Fructose induced endotoxemia in pediatric nonalcoholic Fatty liver disease.

In preclinical studies of fructose-induced NAFLD, endotoxin appears to play an important role. We retrospectively examined samples from three pediatric cohorts (1) to investigate whether endotoxemia is associated with the presence of hepatic steatosis; (2) to evaluate postprandial endotoxin levels in response to fructose beverage in an acute 24-hour feeding challenge, and (3) to determine the change of fasting endotoxin amounts in a 4-week randomized controlled trial comparing fructose to glucose beverages in NAFLD. We found that adolescents with hepatic steatosis had elevated endotoxin levels compared to obese controls and that the endotoxin level correlated with insulin resistance and several inflammatory cytokines. In a 24-hour feeding study, endotoxin levels in NAFLD adolescents increased after fructose beverages (consumed with meals) as compared to healthy children. Similarly, endotoxin was significantly increased after adolescents consumed fructose beverages for 2 weeks and remained high although not significantly at 4 weeks. In conclusion, these data provide support for the concept of low level endotoxemia contributing to pediatric NAFLD and the possible role of fructose in this process. Further studies are needed to determine if manipulation of the microbiome or other methods of endotoxin reduction would be useful as a therapy for pediatric NAFLD.

Keratin 18, Apoptosis, and Liver Disease in Children.

Keratins, a major component of epithelial cell intermediate filaments, provide structural support to the cell and are important for the maintenance of structural integrity. Beyond its role of structural integrity in hepatocytes, keratin 18 (K18) is a known marker of apoptosis and has been proposed as an indicator of progression in chronic liver diseases such as nonalcoholic fatty liver disease (NAFLD). NAFLD is the most common cause of chronic liver disease in children and adolescents in the United States and throughout the world and comprises a wide spectrum of disease ranging from simple steatosis (fatty liver) to nonalcoholic steatohepatitis (NASH) and cirrhosis. While simple steatosis is typically benign in nature, NASH is a more serious condition that may progress to end-stage liver disease and liver failure. Currently, liver biopsy is considered the most reliable method of assessing the histological severity of disease and differentiating between simple steatosis and NASH. Because biopsy is invasive in nature, expensive, and subject to sampling error and/or variability in interpretation, it is not suitable as a screening test. Therefore, it is necessary to examine known mechanisms associated with the progression of liver disease, such as hepatocellular apoptosis, and identify potential biomarkers that could be used as a diagnostic tool in NASH. This review will focus on the role of apoptosis in pediatric liver disease and how K18, an early marker of apoptosis, can be utilized as a noninvasive biomarker to diagnose NASH.

Characterization of apical and basal thiol-disulfide redox regulation in human colonic epithelial cells.

Control of extracellular thiol-disulfide redox potential (E(h)) is necessary to protect cell surface proteins from external oxidative and reductive stresses. Previous studies show that human colonic epithelial Caco-2 cells, which grow in cell culture with the apical surface exposed to the medium, regulate extracellular cysteine/cystine E(h) to physiological values (approximately -80 mV) observed in vivo. The present study tested whether extracellular E(h) regulation occurs on the basal surface of Caco-2 cells and investigated relevant mechanisms. Experiments were performed with confluent, differentiated cells grown on a permeable membrane surface. Cells were exposed to an oxidizing potential (0 mV) using a fixed cysteine-to-cystine ratio, and culture medium was sampled over time for change in E(h). Regulation of extracellular thiol-disulfide E(h) on the basal domain was faster, and the extent of change at 24 h was greater than on the apical surface. Mechanistic studies showed that redox regulation on the basal surface was partially sodium dependent and inhibited by extracellular lysine, a competitive inhibitor of cystine transport by the y(+)L system and by quisqualic acid, an inhibitor of the x(c)(-) system. Studies using the thiol-reactive alkylating agent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid and the glutathione synthesis inhibitor buthionine sulfoximine showed that extracellular redox regulation was not attributable to plasma membrane cysteine/cystine interconversion or intracellular glutathione, respectively. Thus the data show that redox regulation occurs at different rates on the apical and basal surfaces of the polarized Caco-2 epithelial cell line and that the y(+)L and x(c)(-) systems function in extracellular cysteine/cystine redox regulation on the basal surface.

Acetaminophen elimination half-life in humans is unaffected by short-term consumption of sulfur amino acid-free diet.

Sulfation and glutathione (GSH) conjugation are important pathways for elimination of acetaminophen (APAP). Previous studies in rodents show that limitation of dietary sulfur amino acids (SAAs) reduces biosynthesis of 3'-phosphoadenosine-5'-phosphosulfate, the precursor for sulfation, and GSH, the precursor for the mercapturatic acid pathway. The amount of SAA needed for the metabolism of two doses of APAP is equivalent to 62% of the recommended dietary allowance (RDA) for SAA in humans. A decrease in the activity of these metabolic pathways could lead to decreased elimination of the reactive metabolite of APAP and possibly affect risk of APAP use. To determine whether intake of a SAA-deficient diet alters APAP metabolism, a pilot clinical study with a double-blind, cross-over design was performed. Subjects received the RDA for SAA for 3 days for equilibration. After admission to the clinical research unit, subjects were given a chemically defined diet with 100 or 0% of the RDA for SAA for 2 days. On day 3, two doses of APAP (15 mg/kg) or placebo were given successively within a 6-h interval. Plasma samples were collected at baseline and hourly for 12 h, and two 6-h urine aliquots were collected during this time course. The data show that SAA limitation 1) did not change the pattern of APAP metabolites in plasma or urine and 2) did not alter APAP pharmacokinetics. Thus, the results show that 2 days of diet completely devoid of SAA have no effect on APAP metabolism or disposition in healthy humans.

Oxidation of plasma cysteine/cystine and GSH/GSSG redox potentials by acetaminophen and sulfur amino acid insufficiency in humans.

Variations in plasma sulfur amino acid (SAA) pools are associated with disease risks, but little information is available about the factors affecting plasma SAA pools. Drug metabolism by glutathione (GSH) and sulfate conjugation can, in principle, represent a quantitatively important burden on SAA supply. The present study was designed to determine whether therapeutic doses of acetaminophen (APAP) alter SAA metabolism in healthy human adults. A double-blind, crossover design incorporating four treatment periods with diets providing 100% of the recommended dietary allowance (RDA) for SAA without or with APAP (15 mg/kg) and 0% RDA for SAA without or with APAP, in randomized order. After a 3-day equilibration period, chemically defined diets with 100 or 0% RDA for SAA were given for 2 complete days. On day 3, APAP or placebo was given in two successive doses (6-h interval), and timed plasma samples were collected. With SAA intake at 100% RDA, APAP administration oxidized the plasma cysteine/cystine redox potential (E(h)CySS) but not the plasma GSH/GSSG redox potential (E(h)GSSG). The extent of oxidation caused by APAP was similar to that seen with 0% SAA and no APAP. However, APAP administration with 0% SAA did not cause further oxidation beyond APAP or 0% SAA alone. In contrast, an oxidation of the plasma E(h)GSSG was apparent for SAA insufficiency only with APAP. The results suggest a need to evaluate possible effects of APAP in association with SAA insufficiency as a contributing factor in disease risk.

Dietary sulfur amino acid supplementation reduces small bowel thiol/disulfide redox state and stimulates ileal mucosal growth after massive small bowel resection in rats.

Following massive small bowel resection in animal models, the remnant intestine undergoes a dynamic growth response termed intestinal adaptation. Cell growth and proliferation are intimately linked to cellular and extracellular thiol/disulfide redox states, as determined by glutathione (GSH) and GSH disulfide (GSSG) (the major cellular redox system in tissues), and cysteine (Cys) and its disulfide cystine (CySS) (the major redox system in plasma), respectively. The study was designed to determine whether dietary supplementation with sulfur amino acids (SAA) leads to a greater reduction in thiol/disulfide redox state in plasma and small bowel and colonic mucosa and alters gut mucosal growth in an established rat model of short bowel syndrome (SBS). Adult rats underwent 80% jejunal-ileal resection (RX) or small bowel transection (surgical control) and were pair-fed either isonitrogenous, isocaloric SAA-adequate (control) or SAA-supplemented diets (218% increase vs. control diet). Plasma and gut mucosal samples were obtained after 7 d and analyzed for Cys, CySS, GSH, and GSSG concentrations by HPLC. Redox status (E(h)) of the Cys/CySS and GSH/GSSG couples were calculated using the Nernst equation. SAA supplementation led to a greater reduction in E(h) GSH/GSSG in jejunal and ileal mucosa of resected rats compared with controls. Resected SAA-supplemented rats showed increased ileal adaptation (increased full-thickness wet weight, DNA, and protein content compared with RX control-fed rats; increased mucosal crypt depth and villus height compared with all other study groups). These data suggest that SAA supplementation has a trophic effect on ileal adaptation after massive small bowel resection in rats. This finding may have translational relevance as a therapeutic strategy in human SBS.

Glutamine and KGF each regulate extracellular thiol/disulfide redox and enhance proliferation in Caco-2 cells.

Glutamine (Gln) and keratinocyte growth factor (KGF) each stimulate intestinal epithelial cell growth, but regulatory mechanisms are not well understood. We determined whether Gln and KGF alter intra- and extracellular thiol/disulfide redox pools in Caco-2 cells cultured in oxidizing or reducing cell medium and whether such redox variations are a determinant of proliferative responses to these agents. Cells were cultured over a physiological range of oxidizing to reducing extracellular thiol/disulfide redox (Eh) conditions, obtained by varying cysteine (Cys) and cystine (CySS) concentrations in cell medium. Cell proliferation was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation. Gln (10 mmol/l) or KGF (10 microg/l) did not alter BrdU incorporation at reducing Eh (-131 to -150 mV), but significantly increased incorporation at more oxidizing Eh (Gln at 0 to -109 mV; KGF at -46 to -80 mV). Cellular glutathione/glutathione disulfide (GSH/GSSG) Eh was unaffected by Gln, KGF, or variations in extracellular Cys/CySS Eh. Control cells largely maintained extracellular Eh at initial values after 24 h (-36 to -136 mV). However, extracellular Eh shifted toward a narrow physiological range with Gln and KGF treatment (Gln -56 to -88 mV and KGF -76 to -92 mV, respectively; P < 0.05 vs. control). The results indicate that thiol/disulfide redox state in the extracellular milieu is an important determinant of Caco-2 cell proliferation induced by Gln and KGF, that this control is independent of intracellular GSH redox status, and that both Gln and KGF enhance the capability of Caco-2 cells to modulate extremes of extracellular redox.