- Email: [email protected]
Mechanism and regulation of riboflavin uptake by the human-derived liver cells Hep G2
GASTROENTEROLOGY Vol. 114, No. 4
A1330 AASLDABSTRACTS • L0538 THE EPIDEMIOLOGY, TREATMENT PATTERNS AND RESOURCE UTILIZATION OF HEPATITIS C PATIENTS IN A LARGE INSURED NEW ENGLAND POPULATION. Daniel M. Rosenber~, Suzanne F. Cook, Susan C. Eaton, William B. Saunders, Glaxo Wellcome Inc., Research Triangle Park, NC, and Lee L. Lanza, Epidemiology Resources Inc., Newton Lower Falls, MA. The Centers for Disease Control estimates that 1-2% of the U.S. population is affected by HCV. The purpose of this cross-sectional study is to describe the epidemiology, treatment patterns and major health care resources associated with hepatitis C virus (HCV) in a Large Insured New England (LINE) population. This LINE database maintains claims for over 2 million persons, data on patients treated from January 1, 1995 to December 31, 1996 were analyzed. Demographics, type of provider, type of insurance coverage, utilization of outpatient and inpatient services, prescription medications, diagnostic, laboratory, surgical procedures and comorbidities with hepatitis B virus (HBV) and the human immunodeficiency virus (HIV) were examined. Preliminary findings indicate that the prevalence of patients with an HCV diagnosis for whom services were rendered was 49/100,000, HCV/HBV coinfection was 2.8/100,000 and HCV/HIV coinfection was 0.79/100,000. Persons aged 40-49 years had the greatest HCV prevalence (male: 196/100,000 and female: 69/100,000). Nearly 20 percent of patients with only HCV had interferon-alpha therapy. Estimates in this population are subject to underestimation due to the non-inclusion of persons classified with nonspecific viral hepatitis codes, infected persons not presenting for services and institutional differences in diagnostic screening patterns. Further resource utilization and treatment data will be detailed. The prevalence of patients with an HCV diagnosis for whom services were rendered in this LINE population is lower than expected but these data provide important information on treatment patterns and resource utilization. This research was funded by Glaxo Wellcome, Research Triangle Park, NC. • L0539 BODY COMPOSITION AND SUBSTRATE OXIDATION IN LONGTERM SURVIVORS AFTER TRANSPLANTATION OF LIVER OR KIDNEY. A.-E. Roske, H. Hudjetz, T.Schtitz, K. Budde, L. Fritsche, H. Lochs, M. Plauth, Medizinische Klinik, Charit6, Humboldt-Universitfit, Berlin, Germany. Backeround: After liver transplantation (LTX), excessive weight gain is observed in a considerable number of patients. Previously, we found a normalization of fat oxidation rates and a reduction in resting energy expenditure (REE) in patients with normal liver function after LTX. Even in long-term survivors after LTX muscle wasting was prevalent in all patients while fat mass was increased. We therefore investigated whether these findings are unique to patients after LTX or whether they are prevalent also in patients after transplantation of other solid organs, such as kidney (KTX). Methods: In 47 patients after LTX (age 45 -+9ys, median time since LTX 64 months) and 26 patients after KTX (age 46 -+ 10ys, median time since KTX 89 months) REE and substrate oxidation rates were measured by indirect calorimetry (REEcALo) and calculated according to Harris & Benedict (REErm). Food intake was estimated by standardized dietary recall. Body composition was assessed by anthropometry, bioelectric impedance analysis (BIA) and creatinine height index. Number of immunosuppressive drugs per day were recorded. All values are mean -+ sd; statistical evaluation by t-test or Mann-Whitney U-test. Results:
i REEcALo[kcal/d] [ REEcALo/BCMalA[kcal/kg] Fat oxidation rate [%] Non-protein energy intake > 1,3*REErm Protein intake [g/kgBWld] Body mass index [kg/m2] Arm fat area [cm2] Arm fat area > 75 a, percentile Arm muscle area < 10th percentile Creatinine height index [%] Immunosuppressivedrugs [n/d]
LTX
KTX
1601 -+ 274 55,6 -+ 10,1 24,5 -+ 14,5 76% 1,06 -+0,31 27,3 -+ 5,4 33,7 -+ 17,6 57% 100% 104 -+ 15 2,15 -+ 0,65
1654 _+249 56,3 -4--6,0 30,4 -+ 16,6 56% 1,22 -+0,39 26,6 -+4,6 41,7 -+ 13,9 85% 88% 117-+29 2,24 -+0,50
P n.s. n.s. n.s. < 0,05 n.s. < 0,05
n.s. n.s.
Conclusions: In homology to the situation after LTX energy expenditure and substrate oxidation were normal in long term survivors after KTX. Energy intake was increased above requirements more often after LTX while protein intake was significantly higher in patients after KTX. These findings seem to be independent from the immunosuppressive drug regimen. Despite good graft function body composition was grossly abnormal in both groups with an increase in fat mass, particularly after KTX, and a severe reduction in muscle mass. Further studies are needed to clarify whether abnormal body composition after solid organ transplantation is an unavoidable sequel of chronic disease prior to transplantation, whether it is amenable to treatment, and whether it is related to long-term outcome.
• L0540
MECHANISM AND REGULATION OF RIBOFLAVIN UPTAKE BY THE HUMAN-DERIVED LIVER CELLS Hep G2. Hamid M. Said, Alvaro Ortiz and Thomas Y Ma. VA Medical Center, Long Beach, CA, and University of California, Irvine, CA. Riboflavin (RF), a water-soluble vitamin, plays a critical role in many cellular metabolic reactions, and thus, is essential for normal cellular functions, growth and development. The liver plays a central role in normal RF metabolism and is the site of maximum utilization of the vitamin. The mechanism of RF uptake by liver cells has been studied by us and others in animal models, but no information is available about the mechanism of the vitamin uptake in the human situation and about its cellular regulation. In this study, we used the human-derived liver cells Hep G2 as an in vitro cellular model system and 3H-RF to address these issues. Uptake of RF by confluent Hep G2 monolayers was found to be temperature- and energy-dependent; it was, however, Na+-independent in nature. Uptake appeared to involve a carrier-mediated process as indicated by the saturation as a function of increasing the substrate concentration in the incubation media (apparent Km 0.41 -+0.08 pM), and by the ability of the structural analogs lumiflavin and lumichrome (but not the unrelated biotin) to inhibit 3H-RF uptake process [inhibition constant (Ki) of 1.84 and 6.32 pM, respectively]. RF uptake was also found to be sensitive to the inhibitory effect of the -SH group blocker p-chloromercuriphenylsulfonate (p-CMPS) (Ki = 100 uM). Specific modulators of intracellular protein kinase A (PKA)-, protein kinase C (PKC)and protein tyrosine kinase (PTK)-mediated pathways did affect RF uptake by Hep G2 cells. On the other hand, specific inhibitors of the Ca2+/calmodulinmediated pathway significantly inhibited RF uptake process. This effect appeared to be mediated through a decrease in the Vma× of the RF uptake process with no significant changes in the apparent Km. Maintaining Hep G2 cells in a RF-deficient growth medium was associated with a significant upregulation in the substrate uptake. This effect was specific for RF and was again found to be mediated via an increase in the Vmax of the uptake process with no significant changes in the apparent Km. These results describe for the first time the mechanism and cellular regulation of RF uptake by a humanderived liver cellular preparation and shows the involvement of a specialized, carrier-mediated system in the uptake process. Furthermore, the uptake process appears to be regulated by an intracellular Ca2+/calmodulin-mediated pathway, and by substrate level. This work was supported by NIH grant DK47203. L0541 POSTOPERATIVE RECURRENCE OF HEPATOCELLULAR CARCINOMA ACCORDING TO HEPATITIS VIRUS STATUS. A. Saito. K. Takasaki, H. Katsuragawa, S. Katagiri, M. Sakai, T. Ootsubo, T. Tsugita, M. Chiba, E. Miyazaki, N. Hayashi; Institute of Gastroenterology, Tokyo Women's Medical College, Tokyo, Japan. This study was designed clarify differences in hepatocellular carcinoma (HCC) recurrence among 418 patients with Hepatitis B or C virus infection, who underwent HCC resection from 1982-1992. Initially, the patients were divided into B (HBsAg+) and C (HBsAg-) groups but there were no significant differences in survival or recurrence between these two groups. Therefore, we divided the patients into three groups, HBeAg+ (n=18), anti HBe+ (n=45) and C (n=355), for analysis of the nonrecurrence rate and mode of recurrence. The 5 year survival rates were 36.5%, 75.8% and 61.9%, and the 10 year rates 12.2%, 62.2% and 27.5%, respectively, revealing significant differences between the anti HBe+ and C groups (p<0.01). The respective 5 and 10 year non-recurrence rates were 11.1%, 50.1% and 25.7%, and 0%, 31.0% and 10.3%. The differences among the 3 groups were statistically significant (p<0.01). Mode of recurrence was divided into metastatic and multicentric with the rates of each being 64.7% and 23.5% for HBeAg+, 27.2% and 25.0% for anti HBe+, and 25.8% and 44.5% for C group. Metastatic recurrence was more frequent in HBeAg+, whereas multicentric was the most common mode in C group. More than 90% of metastatic recurrences were detected within 3 years postoperatively, while multicentric were seen even after 5 years. Differences in the postoperative recurrence rate and mode of recurrence were demonstrated among the HBeAg+, anti HBe+ and C groups. These findings are important for planning therapy and for determining the type and duration of follow-up. L0542 DISSOCIATION BETWEEN CELLULAR DIFFERENTIATION PROCESS AND INCREASE IN GAP JUNCTION INTERCELLULAR COMMUNICATION (GJIC) OF HUMAN HEPATOMA CELL LINES. H. Saito, F. Kaneko, H. Ebinuma, M. Nakamura, K. Wakabayashi, Y. Saito, and H. Ishii. Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan. Back~,round and aim: We have previously demonstrated that sodium butyrate decreases malignant phenotypes of several human hepatoma ceils without inducing apoptosis. Phenotypic changes induced by this agent suggest that butyrate can alter hepatoma cells into their normal counterparts. The gap junction intercellular communication (GJIC) is known to be important in a
A1330 AASLDABSTRACTS • L0538 THE EPIDEMIOLOGY, TREATMENT PATTERNS AND RESOURCE UTILIZATION OF HEPATITIS C PATIENTS IN A LARGE INSURED NEW ENGLAND POPULATION. Daniel M. Rosenber~, Suzanne F. Cook, Susan C. Eaton, William B. Saunders, Glaxo Wellcome Inc., Research Triangle Park, NC, and Lee L. Lanza, Epidemiology Resources Inc., Newton Lower Falls, MA. The Centers for Disease Control estimates that 1-2% of the U.S. population is affected by HCV. The purpose of this cross-sectional study is to describe the epidemiology, treatment patterns and major health care resources associated with hepatitis C virus (HCV) in a Large Insured New England (LINE) population. This LINE database maintains claims for over 2 million persons, data on patients treated from January 1, 1995 to December 31, 1996 were analyzed. Demographics, type of provider, type of insurance coverage, utilization of outpatient and inpatient services, prescription medications, diagnostic, laboratory, surgical procedures and comorbidities with hepatitis B virus (HBV) and the human immunodeficiency virus (HIV) were examined. Preliminary findings indicate that the prevalence of patients with an HCV diagnosis for whom services were rendered was 49/100,000, HCV/HBV coinfection was 2.8/100,000 and HCV/HIV coinfection was 0.79/100,000. Persons aged 40-49 years had the greatest HCV prevalence (male: 196/100,000 and female: 69/100,000). Nearly 20 percent of patients with only HCV had interferon-alpha therapy. Estimates in this population are subject to underestimation due to the non-inclusion of persons classified with nonspecific viral hepatitis codes, infected persons not presenting for services and institutional differences in diagnostic screening patterns. Further resource utilization and treatment data will be detailed. The prevalence of patients with an HCV diagnosis for whom services were rendered in this LINE population is lower than expected but these data provide important information on treatment patterns and resource utilization. This research was funded by Glaxo Wellcome, Research Triangle Park, NC. • L0539 BODY COMPOSITION AND SUBSTRATE OXIDATION IN LONGTERM SURVIVORS AFTER TRANSPLANTATION OF LIVER OR KIDNEY. A.-E. Roske, H. Hudjetz, T.Schtitz, K. Budde, L. Fritsche, H. Lochs, M. Plauth, Medizinische Klinik, Charit6, Humboldt-Universitfit, Berlin, Germany. Backeround: After liver transplantation (LTX), excessive weight gain is observed in a considerable number of patients. Previously, we found a normalization of fat oxidation rates and a reduction in resting energy expenditure (REE) in patients with normal liver function after LTX. Even in long-term survivors after LTX muscle wasting was prevalent in all patients while fat mass was increased. We therefore investigated whether these findings are unique to patients after LTX or whether they are prevalent also in patients after transplantation of other solid organs, such as kidney (KTX). Methods: In 47 patients after LTX (age 45 -+9ys, median time since LTX 64 months) and 26 patients after KTX (age 46 -+ 10ys, median time since KTX 89 months) REE and substrate oxidation rates were measured by indirect calorimetry (REEcALo) and calculated according to Harris & Benedict (REErm). Food intake was estimated by standardized dietary recall. Body composition was assessed by anthropometry, bioelectric impedance analysis (BIA) and creatinine height index. Number of immunosuppressive drugs per day were recorded. All values are mean -+ sd; statistical evaluation by t-test or Mann-Whitney U-test. Results:
i REEcALo[kcal/d] [ REEcALo/BCMalA[kcal/kg] Fat oxidation rate [%] Non-protein energy intake > 1,3*REErm Protein intake [g/kgBWld] Body mass index [kg/m2] Arm fat area [cm2] Arm fat area > 75 a, percentile Arm muscle area < 10th percentile Creatinine height index [%] Immunosuppressivedrugs [n/d]
LTX
KTX
1601 -+ 274 55,6 -+ 10,1 24,5 -+ 14,5 76% 1,06 -+0,31 27,3 -+ 5,4 33,7 -+ 17,6 57% 100% 104 -+ 15 2,15 -+ 0,65
1654 _+249 56,3 -4--6,0 30,4 -+ 16,6 56% 1,22 -+0,39 26,6 -+4,6 41,7 -+ 13,9 85% 88% 117-+29 2,24 -+0,50
P n.s. n.s. n.s. < 0,05 n.s. < 0,05
n.s. n.s.
Conclusions: In homology to the situation after LTX energy expenditure and substrate oxidation were normal in long term survivors after KTX. Energy intake was increased above requirements more often after LTX while protein intake was significantly higher in patients after KTX. These findings seem to be independent from the immunosuppressive drug regimen. Despite good graft function body composition was grossly abnormal in both groups with an increase in fat mass, particularly after KTX, and a severe reduction in muscle mass. Further studies are needed to clarify whether abnormal body composition after solid organ transplantation is an unavoidable sequel of chronic disease prior to transplantation, whether it is amenable to treatment, and whether it is related to long-term outcome.
• L0540
MECHANISM AND REGULATION OF RIBOFLAVIN UPTAKE BY THE HUMAN-DERIVED LIVER CELLS Hep G2. Hamid M. Said, Alvaro Ortiz and Thomas Y Ma. VA Medical Center, Long Beach, CA, and University of California, Irvine, CA. Riboflavin (RF), a water-soluble vitamin, plays a critical role in many cellular metabolic reactions, and thus, is essential for normal cellular functions, growth and development. The liver plays a central role in normal RF metabolism and is the site of maximum utilization of the vitamin. The mechanism of RF uptake by liver cells has been studied by us and others in animal models, but no information is available about the mechanism of the vitamin uptake in the human situation and about its cellular regulation. In this study, we used the human-derived liver cells Hep G2 as an in vitro cellular model system and 3H-RF to address these issues. Uptake of RF by confluent Hep G2 monolayers was found to be temperature- and energy-dependent; it was, however, Na+-independent in nature. Uptake appeared to involve a carrier-mediated process as indicated by the saturation as a function of increasing the substrate concentration in the incubation media (apparent Km 0.41 -+0.08 pM), and by the ability of the structural analogs lumiflavin and lumichrome (but not the unrelated biotin) to inhibit 3H-RF uptake process [inhibition constant (Ki) of 1.84 and 6.32 pM, respectively]. RF uptake was also found to be sensitive to the inhibitory effect of the -SH group blocker p-chloromercuriphenylsulfonate (p-CMPS) (Ki = 100 uM). Specific modulators of intracellular protein kinase A (PKA)-, protein kinase C (PKC)and protein tyrosine kinase (PTK)-mediated pathways did affect RF uptake by Hep G2 cells. On the other hand, specific inhibitors of the Ca2+/calmodulinmediated pathway significantly inhibited RF uptake process. This effect appeared to be mediated through a decrease in the Vma× of the RF uptake process with no significant changes in the apparent Km. Maintaining Hep G2 cells in a RF-deficient growth medium was associated with a significant upregulation in the substrate uptake. This effect was specific for RF and was again found to be mediated via an increase in the Vmax of the uptake process with no significant changes in the apparent Km. These results describe for the first time the mechanism and cellular regulation of RF uptake by a humanderived liver cellular preparation and shows the involvement of a specialized, carrier-mediated system in the uptake process. Furthermore, the uptake process appears to be regulated by an intracellular Ca2+/calmodulin-mediated pathway, and by substrate level. This work was supported by NIH grant DK47203. L0541 POSTOPERATIVE RECURRENCE OF HEPATOCELLULAR CARCINOMA ACCORDING TO HEPATITIS VIRUS STATUS. A. Saito. K. Takasaki, H. Katsuragawa, S. Katagiri, M. Sakai, T. Ootsubo, T. Tsugita, M. Chiba, E. Miyazaki, N. Hayashi; Institute of Gastroenterology, Tokyo Women's Medical College, Tokyo, Japan. This study was designed clarify differences in hepatocellular carcinoma (HCC) recurrence among 418 patients with Hepatitis B or C virus infection, who underwent HCC resection from 1982-1992. Initially, the patients were divided into B (HBsAg+) and C (HBsAg-) groups but there were no significant differences in survival or recurrence between these two groups. Therefore, we divided the patients into three groups, HBeAg+ (n=18), anti HBe+ (n=45) and C (n=355), for analysis of the nonrecurrence rate and mode of recurrence. The 5 year survival rates were 36.5%, 75.8% and 61.9%, and the 10 year rates 12.2%, 62.2% and 27.5%, respectively, revealing significant differences between the anti HBe+ and C groups (p<0.01). The respective 5 and 10 year non-recurrence rates were 11.1%, 50.1% and 25.7%, and 0%, 31.0% and 10.3%. The differences among the 3 groups were statistically significant (p<0.01). Mode of recurrence was divided into metastatic and multicentric with the rates of each being 64.7% and 23.5% for HBeAg+, 27.2% and 25.0% for anti HBe+, and 25.8% and 44.5% for C group. Metastatic recurrence was more frequent in HBeAg+, whereas multicentric was the most common mode in C group. More than 90% of metastatic recurrences were detected within 3 years postoperatively, while multicentric were seen even after 5 years. Differences in the postoperative recurrence rate and mode of recurrence were demonstrated among the HBeAg+, anti HBe+ and C groups. These findings are important for planning therapy and for determining the type and duration of follow-up. L0542 DISSOCIATION BETWEEN CELLULAR DIFFERENTIATION PROCESS AND INCREASE IN GAP JUNCTION INTERCELLULAR COMMUNICATION (GJIC) OF HUMAN HEPATOMA CELL LINES. H. Saito, F. Kaneko, H. Ebinuma, M. Nakamura, K. Wakabayashi, Y. Saito, and H. Ishii. Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan. Back~,round and aim: We have previously demonstrated that sodium butyrate decreases malignant phenotypes of several human hepatoma ceils without inducing apoptosis. Phenotypic changes induced by this agent suggest that butyrate can alter hepatoma cells into their normal counterparts. The gap junction intercellular communication (GJIC) is known to be important in a