A novel stop codon mutation of the ferrochelatase gene in bovine protoporphyria, a natural animal model of the human disease

April 1998

AASLD A1265

biopsies. Methods: Five patients with coagulopathy due to cirrhosis, regardless of etiology, underwent laparoscopic LB. Ten minutes before insertion of the Veress needle, a single dose of rVIla was given intravenously. LB was then performed with a 16-gauge biopsy needle. Under direct visualization, the time to hemostasis was determined after performance of the LB. The PT response, duration of PT response and hematological safety parameters were assessed. A rescue dose of rVIIa was prepared for each case. Patients were monitored for 23 hours after laparoscopy. Patients and Results: Five patients were included, 3 Child's B and 2 Child's C cirrhotics. The etiology of cirrhosis was: alcoholic cirrhosis (3); autoimmune hepatitis (1); and hepatitis C (1). The male: female ratio was 4:1. Mean age was 47 years (Range 37-55) All patients had prothrombin times > 9 seconds above control. The pre-dose range of prothrombin time was 21.7-31.7 sec. (Control 13.2 see) The mean pre-dose platelet count was 139,600 luL. Patient

Time to Liver Hemostasis (minutes) 10 7 7 7 10

Predose PT (see) 31.7 25 21.7 21.8 23.5

PT 10 min. post rVIIa (sec) 16.7 15.6 12.7 14.5 16.4

PT 30 min. post rVIIa (sec) 21.7 16.1 14.4 15.9 21.8

All patients tolerated laparoscopic LB without complication. No bleeding was seen from either trochar site. The rescue dose of rFVlla was not utilized in any patient. No significant changes in hematocrit or in coagulation parameters were seen at the end of the 23-hour observation period. Conclusion: Laparoscopic LB can be safely performed in severely coagulopathic patients utilizing a single, low dose of rFVIIa. Further studies to evaluate efficacy are ongoing. This research was funded by NovoNordisk, Copenhagen, DK. • L0276 ROUTINE ANALYSIS OF ASCITIC FLUID AT THE TIME OF LARGE VOLUME PARACENTESIS IN ASYMPTOMATIC OUTPATIENTS IS UNNECESSARY. MA Jeffries, MA Stem, NT Gunaratnum, and RJ Fontana Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan. Background: The prevalence of spontaneous bacterial peritonitis(SBP) in cirrhotic patients requiring hospitalization may be as high as 27% with an associated mortality of 30%. The prevalence of SBP in asymptomatic outpatients undergoing therapeutic large volume paracentesis(LVP) however, remains unknown. Therefore, the need for routine ascitic fluid analysis in this setting and the potential impact of antibiotic prophylaxis on culture results remains uncertain. Aim: To prospectively determine the prevalence of SBP in a cohort of asymptomatic outpatients with cirrhotic ascites undergoing LVP, Methods: Between 4/97 and 12/97, 24 consecutive patients with diuretic resistant or intolerant ascites undergoing 101 LVPs were prospectively studied. A brief medical history and exam was obtained and subjects with signs or symptoms of infection, known malignancy, or non-cirrhotic ascites were excluded. LVP was performed using standard sterile technique. Ascitic fluid cell count with differential and bedside inoculation into aerobic/anaerobic blood culture bottles was performed at the time of each LVP. Results: Subject mean age was 55, 19124 were male, and 10/24 were awaiting transplantation. Etiologies of cirrhosis include alcohol (63%), hep C (46%). 4•24 patients had a prior episode of SBP and 7•24 were receiving norfloxacin (400mg/d). Mean LVP volume was 9.5 L(r: 2 to 20.8). Peri-procedural complications included hypotension in 10% and pain in 5%. The SAAG was > 1.1 g/dl in all patients. Fluid Cell counts were < 250 pmn/cc in all instances. A single patient on norfloxacin had two discrete episodes of monomicrobial non-neutrocytic bacterascites (MNB) with Micrococcus and Klebsiella oxytoca. In both instances, fullow-up cell counts and cultures were negative in the absence of treatment. 4•24 patients developed SBP variants (3 MNB, 1 CNNA) associated with symptoms or hospitalization during the study period and were successfully treated with IV antibiotics. One of these 4 patients was receiving norfloxacin. Conclusions: Peritoneal fluid infection at the time of outpatient LVP in cirrhotics is rare (-2%) and of minimal clinical significance. This may relate to the frequent use of antibiotic prophylaxis in high risk patients (47%). Nonetheless, 17% developed serious symptomatic infection during the 8 month interval reflecting the ongoing risk for spontaneous infection. We conclude that with a laboratory cost of $99/case (patient charge=S240) and a low prevalence of infected fluid, routine analysis of ascitic fluid in this setting is not clinically indicated or cost-effective.

• L0277 A NOVEL STOP CODON MUTATION OF THE FERROCHELATASE GENE IN BOVINE PROTOPORPHYRIA, A NATURAL ANIMAL MODEL OF THE HUMAN DISEASE. MM Jenkins 1, RD LeBoeuia, GR Ruth 2, JR Bloomer I. lUniversity of Alabama at Birmingham, 2University of Minnesota, Minneapolis, MN. Protoporphyria (PP) is a genetic disorder in humans in which excess accumulation and excretion of protoporphyrin occurs. The predominant clinical symptom is photosensitivity. Some patients also develop liver failure and the need for liver transplantation due to protoporphyrin-induced liver damage. PP is caused by a deficiency of ferrochelatase (FC), the final enzyme in the heme biosynthesis pathway. FC catalyzes the insertion of ferrous iron into protoporphyrin to produce heme. Bovine are the only species other than man with naturally occurring PP. For expression of the PP phenotype, 2 copies of the mutated gene are necessary in bovine, whereas 1 copy is sufficient in humans. Affected bovine in the USA are expected to be genetically homogeneous because most are direct descendants of a single bull. We report the first potential diseasecausing mutation in the bovine FC gene. The coding region of FC was sequenced from the liver tissue of 2 protoporphyric and one normal bovine. One discrepancy with the published sequence was identified that changed the stop codon to a leucine (TGA-'I'TA) in the protoporphyric bovine. This was not found in the normal bovine. An in-frame stop codon was identified that adds 27 amino acids to the published sequence. To screen other bovine for this G-T transversion and to determine whether it was a polymorphism or a potential disease-causing mutation, cDNAs from 3 normal, 3 heterozygous, and 5 homozygous PP bovine livers were used for allelespeciflc PCR (ASPCR). Phenotypes were determined by each animal's photosensitivity, protoporphyrin levels and/or FC activities. All ASPCR results correlated with the expected phenotypes, supporting the hypothesis that this is the diseasecausing mutation in bovine. Western blots using mitochondria from normal and protoporphyric bovine indicate a slightly larger protein in the protoporphyric animal. These results establish a novel stop codon mutation (X417L) of the FC gene in bovine protoporphyria, a natural animal model of the human disease. Affected animals with PP have a homozygous mutation which leads to marked deficiency of FC and excessive protoporphyrin accumulation. The characterization of this mutation in bovine will help clarify the relationship between the structure and function of the FC gene and its protein product. • L0278 SILYMARIN DECREASES TYPE I PROCOLLOGEN mRNA LEVELS IN RATS WITH SECONDARY BILIARY CIRRHOSIS. J.D. Jia*, G. Boigk*, M. Bauer*, T. Strefeld*, M. Riihl*, E.O. Riecken*, D. Schuppan *#, *Dept. of Gastroenterology and Hepatology, Klinikum B. Franklin, Free University of Berlin, #University of Erlangen, Germany. Background: We have previously reported that silymarin (SIL) reduces collagen accumulation in the livers of rats with secondary biliary cirrhosis (Hepatology 1997; 26:643-9). To elucidate the antifibrotic mechanism, we investigated the in vivo effect of SIL on procollagen txl(I) mRNA steady-state levels by ribonuclease protection assay (RPA). Method: Fifty female Wistar rats (3-months old, weight approx. 250 g) were randomly allocated to 3 groups: 1) Bile duct occlusion by ligation and retrograde injection of Ethibloc (BDO) for 6 weeks; 2) oral SIL treatment at a dosage of 50 mg/kg/d from week 1 to week 6 of BDO, and 3) sham-operated controls receiving SIL at 50 mg/kg/d. Total RNA was isolated from the liver of 4 rats of each group by acidguanidium-phenol-chloroform extraction. Radioactive cRNA probes for rat procollagen cd(I) and glyceraldelhyde phosphate dehydrogenase (GAPDH) were prepared by in vitro transcription, and 20 lag of total RNA was used in RPA. Signal intensities were quantified by phosphor imager and expressed as the ratio of procollagen ~tl(I) and GAPDH mRNA. Results: Compared to the controls (mean, 0.138; 95% CI, 0.062 to 0.214), relative procollagen txl(I) mRNA was increased 20-fold in the BDO group (2.428; 1.381 to 3.583). SIL significantly reduced hepatic procollagen txl(I) mRNA to 1.65 (0.723 to 2.584) (P=0.0386). Conclusion: Downregulation of the steady state level of procollagen I mRNA, which encodes the most abundant ECM component, may be a central mechanism related to the antifibrotic effect of SIL in rat secondary biliary cirrhosis.