Biliary atresia

*041 Neonatal Risk of Parenteral Nutrition-Associated Cholestasis: A Multivariate Analysis of the Effect of Taurine. Ariel U. Spencer, M.D.1, Thomas Tracy, M.D.2, Mostaffa Auothmany, M.D.3, Adolfo Llanos, M.D.4, Morton B. Brown, Ph.D.1, Sunkyung Yu, M.S.1, Marilyn Brown, M.D.4, Robert J. Shulman, M.D.5, Ronald B. Hirschl, M.D.1, Patricia A. DeRusso, M.D.6, Jean Cox, M.S.6, Jacqueline Dahlgren, M.S., M.D.1, Peter Strousse, M.D.1, Daniel H. Teitelbaum, M.D.1 1 University of Michigan, Ann Arbor, MI, USA, 2 Brown University, Providence, RI, USA, 3 St. Vincent's Mercy Children's Hospital, Toledo, OH, USA, 4 Strong Memorial Hospital, Rochester, NY, USA, 5 Baylor College of Medicine, Houston, TX, USA, 6 The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Background: Neonates receiving total parenteral nutrition (TPN) are at risk for parenteral nutrition-associated cholestasis (PNAC) - manifested by elevated conjugated bilirubin (CB). Prevention of PNAC remains a significant problem. Taurine is the major amino acid utilized by the neonate in bile salt conjugation. Despite reports suggesting that taurine prevents neonatal PNAC, no clinical study to date has definitively demonstrated this.

Hypothesis: Taurine-supplemented TPN reduces the incidence of PNAC in premature or critically ill neonates.

Methods: This study was part of a prospective, controlled, randomized, multi-institutional trial conducted between 1996 and 2001, comparing cholecystokinin (CCK) versus placebo in the prevention of PNAC. Enrollment criteria were a requirement for > 50% of nutrition parenterally, and any one of the following diagnoses: either 1) Prematurity (gestational age ≤ 28 weeks at enrollment); 2) Necrotizing enterocolitis (NEC, Grade II or higher); or 3) Surgical requirement for prolonged TPN (including gastroschisis and severe intestinal atresia). Patients were enrolled within two weeks of birth or within seven days after surgery. Exclusion criteria included all causes of hyperbilirubinemia not attributable to TPN; metabolic, biliary, and hepatic diseases. The primary outcome measure was serum CB (assessed weekly). Caloric and protein intake were standardized. However, some institutional and hospital formulary variation in TPN was present, such that 194 subjects received taurine-supplemented TPN, while 39 subjects received TPN without taurine. CCK did not prevent PNAC; however, a secondary analysis of the data was performed to examine the effect of taurine on CB. A multivariate analysis was used, controlling for the effects of diagnosis and gestational age.

Results: Estimated risk of hyperbilirubinemia [95% confidence interval] in premature infants receiving taurine (0.42 [-0.36 to 1.20]) was reduced in comparison to those not receiving taurine (3.37 [1.64 to 5.09]) (p=0.07). Taurine significantly reduced the risk for infants with NEC as well (taurine, estimated risk 3.96 [2.72 to 5.20] vs. no taurine, 8.40 [5.41 to 11.39], p 28 weeks of age (estimated risk: taurine, -0.24 [-1.25 to 0.77] vs. no taurine 5.47 [2.49 to 8.46](p

Conclusions: Taurine-supplemented TPN reduces the levels of CB in premature infants and those with NEC. Though these patients were already at high risk for PNAC, taurine administration reduced the relative risk of PNAC. The etiology of PNAC is likely multifactorial, and thus neonates with surgical conditions may have multiple contributing factors to ongoing liver dysfunction beyond mere taurine deficiency. A differential benefit of taurine is seen with increasing gestational age, which may be attributable to development of hepatic metabolic pathways, and may also suggest the presence of unknown factors in the etiology of PNAC in the extremely premature infant.

*042 Epidermal Growth Factor and/or Growth Hormone Induce Differential, Side-Specific Signal Transduction Protein Phosphorylation. Nelly E. Avissar, PhD, Liana Toia, BS, Harry C. Sax, MD. University of Rochester, School of Medicine and Dentistry, Rochester, NY, USA.

Introduction: EGF plus GH enhance luminal Glutamine transport into rabbit intestine and into human C2^sub BBe^1 (a brushborder expressing enterocyte cell line). EGF can activate different signal transduction pathways when it is applied to the basolateral (Bl) versus the apical (Ap) side of intestinal cells. Our objective was to determine the activation status of signal proteins in C2^sub BBe^1 cells. We studied Ap and/or Bl signal transduction pathways following EGF treatment w/wo Bl GH using a Kinexus Screen.

Methods: C2^sub BBe^1 cells grown on Transwells were treated for 15min w/wo Ap or BI EGF and/or GH. Cell lysates were subjected to Kinexus phospho site-screen (KPSS)-2.1. Two duplicate experiments were performed with almost identical results.

Results: Phosphorylation of EGFR Tyr 1068 (A) was higher with B1 EGF and was not affected by B1 GH. However, B1 GH increased phosphorylation by Ap EGF. A similar pattern was seen for ErbB2 Tyr 1139 phosphorylation (a Grb docking site) (B) excluding both as an optional scaffold for the interaction between B1 EGF + GH. The autophosphorylation sites of Met (the receptor for hepatocyte growth factor) were not phosphorylated by EGF, GH or their combinations (data not shown), but Tyr 1003 (C) (a negative regulatory and binding site for c-Cbl) was phosphorylated by B1 EGF. The interaction between GH and EGF on Tyr 1003 phosphorylation was again evident only with Ap EGF + B1 GH. FAK phosphorylation on serine 722 was increased by Ap EGF or Bl GH and by Ap EGF + B1 GH, although the increase by the combination was not higher than each alone. B1 EGF and B1 EGF+GH decreased the phosphorylation on this serine (D). Synergism through GH activated IGFl can be ruled out, since insulin R/IGF1 R (Tyrs1162/1163), insulin R (Tyr 972), and IRS1 (Tyr 612) were not phosphorylated by GH or by any other conditions (data not shown).

Conclusion: The use of Kinexus Phosphoprotein Screens can aid in determination of pathways involved in the synergistic interaction between EGF and GFI in enhancing nutrient transport.

*043 Dietary Pyrimidines Induce Jejunal Adaptation Following Massive Small Bowel Resection in Rats. Mary E. Evans, Ph.D., Jun-qiang Tian, M.D., Li H. Gu, M.D., Dean P. Jones, Ph.D., Thomas R. Ziegler, M.D. Emory University, Atlanta, GA, USA.

Introduction: Massive small bowel resection (SBR) increases adaptive growth of residual intestine in rats during the early period after SBR. Pyrimidine nucleotides are critical for DNA and RNA synthesis, but no previous study has evaluated whether supplementation of pyrimidines in the diet enhances adaptive gut growth after SBR. This study determined growth responses in jejunal mucosa following 7 days of dietary supplementation with the nucleobase, uracil, or its precursor, orotate, after massive SBR in rats.

Methods: Sprague-Dawley rats (200 g) underwent 80% jejunoileal resection (RX) or ileal transection (TX; control). Rats were pair-fed a purified (AIN-93G) powdered diet supplemented with or without 1% (w/w) orotate or uracil until sacrifice at 7 days post-surgery. Defined jejunal segments were obtained for analysis of mucosal villus height (VH), crypt depth (CD), total mucosal height (TMH), DNA content, protein content, and bromo-deoxyuridine (BrdU) incorporation as measures of intestinal adaptation.

Results: Jejunal VH increased significantly with SBR, as expected, (TX 438±11 vs RX 562±40. µm, respectively), but orotate or uracil did not further stimulate this response (652±39 vs 585±22 µm, respectively). However, jejunal CD increased significantly with both orotate and uracil supplementation compared to resected animals receiving standard diet (TX 99±4, RX+orotale 140±4, RX+uracil 132±10 and RX+standard diet 108±6 µm, respectively; each p

Conclusion: Supplementation of oral diet with the pyrimidine nucleobase uracil and its precursor orotate are associated with significantly increased adaptive jejunal growth after massive SBR. Supplementation with dietary orotate has more potent growth-stimulatory effects than uracil in this animal model of short bowel syndrome.

*044 Impact of Obesity on Histological Severity of Liver Injury in Hepatitis C. Arun Samanta, MD1, Robert RHK Eng, MD2, J Choe, MD2, Lynn Schellhase, RD2, Thomas Chen, MD2, Koneru Baburao, MD1.

1 New Jersey Medical School, UMDNJ, Newark, NJ, USA, 2 VA Medical Center, East Orange, NJ, USA.

Obesity is associated with hepatic steatosis, a risk factor for liver fibrosis that can progress to cirrhosis as seen in non alcoholic steato-hepatitis (NASH). Obesity makes hepatitis C less responsive to antiviral treatment, mechanism of which is not clear. This raises an important question; whether obesity increases the severity of liver injury in obese patients infected with hepatitis C. The impact of combined insult of obesity related hepatic steatosis and HCV infection on the severity of liver injury in such patients is not clear.

Hypothesis: Obesity with hepatic steatosis and its inherent potential of increasing inflammation and fibrosis, facilitates severity of liver injury in obese patients with hepatitis C infection.

Aim: To investigate severity of histologic liver injury in obese and non-obese patients using standard parameters of liver tissue injury. The study also analyzed the role of liver tissue steatosis, necrosis and inflammation, obesity and hyperlipidemia on the fibrosis seen in liver biopsy.

Methods: Sixty-six patients with chronic hepatitis C who had liver biopsies prior to commencement of their treatment were studied. Patients were categorized in two groups comprising of 26 Obese (BMl >30 Kg/m^sup 2^) and 42 Non-obese (BMI 66% liver cells affected). Lipid profile (serum cholesterol, and triglycerides) were obtained.

Results: Steatosis was present in 77% and was grade 1 in 48%, grade 2 in 23% and grade 3 in 6% of the study population. Liver biopsy in obese patients, (mean BMI of 32±3.0) showed significantly greater necrosis and inflammation (score 2.0±0.7), fibrosis (score 2.6±1.5), and staetosis (score 1.6 ± 0.8) than the Non-obese (mean BMI 24.6+3.5) patients with corresponding scores of 1.4_0.8 (p

Conclusions: Obesity is associated with greater histologic liver injury in hepatitis C. Obesity appears to be a cofactor for increasing hepatic necrosis, inflammation, steatosis and fibrosis in such patients infected with hepatitis C virus. This finding coupled with known poor treatment response for hepatitis C in obese patients suggests more aggressive management of obesity to possibly improve treatment outcome of hepatitis C in such patients.

*045 Does Protocol-driven Normo-glycemic Management In Trauma Patients Affect Outcome? Bryan R. Collier, DO. Vanderbilt University Medical Center, Nashville, TN, USA.

Background: Intensive insulin therapy normalizing glucose levels has demonstrated morbidity and mortality reduction among critically ill patients in the surgical intensive care unit. Recent studies have also suggested a direct relationship between morbidity and mortality and blood glucose levels in critically injured trauma patients. The purpose of this study was to determine if protocol-driven normo-glycemic management in the critically ill trauma patient improves outcomes defined as lower glucose levels, ventilator associated pneumonia (VAP), soft tissue infection (STI), and 30-day mortality.

Methods: A prospective, consecutive series, study design evaluated protocol-driven normo-glycemic management among trauma ICU patients admitted from 4/01/02-4/30/04. Inclusion criteria for this study included mechanical ventilation ≥24 hours and age ≥15 years. On 4/01/03 a glycemic-control protocol was instituted for critically ill trauma patients that requires insulin infusion therapy for blood glucose >110 mg/dl with titration of blood glucose to 80-110 mg/dl. Prior to the protocol, hyperglycemia was treated at the physician's discretion. Data were collected for the following variables: trauma demographics, use of total patenteral nutrition (TPN), maximal daily blood glucose levels up to 14 days post trauma admission, VAP, STI, and 30-day mortality.

Results: From 4/01/02-4/30/04, 818 critically injured patients admitted to the trauma ICU met study criteria. During 4/01/023/31/03, 383 patients met criteria and hyperglycemia was managed without protocol. From 4/01/03-4/30/04, 435 patients met criteria and underwent protocol-driven normo-glycemic management. No differences were noted between the groups regarding age, sex, mechanism of injury, Injury Severity Score, Revised Trauma Score, Glasgow Coma Scale, admission lactate or base deficit levels, history of diabetes mellitus, or use of TPN. During the first 72 hours of care no difference in glucose levels was noted. However, post-trauma days 4-8 demonstrated lower mean glucose levels for the protocol group. Subsequently on post-trauma days 9-14 and averaged over 14 days, no difference was noted in glucose levels between the two groups. Both groups had mean glucose levels ≤150 mg/dl after admission. There was also no difference in VAP (31.6% vs 34.5%; p=0.413), STI (5.0% vs 5.7%; p=0.645) or mortality (12.3% vs 13.1%; p=0.722) between the non-protocol and protocol groups. Despite near normalization of blood glucose values, when patients in either group had at least one episode of blood glucose level ≥150 mg/dl (n=638), regardless of post trauma day, outcomes were worse. These patients had higher glucose levels every day for 14 days after admission, higher VAP rates (35.9% vs 23.3%; p=0.002) and increased mortality (14.6% vs 6.1%; P=0.002). Protocol use in these patients was not associated with an outcome change. During the first 6 months of protocol utilization. (4/01/02-10/01/02; n= 261) glucose levels were higher averaged over 14 days when compared with the last 6 months of the study period (11/01/03-4/30/04: n=174) (133.1 ± 11.7 mg/dl vs 122.4 ± 13.7 mg/dl: p=0.03).

Conclusions: 1) Protocol-driven normo-glycemic management decreases glucose levels without change in outcomes. 2) One or more glucose levels >150 mg/dl is associated with worse outcome.3). A learning curve appears to exist for the protocol.

*046 Arginine Depletion By Myeloid Cells is a Cause Of TCell Dysfunction After Trauma. Benjamin M. Matta, B.S., Valeriya P. Makarenkova, M.D., PhD., Vishal Bansal, M.D., Juan B. Ochoa, M.D., FACS. University of Pittsburgh, Pittsburgh, PA, USA.

Introduction: T-cell dysfunction occurs after surgery and trauma and is associated with an impaired immune response and poor clinical outcome. We describe that cells expressing CD11b^sup +^/Gr-1^sup +^ markers invade lymphoid organs shortly after trauma and express high arginase activity that can cause immunosuppression by depletion of L-arginine, an essential amino acid for normal T-cell function.

Methods: Trauma model was established on C57BL/6 mice which underwent laparotomy with teasing of intra-abdominal contents. Spleens were harvested at 6, 12, 24, 48, and 72 hours later and CD11b^sup +^/Gr-1^sup +^ cells were isolated using MACS beads. For immunohistochemical analysis, frozen spleen sections were stained with anti-Integrin αM (CD11b) and anti-Ly-6G (Gr-1) antibodies. CD11b^sup +^/Gr-1^sup +^ cells were also labeled by CFDA-SE fluorescent dye, and i.v. injected into naïve mice. Splenectomy was performed 24 hours later and spleen sections were analyzed under fluorescent microscope. Arginase 1 expression was determined by Western Blot and arginase activity was detected by colorimetric assay. Arginine uptake by CD11b^sup +^/Gr-1^sup +^ cells was measured by ^sup 3^[H]-arginine incorporation. Control and trauma-induced CD11b^sup +^/Gr-1^sup +^ cells were iv injected into naïve mice (in vivo studies) or co-cultured with naïve T-cells in vitro to investigate their effect on T-cell proliferation (by ^sup 3^[H]thymidine incorporation) and IL-2 production (by ELISA) upon stimulation by anti-CD3 and anti-CD28 antibodies. N-COHydroxy-nor-L-Arginine (nor-NOHA) was used for arginase inhibition.

Results: Maximal increase in CD11b^sup +^ cells, expressing the Gr-1 marker, was observed by Flow cytometry 12 hours after trauma (15% vs. 2% in control, p

Conclusions: We have for the first time demonstrated that arginine depletion after trauma occurs in immune tissues by the appearance of specialized myeloid cells (CD11b^sup +^/Gr-1^sup +^) that express arginase 1. These cells co-localize with T cells and cause T cell dysfunction through arginine depletion. Understanding the importance of arginine depletion in creating immune dysfunction explains the beneficial effects of dietary arginine supplementation in surgical patients.

*047 Dietary Restriction Compromises Resistance To Gut Ischemia-reperfusion, Despite Reduction In Circulating Leukocyte Activation. Chikara Ueno1, Kazuhiko Fukatsu2, Etsuko Hara2, Hidetoshi Nagayoshi1, Hoshio Hiraide2, Hidetaka Mochizuki1. 1 National Defense Medical College Department of Surgery 1, Tokorozawa, Japan, 2 National Defense Medical College Division of Basic Traumatology, Tokorozawa, Japan.

Background: Gut ischemia-reperfusion (I/R) accompanying severe surgical insults leads to neutrophil-mediated tissue injury, and is regarded as a triggering event in early MOF. Our previous study demonstrated dietary restriction to downregulate leukocyte activation in peritonitis, which was associated with a poor prognosis. However, inhibition of leukocyte activation with dietary restriction might be beneficial in terms of surviving I/R. The purpose of this study was to examine the effects of diet restriction on survival after I/R. We also evaluated parameters of leukocyte activation and the level of organ glutathione, an antioxidative substance.

Methods:Male ICR mice received chow. 170 (ad libitum), 119 (MR: mild restriction) or 68 (SR: severe restriction) g/kg per day for 7 days.

Exp.1: The mice (n=59) underwent 15 or 45 minutes of gut ischemia and the survival was observed up to 120 h.

Exp.2: The mice (n=73) were killed before or 60 or 120 minutes after 15 minute ischemia. Reactive oxygen intermediate (ROI) production by circulating myeloid cells (neutrophils + monocytes) with or without phorbol myristate acetate (PMA) stimulation and CD11b expression on these cells were determined using flowcytometry. Some mice were assessed for NFkB activation in these cells with laser scanning cytometry. Lungs, liver, kidneys and small intestine were harvested at 120 minutes after reperfusion for histopathological observation. Glutathione levels were also measured in some of the small intestine and liver samples from each group.

Results: Survival was poorer with dietary restriction (Figure). Circulating myeloid cell priming and activation in terms of ROl production and CD11b expression were enhanced in the ad libitum group, but not in the MR or SR group (Table). Intranuclear translocation of NFkB was increased only in the ad libitum group. Histological examination demonstrated the mice with dietary restriction to have slightly more hepatic necrosis than the ad libitum mice. Gut and hepatic glutathione levels were lower in the SR than in the ad libitum group.

Conclusions: Dietary restriction blunts leukocyte priming and activation after gut ischemic insult, but worsens the outcome at least partly by decreasing anti-oxidative property. Clinically, nutritional replenishment may be required to improve the outcome of gut hypoperfusion.

*048 Dietary Carbohydrates and Glycemic Load and the Risk of Gallstone Disease in U.S. Men. Chung-Jyi Tsai, MD, ScD1, Michael Leitzmann, MD, DrPH2, Walter Willett, MD, DrPH2, Edward Giovannucci, MD, ScD2. 1 Division of Digestive Diseases and Nutrition, University of Kentucky, College of Medicine, Lexington, KY, USA, 2 Channing Laboratory, Harvard Medical School, Boston, MA, USA.

Background: Diets with a high glycemic response exacerbate the metabolic consequences of the insulin resistance syndrome. Their effects on the incidence of gallstone disease are not clear, particularly in men.

Methods: Dietary information was collected as part of the Health Professionals Follow-up Study starting in 1986 using a semi-quantitative food frequency questionnaire with follow-up through 1998. The Health Professionals Follow-up Study is a prospective investigation of 51,529 US male health professionals 40 to 75 years of age in 1986 who returned a mailed questionnaire regarding diet, medications and medical history. On biennial questionnaires the participants reported new symptomatic gallstones. For each participant, we derived an average dietary glycemic index value and calculated glycemic load. Age-adjusted relative risks were calculated using the Mantel-Haenszel summary estimator. For multivariate analyses, the relative risks were simultaneously adjusted for potential confounding variables by using a pooled logistic regression model with two-year time increments.

Results: During 12 years of follow-up, we documented 1,810 new cases of symptomatic gallstones. After adjusting for age and other known or suspected risk factors in the multivariate models, the relative risk (RR) for the highest compared with the lowest quintile of carbohydrate intake was 1.59 (95% C.I., 1.25, 2.02, P for trend = 0.002). The RR for the highest compared with the lowest quintile of dietary glycemic load was 1.50 (95% C.I., 1.20, 1.88, P for trend = 0.0008), and was 1.18 for dietary glycemic index (95% C.I., 1.01, 1.39, P for trend = 0.04). Independent positive associations were also seen for intakes of starch, sucrose, and fructose.

Conclusions: Our findings suggest that a high intake of carbohydrate, glycemic load, and glycemic index increases the risk of gallstone disease in men. These results add to the concern that low-fat, high carbohydrate diets may not be an optimal dietary recommendation.

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