HEPATOLOGY Vol. 22, No. 4, Pt . 2, 1995 A A S L D A B S T R A C T S 465A

1433 ELEVATED PROCOLLAGEN-HI-PEPTIDE (P-HI-P) IN HCV- INFECTED BLOOD AND PLASMA PRODUCT RECIPIENTS Transfusion Safety Study Group 1 represented by MJ Nowicki 2 1 Participating institutions, 2 University of Southeru California, Los Angeles, CA, USA.

Background: The amino-terminal peptide level ofprocollagen III (P-III-P) has been proposed as way of munitoring hepatic fibrosis in chronic forms of hepatitis. Chronically infected hemophiliacs need to be compared with blood recipients to determine if connective tissue damage contributes tO the level in the former group. Aim: To compare levels of P-III-P in HCV-infected and uninfected recipients and hemophiliacs. Method: We selected 12 HCV(+) I~V(+) subjects with hemophilia~ 20 who were HCV(+)HIV(-), 20 HCV(-)HIV(-) recipients of HIV(-) blood and 20 age-matched HCV(-)HIV(-) household members as controls. First and second generation EIA's and RIBA2 (Ortho Diagnostics, Raritan, N J, USA) for HCV status and RIA-Gnost P-III-P (Hoechst~ Frankfurt, Germany) were carried out. Results: All hemophiliacs group and blood recipients had higher P-III-P levels than controls. Only HCV(+)I-IIV(+) hemophiliacs had levels significantly higher than HCV(-)HIV(-) hemophiliacs and blood recipients (p<0.001 and p=0.003 respectively).

Group I HCVstatus P-III-P (U/mO p Value Hemophiliacs HIV (+) Positive 1.70 + 0.66 <0.001 Hemophiliacs I-ftV (-) Positive 1.26 + 0.57 <0.001 Hemophiliacs HIV (-) Negative 1.02 + 0.40 <0.001 Recipients of (-) blood Negative 1.06 + 0.58 0.003 Controls Negative 0.62 + 0.13

Conclusions: P-III:P may be a useful assay for monitoring fibrosi s due to chronic viral hepatitis but the contribution of connective tissue damage must be determined. (NHLBI Contracts no. NO1-HB-4-7003 and 9-7074)

1434 APOLIPOPROTEIN A-I GENE EXPRESSION IS MODULATED BY VISCOSITY IN PRIMARY CULTURES OF ADULT RAT HEPATOCYTES P. Nufo-Gonz~ez., T. Mendoza-Figueroa, A. Hem~adez and Panduro A. Institute of Molecular Biology in Medicine, CUCS Universidad de Guadalajara and Hospital Civil de Guadalaj~ra, and Department of Pharmacology and Toxicology, CINVESTAV-IPN, Mexico City.

Previous in vivo studies from our laboratory have shown that increasing plasma viscosity is associated with an augmented expression of the liver-speeific apolipoprotein (apt) A-I gene at the mRNA level. When plasma viscosity exceeds the physiologic r~ge (1.6-1.9), apt A-I gene expression is inhibited posttranscriptionally. The aim of this work was to determine whether this in wvo induction/inhibition of apt A-1 mRNA was reproduced in cultured parenchymal liver cells. Hepatocytes were isolated from male Wistar rats (180-200g) by the collagenase perfusion method, and cultured for 24 h on 100 mm plastic dishes (3 x 10 s cells/dish) with MX,83 serum-free medium supplemented with 5 I.tg/ml insulin, 10 ttg/ml hydrocortisone, and 1 pg/ml transferrin. Then cultures were fed with MX-83 Supplemented medium containing gelatin at concentrations that generated viscosities of !.04, 1.34, 1.60, 1.93, and 2.65. After 24 h incubation hepatocytes were scraped off and immersed immediately in liquid nitrogen; total RNA was extracted and the level of apt A-I mRNA determined by Northern hybridization. Viscosity below or in the physiologic range (from 1.04 to L60) resulted in augmented levels (20-30%) of apt A-I mRNA. However, at viscosity higher than file physiologic rage (from 1.93 to 2.65) an inhibitory effect of up to 50% was observed in apt A-I mRNA levels. To study whether the inhibitory effect at high viscosity was time-dependent, 24 h-cultures were fed with supplemented MX-83 medium with viscosity of 2.65 and after 0, 2, 4, 6, 9, 12 and 24 h of uicubatien hepatocytes were scraped off to determine apt A-I mRNA levels. An initial induction of apt A-I mRNA was observed with a maximum of 13% after 2 h incubation, after which pro~essive inhibition was found, amounting after 24 h incubation to values 70*/o lov,~ than those of the initial level. Our results show that the in vitro model with cultured h e p ~ reproduces the steps of induction/inhibition observed in vivo in the expression of the apt A-1 gane, and suggest that viscosity plays an important role in the posttranacriptional regulation of apt A-1 gene expression. Our results also support the hypothesis that viscosity produces a negative feed-back regulatory effect on apt A- 1 gene expression.

1435 VISCOSITY BUT NOT COLLOIDOSMOTIC PRESSURE MODULATES APOLIPOPROTEIN (apo)A-1 GENE EXPRESSION DURING LIVER REGENERATION. P N~fio..Gonz~laz, T Mendoza-Fioueroa and A Panduro. Institute of Molecular Biology in Medidne, CUCS U. de G. and Hospital Civil de Guadalajara. And Dept. of Pharmacology CINVESTAV, MExico.

Senondaff hypedipidemia occurs dudng liver regeneration and nephrotic syndrome. Colloidusmatic pressure (COP) and viscosity have been implicated as important factors in the induction of the hyperlipidemic state in nephrotic syndrome (NS), but not during liver regener~on. We have previously demostreted that ape A-I gene expression is regulated posttrenscriptionally in both experimental animal models (F_AM). In this study, we analyzed the effect of viscosity and COP in the expression of ape A-I at the mRNA level and its relationship with serum HDL dudng the hyperlipidomic stages (induction- inhibilion). To induce liver regeneration, rats were treated intragestricatly with a single dose of CCI~ (0.5 ml/100 g). The nephrotic syndrome (NS) was originated in young rats with puromycin aminonucleuside (PAN) (15 rag/100 g). Viscosity and COP were determined end analyzed dudng the induction end inhibition stages of the hypedipidemia in both EAM. The steady state levels (SSL) of ape A-I mRNA were determined by the nodhem blot and HDL by enzymatic assays. At difference of the NS, the hypedipidemla presented dudng liver regeneration was not associated with poliuria, hypoproteinemla nor hypoalbuminemia. An inicial hyperlipidemic stage characterized by high levels of total cholesterol and triglyceridos but low levels of HDL was related to a light but not statls~ly significant decrease of serum albumin and PCO. At this stage, a physiological iocrease of the relative plasma viscosity from 1.5 to 1.5 was associated to the induction in the expression of ape A-I gene at the mRNA level. In a second hypedipidomlo stage, the SSL of apo A-I remained elevated, serum HDL increased to values higher than control (from 32 to 72 rag/all) and relative viscosity reached a maximun and ~ significant increase of 1.7 + 0.09. Thereafter, the serum HDL start to coma down to normal values in spite of high levels of ape A-I mRNA. A similar assedation of v'~osity and induction.inhibition of apo A-I gene expression was detected doting the hypedipidomic stages in rats treated with PAN. Hypedipidemia is not associated to proteinuda, hypoproteinemia nor hypoarouminomia in liver regeneration. In both EAM, physiological variation of plasma viscosity were associated to the induction in the exlxeselon of apo A-I gane expression. In contrast, when viscosity reached the highest values, an inhibition of apo A-I gene expression was observed at the tranlallonat or i~ln~sl~imal level,

1436 HCV RNA QUANTITATION: IS PLASMA EQUIVALENT TO SERUM? CB O'Brien 1, B Henzel t, L Wolfe 2 and A Yoder x. ILiver Center, University of Pennsylvania Medical Center, Philadelphia, Pa 19104 and2Roche Molecular Systems, Branchburg, NJ 08876. INTRODUCTION: The detection of HCV RNA by RT-PCR is becoming increasingly important in the diagnosis and management of chronic hepatitis (3. HCV RNA has been detected in serum, plasma, mononuclear Cells and liver tissue. AIM: We wanted to determine if quantitation of HCV RNA by RT-PCR gives equivalent values in plasma and in serum. METHODS: . 10 ml of whole blood were collected into a (SST) serum separator tube and8 ml into a VACUTAINER CPT tube (Becton Dickinson VACUTAINER Systems) for each paired specimen. All tubes were spun at 1500 - 1800 xg withi n 2 hours of collection. Serum aliquots were obtained from the SST tubes. Plasma aliquots were obtained from the VACUTAINER CPT tubes and all specimens were frozen at -70°C. The detection and quantitation of the HCV RNA was performed with the Amplicor TM Monitor assay (Roche Molecular Systems). This uses a single primer pair (5'-untranslated region of HCV, biotinylated downstream primer). A singl e enzyme (rTth DNA polymerase) carries out both RT and DNA polymerase functions. Uracil-N-glycosylase is incorporated to prevent amplicon contamination. Detection was Carried out in a microwell (Avidin-HRP) format. RESULTS: Levels of the HCV RNA in 53 paired serum and plasma samples from 25 patients were compared. HCV RNA was detectable in all samples. The correlations of HCV RNA levels by RT-PCR in serum and plasma are listed below:

Mean Stand. Dev. Plasma 2.3E+05 3.3E+05 Serum 4.2E+05 1.3E+06

CONCLUSION: Quantitation of HCV RNA titer by RT-PCR gives equivalent results in either plasma or serum within I/2 log. Analysis of HCV RNA titers can be performed on plasma derived from the VACUTAINER CPT tube.