Effects of simvastatin on hepatic cholesterol metabolism, bile lithogenicity and bile acid hydrophobicity in patients with gallstones.

BACKGROUND AND AIMS: There is limited information available on the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on hepatic and biliary cholesterol metabolism in patients with gallstones. The aims of this study were to determine the effect of simvastatin on the regulatory elements of cholesterol metabolism that determine the concentrations of cholesterol in plasma and bile. METHODS: Thirty-one gallstone patients were enrolled in the study; 17 were treated with 20 mg simvastatin daily for 3 weeks prior to cholecystectomy and 14 served as controls. Samples of blood, liver, gall-bladder bile and bile from the common bile duct (CBD) were collected and analysed. RESULTS: The plasma cholesterol (-30%), triacylglycerol (-23%) and low-density lipoprotein (LDL) cholesterol (-42%) concentrations were significantly lowered by simvastatin treatment, as was the plasma lathosterol: cholesterol (-70%), which reflects whole-body cholesterol synthesis. Despite these changes, the hepatic LDL receptor protein and LDL receptor activity in circulating mononuclear cells were similar in both groups. There were no differences in the plasma phytosterol: cholesterol, which reflects the intestinal cholesterol absorption capacity or in the activity of hepatic acyl-coenzyme A: cholesterol acyltransferase. There were however, lower cholesterol concentrations in CBD (-68%) and gall bladder (-41%) bile, and decreased lithogenic (-47%) and bile acid hydrophobicity (-22%) indices of CBD bile in the simvastatin group. CONCLUSIONS: These data indicate that simvastatin reduced plasma and biliary cholesterol levels primarily by reducing cholesterol synthesis. The reduction in CBD bile lithogenicity and bile acid hydrophobicity by simvastatin suggests that this agent may be useful for people who have early stages of cholesterol gallstone development and in whom a choleretic effect is required.

Morphology of sporadic colorectal cancer with DNA replication errors.

BACKGROUND: Up to 15% of colorectal cancers are characterised by DNA microsatellite instability (MIN), shown by the presence of DNA replication errors (RERs). AIMS: To identify pathological features that are discriminating for colorectal cancer (CRC) showing extensive MIN. SUBJECTS: A prospective series of 303 patients with CRC and no family history of either familial adenomatous polyposis or hereditary non-polyposis colorectal cancer. METHODS: DNA was extracted from fresh tissue samples and the presence of MIN was studied at nine loci that included TGF beta RII, IGFIIR, and BAX. The 61 cases showing RERs were compared with 63 RER negative cases with respect to a comprehensive set of clinical and pathological variables. Predictive utility of the variables was tested by decision tree analysis. RESULTS: Twenty seven patients with CRC showed extensive RERs (three loci or more) (RER+) and 34 had limited RERs only (28 = one locus; 6 = two loci) (RER+/-), yielding a bimodal distribution. RER+ cancers differed from RER- and RER+/-) cases. Tumour type (adenocarcinoma, mucinous carcinoma, and undifferentiated carcinoma) (p = 0.001), tumour infiltrating lymphocytes (p = 0.001), and anatomical site (p = 0.001) were the most significant of the discriminating variables. Algorithms developed by decision tree analysis allowed cases to be assigned to RER+ versus RER- and +/- status with a global sensitivity of 81.5%, specificity of 96%, and overall accuracy of 93%. CONCLUSIONS: Pathological examination of CRC allows assignment of RER+ status; assignment is specific and relatively sensitive. Conversely RER- and RER+/- CRC are indistinguishable.

Diagnostic value of p53 immunohistochemistry in Barrett's esophagus: an endoscopic study.

The aim of this study was to establish the diagnostic utility of p53 immunohistochemistry in Barrett's esophagus. Three pathologists reviewed endoscopic biopsy specimens and one surgical specimen derived from 102 subjects in a prospective follow-up series. Dysplasia was graded as negative, indefinite, low grade and high grade. p53 staining was assessed as negative, weak or patchy and strong. Kappa coefficients for interobserver agreement for the three combinations of observer pairs were moderate to substantial (0.48, 0.55 and 0.68) for dysplasia and substantial to near perfect (0.77, 0.82 and 0.89) for p53 immunostaining. In the consensus grading achieved for dysplasia, strong p53 staining was recorded in 0/79 samples that were negative or indefinite for dysplasia, 5/15 (33%) examples of low grade dysplasia and 7/8 (87%) examples of high grade dysplasia. Non-dysplastic p53 positive glands were seen in specimens from two subjects harbouring dysplasia or cancer elsewhere. Two p53 positive specimens were, upon review and discussion, re-assigned from low- to high-grade dysplasia. It is concluded that p53 immunohistochemistry facilitates the interpretation of Barrett's epithelium but need only be employed to confirm a suspected diagnosis of dysplasia and assist with the distinction between low- and high-grade dysplasia.

Additional techniques adopted for major vascular anastomoses during orthotopic liver transplantation.

Of 372 patients who underwent liver transplants between January 1985 and March 1995, 7 required variations in vascular anastomoses due to discrepancies in the size-match of the donor and recipient vessels, the presence of a thrombosed hepatic artery or portal vein, or complete absence of the portal vein. The techniques described herein enabled us to perform successful transplantation in all patients. Although rethrombosis of the portal vein developed in one patient after a third transplant, this patient remains clinically well. The use of an operating microscope and the harvesting of an extended length of donor superior mesenteric vein or vascular grafts of the donor iliac or saphenous vessels, for potentially difficult transplants, are invaluable techniques.

A technique for the culture of Barrett's oesophageal cells.

Establishment of cells in tissue culture from Barrett's columnar epithelium has been difficult. The aim of this study was to develop a successful tissue culture method employing a serum-free medium for cultivation of Barrett's epithelial cells. Fragments of Barrett's mucosal tissue were explanted in a 3:1 mixture of Dulbecco's modification of Eagle's medium and Ham's F12, to initiate the outgrowth of epithelial cells. Subsequently, a commercial serum-free medium (formulated for the growth of keratinocytes) was used for the propagation of Barrett's oesophagus cells without fibroblast growth. Cells established in culture retained their epithelial morphology, stained positive for cytokeratin, and contained Alcian blue (pH 2.5) and periodic acid-Schiff reagent-positive/diastase-resistant vacuoles, confirming their origin from Barrett's epithelium. Electron microscopy showed tonofilaments, microvilli and desmosomes. Coating the surface of culture vessels was not required and four cell strains could be passaged up to 20 times with no fibroblast growth, in the keratinocyte serum-free medium.

Endotoxin levels in donors and recipients during orthotopic liver transplantation.

BACKGROUND: The hypothesis being tested in this paper is that endotoxin levels in donors and in recipients during liver transplantation influences postoperative outcome. METHODS: Endotoxin levels in systemic venous and portal venous blood were measured using in 46 adult donors and 44 adult recipients (47 liver transplants) during the period 1992-95. Endotoxin was measured using a modification of the Limulus amoebocyte lysate (LAL) assay. RESULTS: In the donor, systemic endotoxin levels were above normal levels at 10.0 +/- 1.3 pg/mL from the start and rose to 15.8 +/- 2.9 pg/mL after dissection of the hilar structures, but fell to 10.6 +/- 0.8 pg/mL just prior to the removal of the liver (control = 7.8 pg/mL). The mean portal venous endotoxin levels were 18.2 +/- 3.4 pg/mL after dissection of the hilar structures and 12.6 +/- 0.9 pg/mL after cannulation of the portal vein. In the recipients, the highest level in the portal venous blood occurred at the end of the anhepatic phase (46.5 +/- 6.7 pg/mL). The systemic venous samples in the recipients were elevated to start with, but fell rapidly to 19.3 +/- 1.5 pg/mL 24 h postoperatively, and to 13.2 +/- 1.0 pg/mL by day 7. The endotoxin concentrations were higher in recipients who developed complications. CONCLUSIONS: Endotoxin is elevated throughout the recipient transplantation procedure and up to 7 days postoperatively. High levels of endotoxin at induction, the anhepatic phase and at certain time points correlated with patients who developed postoperative complications.

Use of the donor iliac vein to replace the retrohepatic vena cava in pediatric reduced-size liver retransplantation.

The authors describe a new technique that used the donor common iliac vein and its bifurcation into the external iliac and internal iliac veins to replace the retrohepatic vena cava; this was used in a recipient who underwent her second reduced-size transplantation (segments II and III). Anastomosis of the donor hepatic vein to the internal iliac vein, with use of this segment of the venous graft to replace the retrohepatic vena cava, is for patients who have had more than one surgical procedure before liver transplantation.

Liver transplantation in the presence of situs inversus totalis: application of reduced-size graft.

Because of the anatomical features associated with situs inversus, technical difficulties will be encountered during orthotopic liver transplantation. This report describes the case of a patient with situs inversus totalis and end-stage liver disease from biliary atresia who was treated by segmental orthotopic liver transplantation. The segmental graft was safely placed in the left subphrenic space, and a suitable orientation was obtained for anastomoses of the hilar vessels. Chronic rejection necessitated retransplantation, by the same method, 19 months later. This technique has potential advantages in coping with anatomical obstacles encountered in patients with situs inversus.

Endotoxin levels in adult liver donors.

This study was undertaken to determine the levels of endotoxin in a group of adult donors whose livers were procured for transplantation. In the group of 25 adults, endotoxin levels were found to be significantly elevated in the systemic venous blood when compared to control levels. Portal venous endotoxin levels were also elevated following hepatic hilar dissection and after cannulation of the portal vein prior to removing the donor liver.

Identification of human biliary alpha 1-acid glycoprotein as a cholesterol crystallization promoter.

BACKGROUND/AIMS: We have recently outlined the biochemical features of a human 42-kilodalton biliary glycoprotein that shows concentration-dependent cholesterol crystallization-promoting activity. The goal in this work was to establish its identity and to examine some aspects of its biochemical properties relative to its activity. METHODS: Internal amino acid sequencing following tryptic digestion was performed. Based upon this result, immunoreactivity against the 42-kilodalton glycoprotein was examined using a relevant antibody. With the same antibody, the 42-kilodalton glycoprotein was isolated from bile and assayed for activity. Sequential enzymatic deglycosylation of successive terminal glycans of the purified glycoprotein was performed, and the effects on both reductions in molecular radius (M(r)) and on comparative promoter activities were examined. RESULTS: Both amino acid sequence and immunochemical data identify the 42-kilodalton glycoprotein as a biliary form of alpha 1-acid glycoprotein. When purified by immunoaffinity chromatography, potent promoting activity shown was proportionately reduced by successive removal of terminal glycans that also reduced the M(r)s. CONCLUSIONS: The 42-kilodalton cholesterol crystallization-promoting glycoprotein is now identified as a biliary form of alpha 1-acid glycoprotein. Further, some aspects of the important role of glycans in this extensively glycosylated protein have been explored.

Single dose ceftriaxone as prophylaxis for sepsis in colorectal surgery.

During the period May 1986 to July 1989, a prospective, double blind, randomized trial of antibiotic prophylaxis in colorectal surgery was undertaken at the Royal Brisbane Hospital. Three hundred and thirty patients were considered eligible for the trial. Three regimens were compared: a combination of 2 g ceftriaxone and 1 g metronidazole; a single dose of 2 g ceftriaxone; or 1 g cefazolin and 1 g metronidazole, as antibacterial prophylaxis in colorectal surgery. Fifty patients were excluded from analysis. The overall incidence of wound sepsis was 7.9% (22 patients). There was no statistical difference in the incidence of wound infections between the three groups. The presence of drains and the non-performance of a bowel anastomosis at the time of surgery predisposed patients to wound infection. Staphylococcus aureus or Staphylococcus epidermidis were the cause of wound infection in 16 cases. Patients in the cefazolin and metronidazole group had a significantly higher number of postoperative urinary tract and respiratory tract infections than the other two groups combined (P less than 0.01). There did not appear to be any change in sensitivity patterns to ceftriaxone during the 3 year trial. During the 3 year period of the study, ceftriaxone was found to be a safe and effective drug in antibacterial prophylaxis in colorectal surgery.

Inhibition and promotion of cholesterol crystallization by protein fractions from normal human gallbladder bile.

Pooled, normal human gallbladder biles were initially separated on a molecular sieving chromatography column to remove soluble mucin glycoproteins as well as high molecular weight proteins (greater than 200,000). The remaining lower molecular weight proteins and other bile components were then examined by lectin affinity chromatography with four different types of lectin. The separated bound fractions were compared for inhibiting and promoting activities with a newly devised sensitive cholesterol crystal growth assay and for differences in electrophoretic patterns on SDS-gels. Protein factors (presumably glycoproteins) were found to have both inhibiting and promoting activities, even in the absence of cholesterol gallstone disease. The promoting effect was indicated by shortened crystal detection times and increases in crystal growth rate; whereas the inhibiting effect was indicated by decreases in crystal growth rate and reductions in the final crystal concentration as determined by the growth assay. Affinity chromatography mitigated the major problems of removing both lipids and pigment from the glycoproteins. In addition, partial purification of bound fractions with potent cholesterol crystal nucleation-altering activity can be obtained by this technique.

Hepatic acyl-coenzyme A:cholesterol acyltransferase activity is decreased in patients with cholesterol gallstones.

Altered hepatic cholesterol metabolism has been implicated in the etiology of cholesterol gallstones. This hypothesis has been examined by determining acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in liver biopsies from 31 cholesterol gallstone patients and 12 control subjects. Hepatic ACAT activity in gallstone patients was decreased to one-third that in controls (P less than 0.001). No differences in hepatic homogenate or microsomal free and total cholesterol concentrations were observed between the two groups. However, marked increases in free (107%) and total (98%) cholesterol concentrations were found in the cytosolic fraction of liver biopsies from gallstone patients. The total phospholipid concentration of the liver homogenate fraction was unchanged in both groups; however, the microsomal total phospholipid concentration was reduced by 17% (P less than 0.01) in gallstone samples compared with controls. This difference did not result in a significantly increased microsomal cholesterol/phospholipid ratio for the gallstone group (0.180 +/- 0.030) compared with the control group (0.169 +/- 0.042). These results show that hepatic ACAT activity is significantly decreased in cholesterol gallstone patients. These changes in ACAT activity in livers of patients with cholesterol gallstones are consistent with the known increase in the amount of free cholesterol secreted in the bile of these patients. Thus, the changes in ACAT activity may contribute to the pathogenesis of cholesterol gallstones.

Effect of ischaemia on the activities of human hepatic acyl-CoA:cholesterol acyltransferase and other microsomal enzymes.

The effect of ex vivo ischaemia at 37 degrees C on the activities of human hepatic acyl-CoA:cholesterol acyltransferase (ACAT), acyl-CoA hydrolase and carboxylesterase and on the microsomal cholesterol and total phospholipid concentrations was determined in liver tissue from two patients. ACAT activity decreased exponentially with ischaemia, corresponding to half-lives of 59 and 54 min in the two samples. Acyl-CoA hydrolase activity of microsomes was not affected by ischaemia, whereas carboxylesterase activity of microsomes decreased with a corresponding increase in cytosolic carboxylesterase activity. No changes in microsomal cholesterol and total phospholipid concentrations corresponding to the changes in ACAT or carboxylesterase activity were observed. ACAT activity was also determined in microsomes prepared from twenty human liver samples obtained at surgery with in vivo warm ischaemia times ranging from 5-120 min. The data obtained agree well with the ex vivo results, showing a half-life of 57 min for the loss of ACAT activity. Therefore, in comparing ACAT activities in liver samples with different ischaemia times, an appropriate correction must be made for warm ischaemia time.

Oesophageal ruptures and perforations--a review.

Oesophageal ruptures or tears carry a high mortality if they are not recognized and if therapy is delayed. This is so particularly for cases of spontaneous rupture of the oesophagus which carry a higher mortality and morbidity than do cases of iatrogenic injuries. With the widespread use of fibreoptic oesophagogastroscopy, which has been accompanied by the therapeutic manipulation of strictures and tumours, the number of iatrogenic perforations has increased substantially. We report our experience with 23 oesophageal perforations or ruptures that were seen over a 15-year period. The results of both the surgical and the conservative management of such lesions were excellent and were based on the clinical condition of the patient and on the extent of the extravasation of contrast media. Our results show that not all cases of oesophageal perforations require immediate surgical exploration and that the results of surgical treatment are excellent if the diagnosis is made early.