Changes of activity and isoforms of alkaline sphingomyelinase (nucleotide pyrophosphatase phosphodiesterase 7) in bile from patients undergoing endoscopic retrograde cholangiopancreatography.

BACKGROUND: Alkaline sphingomyelinase (NPP7) is an ecto-enzyme expressed in intestinal mucosa, which hydrolyses sphingomyelin (SM) to ceramide and inactivates platelet activating factor. It is also expressed in human liver and released in the bile. The enzyme may have anti-tumour and anti-inflammatory effects in colon and its levels are decreased in patients with colon cancer and ulcerative colitis. Active NPP7 is translated from a transcript of 1.4 kb, whereas an inactive form from a 1.2 kb mRNA was found in colon and liver cancer cell lines. While the roles of NPP7 in colon cancer have been intensively studied, less is known about the function and implications of NPP7 in the bile. The present study examines the changes of NPP7 in bile of patients with various hepatobiliary diseases. METHODS: Bile samples were obtained at endoscopic retrograde cholangiopancreatography (ERCP) in 59 patients with gallstone, other benign disease, tumour, and primary sclerosing cholangitis (PSC). The NPP7 activity was determined. The appearance of the 1.4 and 1.2 kb products in the bile was examined by Western blot. The results were correlated to the diseases and also plasma bilirubin and alkaline phosphatase. RESULTS: NPP7 activity in the tumour group was significantly lower than in the gallstone group (p < 0.05). The activity in the tumour plus PSC group was also lower than in gallstone plus other benign disease group (p < 0.05). Within the tumour group NPP7 activity was lowest in cholangiocarcinoma patients, being only 19% of that in gallstone patients. Bilirubin correlated inversely to NPP7 and was higher in the tumour than in the gallstone group. Western blot identified both the 1.4 kb and the 1.2 kb products in most bile samples. The density ratio for the 1.4/1.2 kb products correlated to NPP7 activity significantly. Two patients (one PSC and one cholangiocarcinoma) lacking NPP7 activity had only the 1.2 kb form in bile. CONCLUSION: NPP7 activity and the ratio of 1.4/1.2 kb products in bile are significantly decreased in malignancy, particularly in cholangiocarcinoma. The implications of the finding in diagnosis of cholangiocarcinoma and 1.2 kb product in hepatobiliary diseases require further investigation.

Reflujo pancreáticobiliar en pacientes con colelitiasis y sin colelitiasis: estudio comparativo / Pancreaticobiliary reflux in patients with cholelithiasis and without cholelithiasis: a comparative study

Background. Pancreaticobiliary reflux is a pathologic phenomenon occurring in patients with gallstones. However, the occurrence of pancreaticobiliary reflux has not been studied in patients without gallstones. The objective of this study was to measure the bile levels of amylase and lipase in patients without gallstones submitted to cholecystectomy as part of another surgical procedure, and to compare these values with patients submitted to cholecystectomy for gallstone disease. Patients and Methods. A prospective observational comparative study was designed. A sample of 136 consecutive patients was included. Amylase and lipase levels were measured in bile. Normal serum amylase levels at our institution are 28-100 U/L and for lipase are 13-60 U/L. There are no established normal levels for pancreatic enzymes in bile. However, we considered elevated the bile amylase and lipase levels whenever they were higher than normal plasma levels. Results. One-hundred three patients (76 percent) had gallstones and 33 (24 percent) liad healthy gallbladders without gallstones. According to normal plasma levels for amylase and lipase, these enzymes in bile were elevated in 83.5 percent patients with gallstones, compared to elevated levels of amylase in 6 percent patients and lipase in 3 percent patients without gallstones. Conclusions. Pancreaticobiliary reflux is a common phenomenon in patients with gallstones and occurs sporadically in patients without gallstones.

Introducción. El reflujo pancreáticobiliar es un fenómeno patológico que ocurre en pacientes con colelitiasis. La ocurrencia de este fenómeno no ha sido estudiada en pacientes sin colelitiasis. El presente estudio tiene por objetivo medir los niveles de amilasa y lipasa en la bilis de pacientes sin colelitiasis, colecistectomizados como parte de otro procedimiento quirúrgico y comparar estos valores con pacientes colecistectomizados por colelitiasis. Pacientes y Métodos. Se diseñó un estudio observacional y comparativo. Una muestra de 136 pacientes consecutivos fue incluida. Se midieron los niveles de amilasa y lipasa en la bilis. En nuestra institución los valores normales para amilasa son 28-100 U/L y para lipasa 13-60 U/L. No se han establecido valores normales de enzimas pancreáticas en la bilis. Para efectos del presente estudio, se consideró como elevados los niveles biliares de amilasa y lipasa cuando fueron mayores a los valores plasmáticos normales. Resultados. 103 pacientes (76 por ciento) tenían colelitiasis y 33 (24 por ciento) tenían vesículas normales sin cálculos. De acuerdo a los valores plasmáticos normales de amilasa y lipasa, estas enzimas se encontraron elevadas en 83,5 por ciento de los pacientes con colelitiasis comparados con valores elevados de amilasa en 6 por ciento en pacientes sin colelitiasis y de lipasa en 3 por ciento de estos pacientes. Conclusiones. El reflujo pancreaticobiliar es un fenómeno común en pacientes con colelitiasis y ocurre esporádicamente en pacientes sin colelitiasis.

Visfatin--a proinflammatory adipokine-in gallstone disease.

BACKGROUND: Visfatin is increasingly associated with several obesity-related diseases. The study is to evaluate if aberrant expression of circulating visfatin occurs in gallstone disease. METHODS: An enzyme-linked immunosorbent assay was used to examine serum visfatin levels in 79 patients with cholesterol gallstones, 71 with pigment gallstones, and 223 healthy controls. The chemical composition of extracted gallstones was determined by Fourier transform infrared spectroscopy. RESULTS: Serum visfatin levels were markedly elevated in the cholesterol and pigment gallstones in comparison with healthy controls. Furthermore, increased visfatin levels were associated with formation of the cholesterol and pigment gallstones. Intriguingly, a significant positive correlation between serum visfation levels and white cell count was noted in the cholesterol gallstones and controls. Moreover, the positive correlation in the cholesterol gallstones was more significant in the body mass index >/=25 subgroup than in the body mass index <25 subgroup. CONCLUSIONS: Gallstone disease is associated with altered circulating visfatin. The proinflammatory effect of circulating visfatin in gallstone disease deserves further investigation.

Gilbert syndrome as a predisposing factor for cholelithiasis risk in the Greek adult population.

We investigated the hypothesis that coinheritance of the common A(TA)(n)TAA promoter mutation at the UGT1A1 locus associated with Gilbert syndrome is a risk factor for gallstone formation in a homogeneous adult population, by conducting a case-control study that included 198 adult patients with cholelithiasis and 152 healthy controls both of Greek origin. Three genotypes were found: 7/7 (17.8% in controls and 23.3% in patients), 6/7 (33.5% in controls and 46.5% in patients), and normal homozygous 6/6 (48.7% in controls and 30.3% in patients). The Gilbert UGT1A1 genotypes 6/7 and 7/7 show significant association (odds ratio 2.225, 95% confidence interval 1.373-3.605, p=0.001, and odds ratio 2.101, 95% confidence interval 1.171-3.770, p=0.013, respectively) with cholelithiasis risk. This association supports the theory that genetic factors are responsible for a fraction of symptomatic gallstone disease; however, further studies are required in different ethnic groups to fully elucidate the involvement of Gilbert syndrome in gallstone disease.

Cholelithiasis in thalassemia major.

OBJECTIVES: Aim of this study was to evaluate prevalence and characteristics of cholelithiasis in a large population of patients with thalassemia major (TM). METHODS: Data from 858 consecutive patients with transfusion-dependent thalassemia at five major Italian centers were analyzed. In these centers, a complete abdomen ultrasonography is performed yearly after the beginning of the transfusion regimen. The role of co-inheriting Gilbert's syndrome genotype was investigated studying the promoter region of the UGT1-A1 gene by automated sequencing. RESULTS: Thirty percent of TM patients had gallstones. The Gilbert's genotype [homozygosity for (TA)(7) motif at UGT1A promoter gene], influenced both the prevalence of cholelithiasis and the age at which it developed. CONCLUSIONS: Cholelithiasis has a remarkable frequency and precocity in patients with TM and especially in those with (TA)(7)/(TA)(7) UGT1-A1 genotype. An early biliary ultrasonography is recommended from childhood and a closer follow-up in patients with thalassemia and associated Gilbert's syndrome may be indicated.

Gilbert's syndrome as a predisposing factor for idiopathic cholelithiasis in children.

The frequency of the (TA)7/(TA)7 promoter genotype of UDP-glucuronosyltransferase gene (UGT1A1) was significantly higher (p<0.05) in a group of 30 children with cholelithiasis than in a control group of 40 healthy children, indicating that this genotype might be an underlying factor for gallstone initiation in otherwise healthy children.

Human cholesterol 7alpha-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype.

Bile acid synthesis plays a critical role in the maintenance of mammalian cholesterol homeostasis. The CYP7A1 gene encodes the enzyme cholesterol 7alpha-hydroxylase, which catalyzes the initial step in cholesterol catabolism and bile acid synthesis. We report here a new metabolic disorder presenting with hyperlipidemia caused by a homozygous deletion mutation in CYP7A1. The mutation leads to a frameshift (L413fsX414) that results in loss of the active site and enzyme function. High levels of LDL cholesterol were seen in three homozygous subjects. Analysis of a liver biopsy and stool from one of these subjects revealed double the normal hepatic cholesterol content, a markedly deficient rate of bile acid excretion, and evidence for upregulation of the alternative bile acid pathway. Two male subjects studied had hypertriglyceridemia and premature gallstone disease, and their LDL cholesterol levels were noticeably resistant to 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. One subject also had premature coronary and peripheral vascular disease. Study of the kindred, which is of English and Celtic background, revealed that individuals heterozygous for the mutation are also hyperlipidemic, indicating that this is a codominant disorder.

Concomitant laparoscopic splenectomy and cholecystectomy as an effective and minimally invasive treatment of pyruvate kinase deficiency with gallstones.

A 4-year-old girl with pyruvate kinase deficiency underwent partial splenic embolization initially. However, even after this procedure, she still had to be transfused every 2 months and then every month. At 5 years of age, she was admitted to our hospital to undergo splenectomy. She underwent laparoscopic splenectomy and concomitant cholecystectomy for gallstones. The hemogram recovered to the normal range after surgery, and her postoperative course was uneventful. Considering the absence of morbidity, the short hospitalization, the quick return to normal activity, the good cosmetic result, and the improved clinical and hematologic results, we consider that simultaneous laparoscopic splenectomy and cholecystectomy is safe and effective for the management of hemolytic anemia resulting from pyruvatre kinase deficiency and associated with cholelithiasis.

Detection of telomerase reverse transcriptase mRNA in biopsy specimens and bile for diagnosis of biliary tract cancers.

Human telomerase reverse transcriptase (hTERT), which codes for the catalytic subunit of telomerase, is essential for telomerase activity. Recent studies revealed that levels of hTERT mRNA as well as telomerase activity are high in neoplasm. The purpose of this study was to correlate the expression of hTERT mRNA with telomerase activity in biopsy specimens and bile from biliary tract cancers and to evaluate the potential diagnostic value of hTERT mRNA analysis for biliary malignancy. We analyzed hTERT mRNA and telomerase activity in biopsy specimens and exfoliated bile cells from patients with cholangiocarcinoma, gallbladder carcinoma and bile duct stones. hTERT was detected by either nested reverse transcriptase-polymerase chain reaction (PCR) or real-time PCR. Telomerase activity was examined by a fluorescence-based telomeric repeat amplification protocol assay. Six of 10 malignant biopsy specimens had detectable hTERT and 7 of 10 had telomerase activity. All cases with hTERT expression had telomerase activity. In bile, 7 of 10 malignant patients had detectable hTERT and 3 of 10 had telomerase activity. Importantly, there were no false positive results in tissue specimens or bile examined in 6 non-cancerous cases. In conclusion, the detection of hTERT mRNA in biopsy specimens and bile cells, in combination with routine histologic and cytologic examination may improve the diagnosis of biliary tract cancers.

Prediction of bile duct stones and complications in gallstone pancreatitis using early laboratory trends.

OBJECTIVES: We aimed to determine whether early trends in the serum pancreatic enzymes and liver tests of patients with gallstone pancreatitis predict persistent common bile duct (CBD) stones and complications. METHODS: Medical records of patients with gallstone pancreatitis were reviewed retrospectively. Serial serum pancreatic enzymes and liver tests were recorded until the time of cholangiography. Laboratory trends were analyzed by comparing initial results obtained in the emergency department to subsequent results obtained 8-24 h, 24-48 h, and 48-72 h after presentation. RESULTS: Of 154 patients with gallstone pancreatitis, 28 (18%) had persistent CBD stones at cholangiography. Complications and death were more frequent in patients with persistent CBD stones than in those without CBD stones (29% and 11% vs 12% and 1%, respectively; p < 0.05). Laboratory trends predicted both persistent CBD stones and complications of pancreatitis. When any laboratory value rose between admission and 24-48 h of hospitalization, persistent CBD stones were present in 31% of cases, versus 8% of those in whom all laboratory values remained constant or fell (p = 0.001). Likewise, complications occurred in 21% of those with any rising laboratory value, versus 8% of those in whom all values remained constant or fell (p < 0.05). CONCLUSIONS: Patients with gallstone pancreatitis and rising serum chemistries had a 4-fold risk of persistent CBD stones and a nearly 3-fold risk of complications compared to patients in whom all chemistry values remained constant or fell. This simple prediction rule may identify patients with biliary pancreatitis who are most likely to benefit from early interventions to diagnose and remove persistent CBD stones.

Pigment gallstone pathogenesis: slime production by biliary bacteria is more important than beta-glucuronidase production.

Pigment stones are thought to form as a result of deconjugation of bilirubin by bacterial beta-glucuronidase, which results in precipitation of calcium bilirubinate. Calcium bilirubinate is then aggregated into stones by an anionic glycoprotein. Slime (glycocalyx), an anionic glycoprotein produced by bacteria causing foreign body infections, has been implicated in the formation of the precipitate that blocks biliary stents. We previously showed that bacteria are present within the pigment portions of gallstones and postulated a bacterial role in pigment stone formation through beta-glucuronidase or slime production. Ninety-one biliary bacterial isolates from 61 patients and 12 control stool organisms were tested for their production of beta-glucuronidase and slime. The average slime production was 42 for biliary bacteria and 2.5 for stool bacteria (P <0.001). Overall, 73% of biliary bacteria and 8% of stool bacteria produced slime (optical density >3). In contrast, only 38% of biliary bacteria produced beta-glucuronidase. Eighty-two percent of all patients, 90% of patients with common bile duct (CBD) stones, 100% of patients with primary CBD stones, and 93% of patients with biliary tubes had one or more bacterial species in their stones that produced slime. By comparison, only 47% of all patients, 60% of patients with CBD stones, 62% of patients with primary CBD stones, and 50% of patients with biliary tubes had one or more bacteria that produced beta-glucuronidase. Most biliary bacteria produced slime, and slime production correlated better than beta-glucuronidase production did with stone formation and the presence of biliary tubes or stents. Patients with primary CBD stones and biliary tubes had the highest incidence of slime production. These findings suggest that bacterial slime is important in gallstone formation and the blockage of biliary tubes.

Mast cell subpopulations in chronic inflammatory hepatobiliary diseases.

AIM/BACKGROUND: In various hepatobiliary diseases mast cells have been found to be associated with fibrogenesis. However, mast cell subpopulations have not been investigated in the human liver in normal subjects or in disease. Human mast cells are categorized into mast cells positive for tryptase (MC(T)) only and mast cells positive for both tryptase and chymase (MC(TC)). METHODS: In this study we investigated mast cell subpopulations (MC(T) and MC(TC)) by double immunostaining for mast cell tryptase and chymase as well as by a computer-aided quantitative morphometry in 13 normal livers and in 193 liver tissue specimens comprising of primary biliary cirrhosis (n=43), autoimmune hepatitis (n=11), chronic hepatitis B (n=37), chronic hepatitis C (n=41), alcoholic liver disease (n=40) and hepatolithiasis (n=21). RESULTS: The densities of MC(T) and MC(TC) per 1 mm2 stroma were low in normal livers but high in chronic liver diseases, and correlated positively with the degree of fibrosis. The percentages of MC(T) and MC(TC) subpopulations were 25% and 75%, respectively. The percentage was almost the same in normal livers and various hepatobliliary diseases, as well as between less fibrotic cases and more fibrotic cases in liver diseases. CONCLUSIONS: These results suggest that MC(T) and MC(TC) subpopulations in healthy and diseased livers do not change during liver fibrosis of any etiology.

[Changes of lipoprotein lipase and hepatic lipase and their significance in gallstone formation in rabbit model].

This experiment was made to investigate the changes of lipoprotein lipase(LPL) and hepatic lipase (HL) activity and their effects on gallstone formation in rabbit model in which the stones were induced by high cholesterol diet. Activities of plasma LPL and HL were determined; other data including concentration of plasma lipoprotein cholesterol, concentration of bile cholesterol and bile acids were also obtained. The results showed that with the rabbits continuously fed on high cholesterol diet, LPL activity heightened markedly (P < 0.05), and HL activity increased gradually (3 and 4 weeks groups vs control group, P < 0.05). The changes of concentration of plasma VLDL-C and LDL-C were the same as that of LPL activity, but the concentration of plasma HDL-C, HDL2-C, HDL3-C and bile acids showed no significant changes (P > 0.05). These results suggest that the heightened activities of LPL and HL might make the liver take up more cholesterol, secrete it into the bile duct and hence accelerate gallstone formation.

Does laparoscopic cholecystectomy influence peri-sinusoidal cell activity?

BACKGROUND/AIMS: To investigate the influence of laparoscopic procedures on perisinusoidal cell function. METHODOLOGY: In 31 patients who underwent laparoscopic cholecystectomy for symptomatic cholelithiasis, the serum levels of beta-N-acetyl hexosaminidase (beta-NAH) and hyaluronic acid (HA) were measured. Six female patients, who underwent other laparoscopic procedures, not involving the hepatobiliary system, served as controls. RESULTS: HA serum levels increased significantly in both study and control groups, while beta-NAH serum levels remained within normal values. Post-operative AST and ALT serum levels increased significantly only in the study group. No elevation of serum ALP was noted in any of our patients, and post-operative bilirubin levels did not increase in patients with normal pre-operative levels. CONCLUSIONS: Laparoscopic procedures caused detectable damage to Kupffer and endothelial cells as reflected by elevation of post-operative HA serum levels. The damage to the liver hepatocytes and perisinusoidal cells has no clinical significance and the laparoscopic procedure itself is the probable cause of the Kupffer and endothelial cells damage, while other mechanisms caused damage to the hepatocytes.

Usefulness of alanine and aspartate aminotransferases in the diagnosis of microlithiasis in idiopathic acute pancreatitis.

CONCLUSION: Serum increases of aminotransferases, especially alanine aminotransferase (ALT), were suggestive of microlithiasis in idiopathic acute pancreatitis, particularly when assessed early after the onset of abdominal pain. BACKGROUND: It has been shown that biochemical laboratory values only are useful parameters in distinguishing gallstone from nongallstone acute pancreatitis. We assessed the diagnostic usefulness of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for identification of occult microlithiasis in idiopathic acute pancreatitis. METHODS: Ninety-one patients with idiopathic acute pancreatitis who underwent microscopic examination of stimulated duodenal bile sediments were retrospectively studied. According to earliness of ALT and AST assay after the onset of abdominal pain, patients were divided into two groups: group A, within the first 24 h (n = 56) and group B, between 24 and 72 h (n = 35). RESULTS: ALT and AST values expressed as number of elevations of the upper limits of normal were higher in group A patients with positive biliary drainage than in group B. Median (range) ALT and AST values were 2.5 (0.1-18.1) vs 0.4 (0.1-8.6) and 3 (0.3-17.4) vs 0.5 (0.3-11.9), respectively. In the univariate analysis and receiver operating characteristic (ROC) curves, ALT within the first 24 h showed a sensitivity of 73%, specificity of 86%, and positive predictive value of 92% for a cutoff of 1.2 elevations of the upper limit of normal. These values were slightly higher, although without statistically significant differences, than those of AST (73, 80, and 89%, respectively).

The level of 7-dehydrocholesterol in plasma reflects the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the human liver.

Plasma levels of the cholesterol precursor 7-dehydrocholesterol (7-DHC) were compared with activities of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase assayed in liver biopsies from patients undergoing cholecystectomy. Some patients were treated with cholestyramine, deoxycholic acid or chenodeoxycholic acid prior to surgery in order to alter the activity of the enzyme. The median level of 7-DHC and the activity of HMG-CoA reductase in the untreated group were 55 ng/ml and 98 pmol/min/mg protein, respectively. The sterol levels and enzyme activities were increased in patients treated with cholestyramine (85 ng/ml and 439 pmol/min/mg protein) and deoxycholic acid (86 ng/ml and 173 pmol/min/mg protein) and decreased in patients treated with chenodeoxycholic acid (38 ng/ml and 51 pmol/min/mg protein). There was a strong positive correlation (rs=0.75, P<0.0005) between the plasma levels of 7-DHC and the activities of hepatic HMG-CoA reductase in these patients. This correlation was further improved when the plasma levels of 7-DHC were expressed relative to those of cholesterol (rs=0.90, P<0.0001). The results show that the level of 7-DHC in plasma reflects the activity of HMG-CoA reductase in the liver.

[Alpha-L-fucosidase activity in patients with ductal cholelithiasis]. / Alfa-L-fukozydaza u chorych z przewodowymi postaciami kamicy zólciowej.

Alpha-L-fucosidase (ALF), AST, ALT and GGT activities were measured in blood serum of 36 patients with recurrent cholelithiasis (group I), 32 patients with ductal and/or bladder cholelithiasis (group II), 24 patients with focal changes in the liver (group III) and in 22 patients without disturbances of gastrointestinal tract (control group). A statistically significant increase in ALF activity was found in the patients with recurrent cholelithiasis as compared to the control group (p < 0.001), the patients of group II (p < 0.001) and the patients of group III (p < 0.05). The AST and ALT activities were higher both in group I and in group II than in the control group (p < 0.001 and p < 0.02 for group I and group II, respectively), whereas the mean GGT activity was significantly higher in all three patients groups as compared to the control group (p < 0.001 in all cases). On the basis of the observed differences in the activities of the enzymes studied it was postulated that the determination od ALF activity in blood serum of patients with cholelithiasis may provide the means for early diagnosis of the predisposition to recurrent stones.