Quantification of Serum Matrix Metallopeptide 7 Levels May Assist in the Diagnosis and Predict the Outcome for Patients with Biliary Atresia.

OBJECTIVE: To assess the diagnostic and prognostic usefulness of the serum matrix metallopeptidase-7 (MMP-7) level for biliary atresia in infants with cholestasis after hepatoportoenterostomy. STUDY DESIGN: We enrolled 100 infants with cholestasis (age, 43.56 ± 1.97 days; 62 males) with a direct bilirubin level of >1 mg/dL, of whom 36 (36%) were diagnosed with biliary atresisa. The MMP-7 levels in serum samples collected during the cholestasis workup and 6 months after hepatoportoenterostomy were assessed by enzyme-linked immunosorbent assay. We quantified liver fibrosis by Picro Sirius red staining of collagen in specimens from the 81 infants with cholestasis. RESULTS: Infants with biliary atresisa had a significantly higher serum MMP-7 level than that of non-biliary atresisa infants with cholestasis of equivalent age (P < .0001). Receiver operating characteristic analysis showed that a serum MMP-7 level of >1.43 ng/mL was predictive of biliary atresisa in infants with cholestasis (diagnostic accuracy, 88%). There was a positive correlation between the serum MMP-7 level and the severity of liver fibrosis (P = .0002). Survival analysis showed that the frequency of liver transplantation was significantly higher in infants with biliary atresisa with a serum MMP-7 level of >10.30 ng/mL compared with a serum MMP-7 level of ≤10.30 ng/mL after hepatoportoenterostomy (hazard ratio, 4.22; P = .02). CONCLUSIONS: The serum MMP-7 level, which reflects the severity of liver fibrosis and can be determined noninvasively, may facilitate the diagnosis of biliary atresisa among infants with cholestasis. Moreover, the serum MMP-7 level after hepatoportoenterostomy is associated with a need for liver transplantation in infants with biliary atresisa.

Heme oxygenase-1 induction by hemin prevents oxidative stress-induced acute cholestasis in the rat.

We previously demonstrated in in vitro and ex vivo models that physiological concentrations of unconjugated bilirubin (BR) prevent oxidative stress (OS)-induced hepatocanalicular dysfunction and cholestasis. Here, we aimed to ascertain, in the whole rat, whether a similar cholestatic OS injury can be counteracted by heme oxygenase-1 (HO-1) induction that consequently elevates endogenous BR levels. This was achieved through the administration of hemin, an inducer of HO-1, the rate-limiting step in BR generation. We found that BR peaked between 6 and 8 h after hemin administration. During this time period, HO-1 induction fully prevented the pro-oxidant tert-butylhydroperoxide (tBuOOH)-induced drop in bile flow, and in the biliary excretion of bile salts and glutathione, the two main driving forces of bile flow; this was associated with preservation of the membrane localization of their respective canalicular transporters, bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2), which are otherwise endocytosed by OS. HO-1 induction counteracted the oxidation of intracellular proteins and membrane lipids induced by tBuOOH, and fully prevented the increase in the oxidized-to-total glutathione (GSHt) ratio, a sensitive parameter of hepatocellular OS. Compensatory elevations of the activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) were also prevented. We conclude that in vivo HO-1 induction protects the liver from acute oxidative injury, thus preventing consequent cholestasis. This reveals an important role for the induction of HO-1 and the consequently elevated levels of BR in preserving biliary secretory function under OS conditions, thus representing a novel therapeutic tool to limit the cholestatic injury that bears an oxidative background.

Pyrimidine-5'-nucleotidase Campinas, a new mutation (p.R56G) in the NT5C3 gene associated with pyrimidine-5'-nucleotidase type I deficiency and influence of Gilbert's Syndrome on clinical expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Bone marrow mononuclear cell transplantation increases metalloproteinase-9 and 13 and decreases tissue inhibitors of metalloproteinase-1 and 2 expression in the liver of cholestatic rats.

Liver fibrosis results from chronic injury followed by activation of macrophages and fibrogenic cells like myofibroblasts and activated hepatic stellate cells. These fibrogenic cells express α-smooth muscle actin (α-SMA) and produce excessive extracellular matrix (ECM), with disorganization and loss of function of hepatic parenchyma. It is known that increased levels of metalloproteinases (MMPs) in liver fibrosis are associated with reduction of the pathologic ECM and fibrosis resolution. Recently, it has been shown that bone marrow mononuclear cells (BMMNCs) may reduce collagen and α-SMA expression, and ameliorate liver function in cholestatic rats. Therefore, this study aimed to analyze MMP-2, MMP-9 and MMP-13, and tissue inhibitors of MMPs (TIMPs)-1 and TIMP-2 in the liver of cholestatic rats transplanted with BMMNC. Animals were divided into normal rats, cholestatic rats obtained after 14 and 21 days of bile duct ligation (BDL), and rats obtained after 14 days of BDL that received BMMNCs and were killed after 7 days. MMP and TIMP expression was assessed by Western blotting, along with α-SMA, CD68 and CD11b expression by confocal microscopy. Western blotting analysis showed that 14-day BDL animals had significantly reduced amounts of MMP-2 and MMP-13, but increased amounts of MMP-9 compared to normal rats. After 21 days of BDL, overall MMP amounts were decreased and TIMPs were increased. BMMNC transplantation significantly increased MMP-9 and MMP-13, and decreased TIMP expression. Increased MMP activity was confirmed by zymography. MMP-9 and MMP-13 were expressed by macrophages near fibrotic septa, suggesting BMMNC may stimulate MMP production in fibrotic livers, contributing to ECM degradation and hepatic regeneration.

Differential diagnosis of neonatal cholestasis: clinical and laboratory parameters.

OBJECTIVE: To evaluate if clinical and laboratory parameters could assist in the differential diagnosis of intra and extra-hepatic neonatal cholestasis (NC). METHODS: Retrospective study of NC patients admitted at the Pediatric Hepatology Outpatient Clinic of the teaching hospital of Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil, between December 1980 and March 2005. The approach to the diagnosis of NC was standardized. According to diagnosis, patients were classified into two groups: I (intra-hepatic neonatal cholestasis) and II (extra-hepatic neonatal cholestasis). In order to verify if there was association with the categorical variable, the chi-square and Mann-Whitney tests were used, with corrections for age for the covariance analysis (ANCOVA). The determination of accuracy of the clinical and laboratory variables for differentiation of the groups was made using the analysis of the ROC curve. RESULTS: One hundred and sixty-eight patients were evaluated (group I = 54.8% and group II = 45.2%). In the patients with less than 60 days of life there was predominance of intra-hepatic causes, whereas, in those older than 60 days, there was predominance of extra-hepatic etiology (p < 0.001). Median birth weight was lower in group I (p = 0.003), as well as length at birth (p = 0.007). Median values of direct bilirubin were higher in group II (p = 0.006). Values of gamma-glutamyltransferase (GGT) (10 times higher than the limit of normality) presented sensitivity of 56.3%, specificity of 91.5%, and accuracy of 75.7% for the diagnosis of extra-hepatic cholestasis. CONCLUSION: In the present study, extra-hepatic NC presented greater weight and length at birth, fecal hypocholia/acholia, choluria, hepatomegaly, increase in GGT (10.8 times higher than the limit of normality), and a delay for investigation in the tertiary center.

Diagnóstico diferencial de colestase neonatal: parâmetros clínicos e laboratoriais / Differential diagnosis of neonatal cholestasis: clinical and laboratory parameters

Objetivo: Avaliar se os parâmetros clínicos e laboratoriais poderiam auxiliar no diagnóstico diferencial da colestase neonatal (CN) intra- e extra-hepática. Métodos: Estudo retrospectivo de pacientes com CN hospitalizados na Clínica de Hepatologia Pediátrica do Hospital de Clínicas da Universidade Estadual de Campinas (UNICAMP), Campinas (SP), entre dezembro de 1980 e março de 2005. A abordagem para o diagnóstico da CN foi padronizada. De acordo com o diagnóstico, os pacientes foram classificados em dois grupos: I (colestase neo natal intra-hepática) e II (colestase neonatal extrahepática). Para verificar se havia associação com a variável categórica, os testes de qui-quadrado e Mann-Whitney foram utilizados com correções para idade para a análise de covariância (ANCOVA). A determinação da precisão das variáveis clínicas e laboratoriais para a diferenciação dos grupos foi realizada através da análise da curva ROC. Resultados: Cento e sessenta e oito pacientes foram avaliados (grupo I = 54,8 por cento e grupo II = 45,2 por cento). Nos pacientes com menos de 60 dias de vida, houve predominância de causas intra-hepáticas, enquanto que naqueles com mais de 60 dias, houve predominância de etiologia extrahepática (p < 0,001). A mediana de peso ao nascer foi mais baixa no grupo I (p = 0,003), assim como o comprimento ao nascer (p = 0,007). Os valores da mediana de bilirrubina direta foram mais altos no grupo II (p = 0,006). Os valores de gama glutamil transferase (GGT) (10 vezes mais altos do que o limite de normalidade) apresentaram sensibilidade de 56,3 por cento, especificidade de 91,5 por cento e acurácia de 75,7 por cento para o diagnóstico de colestase extra-hepática. Conclusão: No presente estudo, a CN extra-hepática apresentou maior peso e comprimento ao nascer, hipocolia/acolia fecal, colúria, hepatomegalia, aumento de GGT (10,8 vezes mais alto do que o limite de normalidade) e um atraso no encaminhamento para a investigação no hospital terciário.

Objective: To evaluate if clinical and laboratory parameters could assist in the differential diagnosis of intra and extra-hepatic neonatal cholestasis (NC). Methods: Retrospective study of NC patients admitted at the Pediatric Hepatology Outpatient Clinic of the teaching hospital of Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil, between December 1980 and March 2005. The approach to the diagnosis of NC was standardized. According to diagnosis, patients were classified into two groups: I (intra-hepatic neonatal cholestasis) and II (extra-hepatic neonatal cholestasis). In order to verify if there was association with the categorical variable, the chi-square and Mann-Whitney tests were used, with corrections for age for the covariance analysis (ANCOVA). The determination of accuracy of the clinical and laboratory variables for differentiation of the groups was made using the analysis of the ROC curve. Results: One hundred and sixty-eight patients were evaluated (group I = 54.8 percent and group II = 45.2 percent). In the patients with less than 60 days of life there was predominance of intra-hepatic causes, whereas, in those older than 60 days, there was predominance of extra-hepatic etiology (p < 0.001). Median birth weight was lower in group I (p = 0.003), as well as length at birth (p = 0.007). Median values of direct bilirubin were higher in group II (p = 0.006). Values of gamma-glutamyltransferase (GGT) (10 times higher than the limit of normality) presented sensitivity of 56.3 percent, specificity of 91.5 percent, and accuracy of 75.7 percent for the diagnosis of extra-hepatic cholestasis. Conclusion: In the present study, extra-hepatic NC presented greater weight and length at birth, fecal hypocholia/acholia, choluria, hepatomegaly, increase in GGT (10.8 times higher than the limit of normality), and a delay for investigation in the tertiary center.

Expression of kidney and liver bilitranslocase in response to acute biliary obstruction.

BACKGROUND/AIM: It has been recently demonstrated that acute obstructive jaundice is associated with modifications in the renal expression and function of organic anion transporters such as Oat1, Oat3, Oatp1 and Mrp2. This study examined the expression and function of bilitranslocase in liver and kidney from rats with bile duct ligation (BDL). METHODS: Bilitranslocase expression was evaluated in renal homogenates (H), renal basolateral plasma membranes (KBLM) and liver plasma membranes (LPM) by immunoblotting. Bilitranslocase function was studied by measuring the kinetic parameters of electrogenic bromosulfophthalein (BSP) uptake in KBLM and LPM by a spectrophotometric technique. RESULTS: An increased abundance of bilitranslocase in KBLM without modifications in renal H and in LPM from BDL rats was observed compared with Sham rats. BDL rats showed a higher V(max) for BSP uptake in KBLM. No differences between groups were observed for Michaelis-Menten parameters in LPM. CONCLUSION: The higher renal expression and function of bilitranslocase in renal basolateral membranes from rats with obstructive cholestasis might also contribute to the dramatic increase in BSP renal excretion observed in this experimental model. This would be another compensation mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

Ascorbic acid supplementation has a cytoprotective effect on secondary biliary cirrhosis: experimental study in young rats.

OBJECTIVE: To test whether ascorbic acid supplementation has any cytoprotective effect on a model of secondary biliary cirrhosis in young rats. METHODS: We studied 40 Wistar rats weaned at the 21st postnatal day. Each group of 10 was subjected to one of the following four treatments, until 49th postnatal day, when they suffered euthanasia: 1) LC-double ligature and resection of the common bile duct and daily administration of ascorbic acid [100 mg/g of body weight (bw)]; 2) LA-double ligature and resection of the common bile duct and daily administration of aqueous vehicle (1 mL/g bw); 3) SC-sham operation and daily administration of ascorbic acid (100 mg/g bw); 4) SA-double ligature and resection of the common bile duct and daily administration of aqueous vehicle (1 mL/g bw). The rats were weighed daily. On the 27th day after the operation they received an intra-peritoneal injection of 1.5 mg/g bw of sodium pentobarbital, and the pentobarbital sleeping time was measured. Blood was collected for serum alanine aminotransferase and aspartate aminotransferase activity measurements, serum albumin and globulin concentrations, and the liver was assessed for liver water and fat content. Data were submitted to two-way ANOVA and paired comparisons between groups were tested using the SNK method. Significance level was set at 0.05. RESULTS: Ascorbic acid supplementation attenuated the effects of cholestasis: decreased the pentobarbital sleeping time, serum globulin, and the liver fat content. CONCLUSIONS: Our results corroborate the hypothesis that ascorbic acid supplementation has a cytoprotective effect in secondary biliary cirrhosis.

Suplementação com ácido ascórbico tem efeito citoprotetor na cirrose biliar secundária: estudo experimental em ratos jovens / Ascorbic acid supplementation has a cytoprotective effect on secondary biliary cirrhosis: experimental study in young rats

OBJETIVO: Testar se a suplementação com ácido ascórbico tem algum afeito citoprotetor em um modelo de cirrose biliar secundária em ratos jovens. MÉTODOS: Foram estudados 40 ratos Wistar desmamados no 21º dia pós-natal. Cada grupo de 10 foi submetido a um dos seguintes quatro tratamentos, até o 49º dia pós-natal, quando foram submetidos a eutanásia: 1) LC - ligadura dupla e ressecção do ducto biliar comum e administração diária de ácido ascórbico [100 mg/g de peso corporal (pc)]; 2) LA - ligadura dupla e ressecção do ducto biliar comum e administração diária de veículo aquoso (1 mL/g pc); 3) SC - operação simulada e administração diária de ácido ascórbico (100 mg/g pc); 4) SA - ligadura dupla e ressecção do ducto biliar comum e administração diária de veículo aquoso (1 mL/g pc). Os ratos eram pesados diariamente. No 27º dia pós-operatório, eles receberam injeção intraperitoneal de 1,5 mg/g pc de pentobarbital sódico, e o tempo de sono induzido pelo pentobarbital foi medido. Coletou-se sangue para determinação de atividade sérica de alanina aminotransferase e de aspartato aminotransferase, níveis de albumina e globulina séricas, e o fígado foi analisado quanto à conteúdo de água e gordura. Os dados foram submetidos à ANOVA two-way, e comparações pareadas entre grupos foram testadas com o método de SNK. O nível de significância foi estabelecido em 0,05. RESULTADOS: A suplementação com ácido ascórbico atenuou os efeitos da colestase: reduziu o tempo de anestesia pelo pentobarbital, globulina sérica e o conteúdo de gordura no fígado. CONCLUSÕES: Nossos resultados corroboram a hipótese de que a suplementação com ácido ascórbico tem um efeito citoprotetor na cirrose biliar secundária.

OBJECTIVE: To test whether ascorbic acid supplementation has any cytoprotective effect on a model of secondary biliary cirrhosis in young rats. METHODS: We studied 40 Wistar rats weaned at the 21st postnatal day. Each group of 10 was subjected to one of the following four treatments, until 49th postnatal day, when they suffered euthanasia: 1) LC-double ligature and resection of the common bile duct and daily administration of ascorbic acid [100 mg/g of body weight (bw)]; 2) LA-double ligature and resection of the common bile duct and daily administration of aqueous vehicle (1 mL/g bw); 3) SC-sham operation and daily administration of ascorbic acid (100 mg/g bw); 4) SA-double ligature and resection of the common bile duct and daily administration of aqueous vehicle (1 mL/g bw). The rats were weighed daily. On the 27th day after the operation they received an intra-peritoneal injection of 1.5 mg/g bw of sodium pentobarbital, and the pentobarbital sleeping time was measured. Blood was collected for serum alanine aminotransferase and aspartate aminotransferase activity measurements, serum albumin and globulin concentrations, and the liver was assessed for liver water and fat content. Data were submitted to two-way ANOVA and paired comparisons between groups were tested using the SNK method. Significance level was set at 0.05. RESULTS: Ascorbic acid supplementation attenuated the effects of cholestasis: decreased the pentobarbital sleeping time, serum globulin, and the liver fat content. CONCLUSIONS: Our results corroborate the hypothesis that ascorbic acid supplementation has a cytoprotective effect in secondary biliary cirrhosis.

Nitric oxide and inducible nitric oxide synthase expression are downregulated in acute cholestasis in the rat accompanied by liver ischemia.

Hepatic blood flow decreases under cholestasis and there is evidence that NO regulates liver microvascular perfusion. Thus, the aim of the present study was to evaluate NO synthesis in cholestasis. Cholestasis was induced by bile-duct ligation (BDL) in male Wistar rats. Bilirubins and enzyme activities were measured in serum. Lipid peroxidation, GSH, GSSG and glycogen were determined in liver. Histopathological analysis was performed. Serum NO2- + NO3- concentration was measured by the Gries reaction. iNOS immunoblot analysis was carried out using an iNOS polyclonal antibody. After 7 days of BDL lipid peroxidation increased while GSH/GSSG ratio decreased. Serum NO2- + NO3- and liver iNOS protein were reduced, accompanied by ischemia as revealed by the histopathological analysis. GSH upregulates NO synthesis by increasing iNOS mRNA levels and iNOS activity, thus the reduction of GSH/GSSG ratio may be responsible for the downregulation of iNOS protein and NO synthesis, which in turn may explain the observed ischemia and the decreased hepatic blood perfusion in cholestasis reported by others.

Enhanced biliary excretion of canalicular membrane enzymes in ethynylestradiol-induced cholestasis. Effects of ursodeoxycholic acid administration.

Cholestasis is associated with a marked increase in the release of canalicular membrane enzymes into bile. This phenomenon has been related to an increased lability of these canalicular membrane integral proteins to the solubilizing effects of secreted bile salts. To further characterize the effects of oral ursodeoxycholic acid (UDCA) administration on ethynylestradiol (EE)-induced cholestasis, the influence of this bile acid on changes in biliary excretion of membrane-bound enzymes was investigated. Bile flow, basal bile salt and biliary lipid secretory rates, the maximum secretory rate of taurocholate (TC SRm), and the biliary excretion of the canalicular membrane-bound ectoenzymes alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGT) were measured in rats after EE and/or UDCA administration. The activities of ALP, GGT and Na+,K(+)-ATPase in purified isolated canalicular and sinusoidal membrane fractions and the ultrastructure of hepatic acinus, including histochemical studies of ALP distribution, were also examined. EE significantly reduced bile flow, bile salt and biliary lipid secretory rates, and TC SRm, and caused dilatation and loss of microvilli at the canalicular pole of hepatocytes. Biliary excretion of ALP increased 2-fold, whereas biliary excretion of GGT was unchanged. The relationship between biliary excretion of ALP or GGT and bile salt secretion (units of enzyme activity secreted per nanomole of bile salt) was greater in EE-treated rats compared with controls (2.1- and 1.5-fold greater for ALP and GGT, respectively), indicating that in EE-induced cholestasis more enzyme was released into bile per nanomole of bile salt. Na+,K(+)-ATPase activity in sinusoidal membrane fraction was reduced significantly, whereas ALP activity increased in both membrane fractions in EE-treated rats. The histochemical distribution of ALP in the acinus showed a strong reaction in acinar zone 3 and at both the canalicular and sinusoidal membranes. Oral administration of UDCA prevented EE-induced bile secretory failure by normalizing bile flow, bile salt and biliary phospholipid secretory rates, and TC SRm. UDCA also prevented the EE-induced changes in the biliary excretion of enzymes. On the contrary, UDCA did not modify either the enzyme activity in isolated membrane fractions or the morphological or ALP histochemical changes associated with EE administration. These data indicate that in EE-induced cholestasis changes occur at the canalicular membrane, enabling this portion of the plasma membrane to be more susceptible to the solubilizing effect of bile salt, and that oral administration of UDCA prevents bile secretory failure and changes in the biliary excretion of ALP and GGT in EE-treated rats.

Liver microsomal phospholipid fatty acids behavior and its relationship to bilirubin UDP-glucuronyltransferase activity in bile duct ligated rats.

Phospholipid fatty acid composition and bilirubin UDP-glucuronyltransferase activity from liver microsomal membrane were studied in normal and in bile duct ligated rats. Incubation of normal microsomes with 15 microM bilirubin (considered as physiological concentration) yielded 60% bilirubin diglucuronide; in 2 days post-cholestatic rats, they showed 20% bilirubin diglucuronide which was undetectable in 8 days post-cholestatic group. When compared to controls, after 2 days of cholestasis, microsomal phospholipids showed a clear decrease in linoleic and arachidonic acids and an increment in palmitic and stearic acids. 8 days post-cholestatic rats presented a marked increase in palmitic, oleic and docosaexaenoic acids, while linoleic and arachidonic acids decreased. Cholestasis produced disturbances in microsomal phospholipids fatty acid composition; but these changes are unable to explain entirely the severe damage observed in bilirubin diglucuronide formation.

Galactosemia como causa de colestasis neonatal prolongada / Prolonged neonatal cholestasis due to galactosemia: a report of four cases

Se presentan cuatro casos de galactosemia clásica cuya manifestación clínica inicial fue colestasis neonatal prolongada, atendidos en el Servicio de Gastroenterología del Instituto Nacional de Pediatría de la Ciudad de México, durante el período comprendido de 1987 a 1990. El diagnóstico definitivo se hizo mediante la medición de la actividad de la enzima galactosa 1-fosfato uridil transferasa en eritrocitos (prueba de Beutler). El manejo de los pacientes consistió básicamente en la restricción de galactosa en la dieta. Dos de los pacientes tuvieron una evolución clínica desfavorable, presentamos uno de ellos secuelas neurológicas importantes y el otro paciente falleció (septicemia)

Caracterización de formas macroenzimáticas de la fosfatasa alcalina: descripción de un nuevo método / A new method for the characterization of alkaline phosphatase macroenzymes

Se describe un nuevo método para la caracterización de macroenzimas de la fosfatasa alcalina. El método combina una separación previa de las formas isoenzimáticas por gel filtración en capa fina de Sephadex G-200, y el posterior revelado de la correspondiente actividad enzimática in situ. Entre otras ventajas, este sistema no sólo separa adecuadamente las formas macroenzimáticas, sino que mantiene todas las isoenzimas en estado nativo, a diferencia de las técnicas con desnaturalizantes, por lo que es posible su detección y eventual aislamiento. Permite además la resolución simultánea de un gran número de muestras, así como la inclusión de controles de distinto peso molecular (PM) dentro de una misma corrida, lo que facilita en forma significativa la posterior interpretación del perfil obtenido, aportando una gran ventaja con respecto a la técnica de gel filtración en columna. El método propuesto se presenta como una técnica relativamente simple y rápida para el screening de macroenzimas en el laboratorio clínico

Caracterización de formas macroenzimáticas de la fosfatasa alcalina: descripción de un nuevo método / A new method for the characterization of alkaline phosphatase macroenzymes

Se describe un nuevo método para la caracterización de macroenzimas de la fosfatasa alcalina. El método combina una separación previa de las formas isoenzimáticas por gel filtración en capa fina de Sephadex G-200, y el posterior revelado de la correspondiente actividad enzimática in situ. Entre otras ventajas, este sistema no sólo separa adecuadamente las formas macroenzimáticas, sino que mantiene todas las isoenzimas en estado nativo, a diferencia de las técnicas con desnaturalizantes, por lo que es posible su detección y eventual aislamiento. Permite además la resolución simultánea de un gran número de muestras, así como la inclusión de controles de distinto peso molecular (PM) dentro de una misma corrida, lo que facilita en forma significativa la posterior interpretación del perfil obtenido, aportando una gran ventaja con respecto a la técnica de gel filtración en columna. El método propuesto se presenta como una técnica relativamente simple y rápida para el screening de macroenzimas en el laboratorio clínico (AU)

Effects of ursodeoxycholic acid on liver function in patients with cystic fibrosis and chronic cholestasis.

Ursodeoxycholic acid, 10 to 20 mg/kg per day, was administered for 1 year to 22 patients with cystic fibrosis and chronic cholestasis, resulting in significantly improved liver enzyme values. However, evidence of cholestasis continued, as shown by the pattern of alkaline phosphatase isoenzymes.

Inhibition of carnitine acyltransferase activities by bile acids in rat liver peroxisomes.

We recently reported that purified carnitine acetyltransferase is competitively inhibited by bile acids (Sekas, G. and Paul, H.S. (1989) Anal. Biochem. 179, 262-267). In the present study, we initially investigated the effect of bile acids on carnitine acyltransferases in rat hepatic peroxisomes. Activities of carnitine acetyltransferase, carnitine octanoyltransferase, and carnitine palmitoyltransferase were progressively inhibited by increasing concentrations of chenodeoxycholic acid. Kinetic studies revealed that the inhibition by chenodeoxycholic acid was competitive with respect to carnitine with an apparent Ki of 890 microM for carnitine acetyltransferase, 650 microM for carnitine octanoyltransferase and 600 microM for carnitine palmitoyltransferase. We then investigated whether bile acids inhibit the activities of these enzymes ex vivo. The hepatic concentration of bile acids was increased by inducing cholestasis by bile duct ligation. Cholestasis reduced the activity of carnitine acetyltransferase, carnitine octanoyltransferase, and carnitine palmitoyltransferase to 66 +/- 2%, 64 +/- 3%, and 40 +/- 2%, of the control, respectively. The inhibition for each of these enzymes was proportional to the degree of cholestasis. The effect of cholestasis appeared specific for carnitine acyltransferases since the activity of catalase, another peroxisomal enzyme, was not affected by cholestasis. We conclude that bile acids inhibit the activities of carnitine acyltransferases in hepatic peroxisomes. This inhibition by bile acids may be of significance in cholestatic liver disease.

[Dyslipidemia in cholestasis]. / Dislipidemia en colestasis.

Lipoprotein-x (Lp-x) was tested by electrophoresis technique and its cuali and quantitative determination was realized on the sera of thirty five patients with cholestasis compared with a control group without the presence of lipoprotein. The results of our study set up that this lipoprotein means an improvement in the cholestasis diagnosis. We point up its importance as a diagnostic proof with the classic determinations.