella penumoniae, Eschericha coli, Streptococcus faecalis, Enterobacter cloacae, Klebsiella oxytoca, Streptococcus a-haemolitico. In the gallbladder bile bacteria were isolated in 7 patients: Klebsie/la pneumoniae, Escherichia co/i, Streptococcus faecalis, Enterobacter cloacae. There was agreement between the results of the common duct and gallbladder bile in 97,7% o f the cases. The culture o f the hepatic biopsy was positiva in two cases. In one, it was isolated Staphi/ococcus epidermidis , were probably there was externai contamination. In the other one, it was isolated Klebsiella penumoniae, which had also been found in the common duct bile of the same patient. In that patient it was isolated Klebsiella in the hepatic aspirated. lt was the only case of positive culture in the hepatic aspirated. The hepatic biopsy culture had a sensibility of O, 11, a specificity of 0,98, a predictive positiva value of 0,50 and a predictive negative value of 0,83. The hepatic aspirated culture showed a sensibility of O, 11, a specificity of 1 ,O, a predictive positive value of 1 ,O and a predictive negative value of 0,84. We concluded that the culture of the biopsy and hepatic aspirated, with the bacteriological technique used in the study, showed a low diagnostic accuracy. lt has a low clinicai use in the identification of patients with biliary tract colonization.

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Abstract Fatty acid degradation in living organisms occurs mainly via the β-oxidation pathway. When this work was started, it was known that the hydration and dehydrogenation reactions in mammalian peroxisomal β-oxidation were catalyzed by only multifunctional enzyme type 1 (MFE-1; Δ2-Δ3-enoyl-CoA isomerase/2-enoyl-CoA hydratase 1/(3S)-hydroxyacyl-CoA dehydrogenase) via the S-specific pathway, whereas in the yeast peroxisomes via the R-specific pathway by multifunctional enzyme type 2 (MFE-2; 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl-CoA dehydrogenase). The work started with the molecular cloning of the rat 2-enoy-CoA hydratase 2 (hydratase 2). The isolated cDNA (2205 bp) encodes a polypeptide with a predicted molecular mass of 79.3 kDa, which contains a potential peroxisomal targeting signal (AKL) in the carboxyl terminus. The hydratase 2 is an integral part of the cloned polypeptide, which is assigned to be a novel mammalian peroxisomal MFE-2. The physiological role of the mammalian hydratase 2 was investigated with the recombinant hydratase 2 domain derived from rat MFE-2. The protein hydrates a physiological intermediate (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-enoyl-CoA to (24R, 25R)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl-CoA in bile acid synthesis. The sequence alignment of human MFE-2 with MFE-2(s) of different species reveals 12 conserved protic amino acid residues, which are potential candidates for catalysis of the hydratase 2. Each of these residues was replaced by alanine. Complementation of Saccharomyces cerevisiae fox-2 (devoid of endogenous MFE-2) with human MFE-2 provided a model system for examing the in vivo function of the variants. Two protic residues, Glu366 and Asp510, of the hydratase 2 domain of human MFE-2 have been identified and are proposed to act as a base and an acid in catalysis. Mammalian MFE-2 has a (3R)-hydroxyacyl-CoA dehydrogenase domain, whereas the yeast MFE-2 has two dehydrogenase domains, A and B. The present work, applying site-directed mutagenesis to dissect the two domains, shows that the growth rates of fox-2 cells expressing a single functional domain are lower than those of cells expressing S. cerevisiae MFE-2. Kinetic experiments with the purified proteins demonstrate that domain A is more active than domain B in catalysis of medium- and long-chain (3R)-hydroxyacyl-CoA, whereas domain B is solely responsible for metabolism of short-chain substrates. Both domains are required when yeast cells utilize fatty acids as the carbon source.