April 1995 AASLD A1201

THE CRYSTAL STRUCTURE OF POTASSIUM CHOLATE; COM- PARISON WITH 9ODIUM AND CESIUM CHOLATE. J-G Wu, R.D. Solowav, D-F Xu, Z-J Liu, G-D Yang. Institute of Chemistry and Molecuiar Engineering, Peking University, Beijing, China and Division of Gastroenterology, University of Texas Medical Branch, Galveston TX.

The ability to grow single crystals of bile salts enables exact determination of all the intra- and intermolecular atomic coordination relationships in the solid state. As with protein structure, these studies give added insight into relationships in the liquid state. The aim of this study was to define the atomic coordination relationships of a recently synthesized single crystal of potassium cholate (KC) KC2405H37 and to compare the coordination structure with structures previously determined for sodium cholate (NaC) and cesium chelate (CsC). The monoclinic crystal was grown in dimethylformamide solution by reacting cholic acid with KOH. Its x-ray diffraction pattern was determined using a Syntex-R3 four-circle diffractiometer. Conclusions: Monovalent metals bind to chelate (table) to form a 1:1 complex

NaC KC CsC nH20 1 0 0 coordination number 5 6 7 coordination bonds 3ctOH 3otOH 3ctOH

7ctOH 7ctOH 7aOH 12otOH 12~zOH 12ctOH C-O C-O(4) 12cxOH H20 C-O(4') C-O(4)

c-o(5) c-o(4),c-o(5) but are coordinated to a variable number of carboxyl and ring hy- droxyl groups. As the ionic radius increases, the coordination number increases. Among these monovalent ions studied, only sodium has 1 molecule of hydrated water. The cholate ion is bound to 5 potassium and vice versa. One potassium is bound to hydroxyl oxygens from 3 different chelate molecules while the carboxyl oxygen bonds are from 2 additional cholate molecules. When an O atom forms 2 coordination bonds, the first is less than 2A ° and is in the inner coordination sphere while the second is longer than 2A ° and is in the second coordination sphere of the metal ion.

• EXPRESSION OF cRAS IN HEPATOCELLUI~R CARCINOMA (HCC) Wu PC, Lau VKT t, Lai CL 2 Lau JYN 1 Departments of Pathology ![ and Mledicine2 , University of Hong Kong, Hong Kong; Section of Hepatobiliary Diseases , Division of Gastroenterology, Hepatology and Nutrition. Department of Medicine, University of Florida, Gainesville, FL.

Background The normal ras protein (p21) plays an important role in signal transduction. There are three members of the c-ras family, H-ras, K-ras and N-ras. II has been shown that when the c-ras genes were overexpressed, they could transform normal cells to tumor cells. Activated and mutant c-ras gene products have been found in a wide variety of tumors. Hypothesis The c-ras genes play a role in HCC tumorogenesis. Aim_ To determine the expression of c-ras gene products (p21) in HCC and the adjacent liver tissue. Methods 149 Chinese patients with HCC were studied (M:F 129:20. age 14-83 yrs, histological evidence of cirrhosis in 68/89 specimens with adequate non-tumorous tissue, HBsAg+ 86.3%. survival 1-212 weeks). Formalin-fixed. paraffin-embedded liver sections were employed for the detection of c-ras gene product (p21 immunohistochemistry, Oncogene Science: Uniondale, NY). proliferation markers [immunohistochemistry, PCNA, Ki67 (with citrate buffer-microwave retrieval), Dako, Carpentia, CA], alphafetoprotein (AFP. immuuohistochemistry, Dako), and oncogene products (p53, TGFct. immunohistochemistry, Oncogene Science). The biliary markers (AE1/AE3 and cytokeratin 19. ICN. Costa Mesa. CA) were also assessed by immunohistochemistry. Expression of proliferation, oucogene, and biliary markers were assessed semi-quantitatively (0 = 0%, 1 = 1-24%, 2 = 25-49%, 3 =50-74%, 4..~> 75% "+" cells). The expressmn of e-ras in adjacent liver tissue was also examined where allowed. All histological evaluation was performed blindly without prior knowledge of the clinical information. Results c-gas product was detected in 106/149 (71.1%) HCC (median 2+, range: 0-4+ ) and in the adjacent liver tissue in 53/75 (70.7%, median 1% range 0-4+) patients where there was normal liver tissue in the biopsy for assessment. There was a good correlation between the expression of e-ras in HCC and the adjacent liver tissue (r = 0.633. n = 75, p < 0.001). The expression of c-ras was associated with a younger age (r =0.181, n= 147, p=0.029) and a trend towards AFP expression (r=0.169, n= l l5 , p=0.07), but not with gender, presence/absence of cirrhosis, HBsAg positivity, histological differentiation of the HCC, presence/absence of biliary cell markers in the HCC. cell proliferation markers (PCNA, Ki67), expression of oncogene products (p53, TGFo), or patient survival. Conclusions These data indicate that (l) the e-ras gene is activated in both the HCC and the adjacent liver tissue, and (2) c-ras gene product is more common in young patients with HCC. Whether this increase in c-ras gene product represents activation of the normal c-ras gene or a mutant c-ras gene which accumulates its gene products within the tumor cells remains to be established.

• TREATMENT OF THE CHOLESTEROL SYNTHETIC DEFECT IN A RAT MODEL OF THE SMITH-LEMLI-OPrI-Z SYNDROME. G Xu, G Salen, S Shefer, G Ness, T Chen, GS Tint. GI Lab, VA Medical Center, East Orange, NJ 07018. The biochemical abnormalities of decreased plasma and tis- suse cholesterol with increased 7-dehydrocholesterol (7DHC) levels as seen in homozygotes with the Smith-Lemli-Opitz syndrome can be reproduced in rats by BM 15.766, an in- hibitor of 7DHC-zxZ-reductase. We investigated the effect of cholesterol, cholic acid, and Iovastatin feeding alone or in combination on abnormal cholesterol biosynthesis in rats. Plasma cholesterol and 7DHC concentrations were related to the hepatic activities and mRNA levels for HMG-CoA reductase (HMG) and cholesterol 7a-hydroxylase (Ch7,,). Results: After 2 weeks of inhibitor treatment, plasma cholesterol fell 67% and 7DHC rose from trace to 17 mg/dl, and hepatic HMG activity and mRNA levels were stimulated 73% and 2 times respective- ly. When cholesterol was given for 1 week to BM 15.766 treated rats, plasma concentrations increased 2.9 times and 7DHC levels decreased 49%, while elevated HMG activity and mRNA levels were inhibited 74% and 49% respectively. In com- parison, cholic acid plus BM 15.766 increased plasma choles- terol 94% without reducing 7DHC levels. The combination of cholic acid with cholesterol enhanced plasma cholesterol 10.5 times also without decreasing 7DHC levels. HMG activity and mRNA levels were suppressed 49% and 61% by cholic acid alone and 81% and 74% respectively when cholesterol was combined with cholic acid. Adding Iovastatin to BM 15.766 treated rats depressed plasma cholesterol levels 57% further without reducing 7DHC levels. Ch7e activity was not affected by BM 15.766 but was stimulated by cholesterol feeding and inhibited by cholic acid. Ch7e mRNA levels rose almost 2 fold with BM 15.766 treatment and were not further increased by cholesterol feeding but were markedly inhibited by cholic acid. Conclusion: Cholesterol is essential to correct abnormal cho- lesterol synthesis induced by the inhibition of 7DHC-zx 7- reductase in rats. Plasma levels of cholesterol are replenished and 7DHC decline because absorbed cholesterol expands the pool and exerts feedback inhibition on HMG. Neither cholic acid nor Iovastatin separately are effective but cholic acid plus cholesterol would offer some additional benefit.

A NEW METHOD TO STORE RAT LIVER IN COLD SALINE FOR 12 H O U R S W I T H THE RECIPIENT SURVIVAL. H.S. Xu. W.C. Stevenson, and R.S. Jones. Department o f Surgery, University of Virginia Health Sciences Center, Charlottesville, Virginia.

Currently, donor livers are flushed with cold peffusates via the aorta and portal vein during harvesting and stored in the same solution to prevent liver iscbemic injury. The maximum time of rat liver cold storage in the chilled saline with the recipient survival is about 6 hours. This study investigated a new method for donor liver preservation. The experiment was carried out in 5 groups of fiver-transplanted rats using current fiver perfusion method and 5 experimental groups using new method to store livers for 6, 8, 10, 12, and 16 hours. Cold saline at 2-3 oC was used to flush and store the liver as perfusate in this study. The new method includes: (1) donor heparinization (380 mg/kg); (2) drainage of fiver blood flow through the suprabepatic vena cava by clamping the infrahepatic vena cava and portal vein without the liver cold perfusion; (3) liver surface cooling; (4) cold store of the isolated liver; (5) flushing the portal vein after long-term cold storage. Serum enzymes and monoethylglycinexylidide (MEGX) were measured every day following surgery. Surviving rats were sacrificed on day 7 for liver histologic analysis.,

COLD ISCHEMIC TIME SURVIVAL RATE (>7 DAYS) (hours) CONTROL EXPERIMENTAL

6 50.0% (3/6) 100.0% (6/6) * 8 0 (0/6) 66.7% (4/6) *

10 0 (0/6) 50.0% (3/6) * 12 0 (0/6) 16.7% (1/6) 16 0 (0/6) 0 (0/6)

* p<0.05 compared with the paired control;

The new method significantly increased recipient survival rate in experimental groups with 6, 8, and 10 hours o f cold ischemia and extended maximum time for safe rat liver preservation from 6 hours in the control group to 12 hours in the new method group. Meanwhile, serum aspartate transferase and lactate dehydrogenase levels in the recipients following surgery decreased significandy in the experimental groups compared to each paired control. The beneficial effects o f the new method may result from the protection o f fiver endothelial cell damage f rom cold ischemia. However, cold saline is not an ideal solution for the liver storage, the effects of the reported method may give a new approach for liver cold preservation.