Charles H. Halsted

Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism

Autism is a behaviorally defined neurodevelopmental disorder usually diagnosed in early childhood that is characterized by impairment in reciprocal communication and speech, repetitive behaviors, and social withdrawal. Although both genetic and environmental factors are thought to be involved, none have been reproducibly identified. The metabolic phenotype of an individual reflects the influence of endogenous and exogenous factors on genotype. As such, it provides a window through which the interactive impact of genes and environment may be viewed and relevant susceptibility factors identified. Although abnormal methionine metabolism has been associated with other neurologic disorders, these pathways and related polymorphisms have not been evaluated in autistic children. Plasma levels of metabolites in methionine transmethylation and transsulfuration pathways were measured in 80 autistic and 73 control children. In addition, common polymorphic variants known to modulate these metabolic pathways were evaluated in 360 autistic children and 205 controls. The metabolic results indicated that plasma methionine and the ratio of S‐adenosylmethionine (SAM) to S‐adenosylhomocysteine (SAH), an indicator of methylation capacity, were significantly decreased in the autistic children relative to age‐matched controls. In addition, plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, an indication of antioxidant capacity and redox homeostasis, were significantly decreased. Differences in allele frequency and/or significant gene–gene interactions were found for relevant genes encoding the reduced folate carrier (RFC 80G > A), transcobalamin II (TCN2 776G > C), catechol‐O‐methyltransferase (COMT 472G > A), methylenetetrahydrofolate reductase (MTHFR 677C > T and 1298A > C), and glutathione‐S‐transferase (GST M1). We propose that an increased vulnerability to oxidative stress (endogenous or environmental) may contribute to the development and clinical manifestations of autism.

Metabolic Interactions of Alcohol and Folate

The goals and objectives of these studies, conducted over the past 30 y, were to determine: a) how chronic alcoholism leads to folate deficiency and b) how folate deficiency contributes to the pathogenesis of alcoholic liver disease (ALD). The intestinal absorption of folic acid was decreased in binge drinking alcoholics and, prospectively, in volunteers fed alcohol with low folate diets. Monkeys fed alcohol for 2 y developed decreased hepatic folate stores, folic acid malabsorption and decreased hepatic uptake but increased urinary excretion of labeled folic acid. Micropigs fed alcohol for 1 y developed features of ALD in association with decreased translation and activity of intestinal reduced folate carrier. Another study in ethanol-fed micropigs demonstrated abnormal hepatic methionine and DNA nucleotide imbalance and increased hepatocellular apoptosis. When alcohol feeding was combined with folate deficiency, micropigs developed typical histological features of ALD in 14 wk, together with elevated plasma homocysteine levels, reduced liver S-adenosylmethionine and glutathione and increased markers for DNA and lipid oxidation. In summary, chronic alcohol exposure impairs folate absorption by inhibiting expression of the reduced folate carrier and decreasing the hepatic uptake and renal conservation of circulating folate. At the same time, folate deficiency accelerates alcohol-induced changes in hepatic methionine metabolism while promoting enhanced oxidative liver injury and the histopathology of ALD.

Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo.

Hepatic stellate cell activation is a main feature of liver fibrogenesis. We have previously shown that phagocytosis of apoptotic bodies by stellate cells induces procollagen α1 (I) and transforming growth factor beta (TGF‐β) expression in vitro. Here we have further investigated the downstream effects of phagocytosis by studying NADPH oxidase activation and its link to procollagen α1 (I) and TGF‐β1 expression in an immortalized human stellate cell line and in several models of liver fibrosis. Phagocytosis of apoptotic bodies in LX‐1 cells significantly increased superoxide production both in the extracellular and intracellular milieus. By confocal microscopy of LX‐1 cells, increased intracellular reactive oxygen species (ROS) were detected in the cells with intracellular apoptotic bodies, and immunohistochemistry documented translocation of the NADPH oxidase p47phox subunit to the membrane. NADPH oxidase activation resulted in upregulation of procollagen α1 (I); in contrast, TGF‐β1 expression was independent of NADPH oxidase activation. This was also confirmed by using siRNA to inhibit TGF‐β1 production. In addition, with EM studies we showed that phagocytosis of apoptotic bodies by stellate cells occurs in vivo. In conclusion, these data provide a mechanistic link between phagocytosis of apoptotic bodies, production of oxidative radicals, and the activation of hepatic stellate cells. (HEPATOLOGY 2006;43:435–443.)

Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig

Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM/S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2′-deoxyguanosine [oxo(8)dG]/mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM/SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury.

Alcohol and the Gastrointestinal Tract

The effects of acute and chronic ethanol ingestion on esophageal motility and the potential complications of these alterations are reviewed. Injury to the gastric mucosa and the small intestine and alterations in intestinal absorption can also result from alcohol abuse.

Intestinal Malabsorption In Folate-Deficient Alcoholics

Malnutrition with folate deficiency is frequently found among alcoholics, and could be caused in part by decreased intestinal absorption. To evaluate the relationship of nutrition and absorption in alcoholics, intestinal absorption was studied in patient volunteers placed on a folate-deficient diet with ethanol. Intestinal absorption was measured by conventional means and by the technique of triple lumen perfusion of the jejunum. In 2 patients who ingested ethanol, 200 g per day with a low folate diet, the dietary induction of folate deficiency was followed by decreased absorption of d-xylose, labeled folic acid (3H-pteroylglutamic acid), glucose, fluid, and sodium. Mild net secretion of fluid and sodium into the intestinal lumen was observed in a patient who remained sober on the low folate diet and in a patient who ingested ethanol, 300 g per day, with a regular diet. The morphology of the jejunal mucosa was not affected. These preliminary data suggest that the combination of dietary folate deficiency and prolonged ethanol intake results in intestinal malabsorption of several water-soluble substances, which may account in part for the poor nutrition often found in binge drinkers.

Human immunodeficiency virus infection of enterocytes and mononuclear cells in human jejunal mucosa

Intestinal malabsorption is a recognized cause of malnutrition in patients infected with human immunodeficiency virus. However, the relationships among human immunodeficiency virus infection, morphological changes in the intestine, and development of intestinal malabsorption are not well established. Nine patients infected with human immunodeficiency virus underwent tests of intestinal absorption and jejunal biopsies for morphometric measurements, enzyme assays, and virus detection by in situ hybridization. Steatorrhea and low lactase activities were found in more than 85% of the patients. All biopsy specimens were abnormal with reversal of the ratio of villus length to crypt depth in seven and enlarged enterocyte nuclear size in nine. Human immunodeficiency virus was detected in five jejunal biopsy specimens, within villus enterocytes of one patient who had the most severe malabsorption of the group and in four other biopsy specimens in mononuclear infiltrating cells of the lamina propria. These results suggest that human immunodeficiency virus infection of the small intestinal mucosa is an early event that is associated with altered enterocyte differentiation and function.

Folylpoly-γ-glutamate Carboxypeptidase from Pig Jejunum MOLECULAR CHARACTERIZATION AND RELATION TO GLUTAMATE CARBOXYPEPTIDASE II

Jejunal folylpoly-γ-glutamate carboxypeptidase hydrolyzes dietary folates prior to their intestinal absorption. The complete folylpoly-γ-glutamate carboxypeptidase cDNA was isolated from a pig jejunal cDNA library using an amplified homologous probe incorporating primer sequences from prostate-specific membrane antigen, a protein capable of folate hydrolysis. The cDNA encodes a 751-amino acid polypeptide homologous to prostate-specific membrane antigen and rat brain N-acetylated α-linked acidic dipeptidase. PC3 transfectant membranes exhibited activities of folylpoly-γ-carboxypeptidase and N-acetylated α-linked acidic dipeptidase, while immunoblots using monoclonal antibody to native folylpoly-γ-glutamate carboxypeptidase identified a glycoprotein at 120 kDa and a polypeptide at 84 kDa. The kinetics of native folylpoly-γ-carboxypeptidase were expressed in membranes of PC3 cells transfected with either pig folylpoly-γ-carboxypeptidase or human prostate-specific membrane antigen. Folylpoly-γ-carboxypeptidase transcripts were identified at 2.8 kilobase pairs in human and pig jejunum, human and rat brain, and human prostate cancer LNCaP cells. Thus, pig folylpoly-γ-carboxypeptidase, rat N-acetylated α-linked acidic dipeptidase, and human prostate-specific membrane antigen appear to represent varied expressions of the same gene in different species and tissues. The discovery of the jejunal folylpoly-γ-carboxypeptidase gene provides a framework for future studies on relationships among these proteins and on the molecular regulation of intestinal folate absorption.

Hepatic transmethylation reactions in micropigs with alcoholic liver disease.

Alcoholic liver disease is associated with abnormal hepatic methionine metabolism, including increased levels of homocysteine and S‐adenosylhomocysteine (SAH) and reduced levels of S‐adenosylmethionine (SAM) and glutathione (GSH). The concept that abnormal methionine metabolism is involved in the pathogenesis of alcoholic liver disease was strengthened by our previous findings in a micropig model where combining dietary folate deficiency with chronic ethanol feeding produced maximal changes in these metabolites together with early onset of microscopic steatohepatitis and an eightfold increase in plasma aspartate aminotransferase. The goal of the present study was to determine potential mechanisms for abnormal levels of these methionine metabolites by analyzing the transcripts and activities of transmethylation enzymes in the livers of the same micropigs. Ethanol feeding or folate deficiency, separately or in combination, decreased transcript levels of methylenetetrahydrofolate reductase (MTHFR), methionine adenosyltransferase (MAT1A), glycine‐N‐methyltransferase (GNMT) and S‐adenosylhomocysteine hydrolase (SAHH). Ethanol feeding alone reduced the activities of methionine synthase (MS) and MATIII and increased the activity of GNMT. Each diet, separately or in combination, decreased the activities of MTHFR and SAHH. In conclusion, the observed abnormal levels of methionine metabolites in this animal model of accelerated alcoholic liver injury can be ascribed to specific effects of ethanol with or without folate deficiency on the expressions and activities of hepatic enzymes that regulate transmethylation reactions. These novel effects on transmethylation reactions may be implicated in the pathogenesis of alcoholic liver disease. (HEPATOLOGY 2004;39:1303–1310.)

Evaluation of genetic variants in the reduced folate carrier and in glutamate carboxypeptidase II for spina bifida risk

Genetic variants in folate metabolism have been reported to increase risk for neural tube defects (NTD). The first such sequence change was the 677C-->T substitution in methylenetetrahydrofolate reductase (MTHFR), but additional sequence changes have been identified in enzymes or transporters for folates. Two recently identified variants are the 1561C-->T (H475Y) mutation in glutamate carboxypeptidase II (GCPII) and the 80A-->G (H27R) change in the reduced folate carrier RFC-1. We examined a group of mothers of spina bifida offspring, and a group of control women, for the above polymorphisms to assess their impact on NTD risk as well as on homocysteine and nutrient (RBC folate, serum folate, and serum cobalamin) levels. The GCPII variant (in the heterozygous state) did not influence NTD risk or metabolite levels; homozygous mutant (YY) women were not observed in our study group. The homozygous mutant (RR) genotype for the RFC-1 gene was not associated with a significant difference in NTD risk (OR=1.39, 95% CI=0.55-3.54), but there was a borderline significant (p=0.065) decrease in RBC folate levels, compared with the HH genotype. However, the combination of the RR genotype for RFC-1 and low RBC folate was associated with a significant 4.6-fold increase in NTD risk (OR=4.6, 95% CI=1.47-14.37). Since this small study is the first to demonstrate increased risk for women with the RFC-1 variant for having a child with a NTD, additional larger studies are required to confirm this change as another potential genetic modifier for spina bifida risk.