Variations in hepatic lipid species of age-matched male mice fed a methionine-choline-deficient diet and housed in different animal facilities.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a common disease and feeding mice a methionine-choline-deficient (MCD) diet is a frequently used model to study its pathophysiology. Genetic and environmental factors influence NASH development and liver lipid content, which was studied herein using C57BL/6 J mice bred in two different animal facilities. METHODS: Age-matched male C57BL/6 J mice bred in two different animal facilities (later on referred to as WT1 and WT2) at the University Hospital of Regensburg were fed identical MCD or control chows for 2 weeks. Hepatic gene and protein expression and lipid composition were determined. RESULTS: NASH was associated with increased hepatic triglycerides, which were actually higher in WT1 than WT2 liver in both dietary groups. Cholesterol contributes to hepatic injury but was only elevated in WT2 NASH liver. Ceramides account for insulin resistance and cell death, and ceramide species d18:1/16:0 and d18:1/18:0 were higher in the NASH liver of both groups. Saturated sphingomyelins only declined in WT1 NASH liver. Lysophosphatidylcholine concentrations were quite normal in NASH and only one of the 12 altered phosphatidylcholine species declined in NASH liver of both groups. Very few phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol species were comparably regulated in NASH liver of both animal groups. Seven of these lipid species declined and two increased in NASH. Notably, hepatic mRNA expression of proinflammatory (F4/80, CD68, IL-6, TNF and chemerin) and profibrotic genes (TGF beta and alpha SMA) was comparable in WT1 and WT2 mice. CONCLUSIONS: Mice housed and bred in different animal facilities had comparable disease severity of NASH whereas liver lipids varied among the groups. Thus, there was no specific lipid signature for NASH in the MCD model.

p16 deficiency promotes nonalcoholic steatohepatitis via regulation of hepatic oxidative stress.

Nonalcoholic steatohepatitis (NASH) is characterized by excess accumulation of lipids in liver, accompanied with hepatocyte injury, cell death and inflammation. Although p16 is known as tumor suppressor in multiple cancer types, it remains unclear whether p16 plays a critical role in NASH. To determine whether p16 could play a role in the pathogenesis of NASH, wild-type mice and p16-/- mice were fed on a methionine and choline-deficient (MCD) diet for 3 weeks, and liver steatosis, fibrosis, and inflammation were evaluated. Our data show that p16-/- mice fed with MCD diet displayed more significant hepatic steatosis, hepatocyte damage, increased oxidative stress and inflammatory cell infiltration compared to MCD-fed WT mice. It was also clear that the increased ROS and the accumulation of lipid in BEL-7402 cells occurred when p16 expression was depleted with siRNA. These findings indicate that p16 may play a critical role in the development of NASH by reining in ROS production and by inhabiting inflammatory response.

Elevation of liver endoplasmic reticulum stress in a modified choline-deficient l-amino acid-defined diet-fed non-alcoholic steatohepatitis mouse model.

Endoplasmic reticulum (ER) stress caused by accumulation of misfolded proteins is observed in several kinds of diseases. Since ER stress is reported to be involved in the progression of non-alcoholic steatohepatitis (NASH), highly sensitive and simple measurement methods are required for research into developing novel therapy for NASH. To investigate the involvement of ER stress in NASH pathogenesis in a mouse model, an assay for liver ER stress was developed using ER stress activated indicator-luciferase (ERAI-Luc) mice. To establish the assay method for detection of ER stress in the liver, tunicamycin (TM) (0.3 mg/kg i. p.) was administered to ERAI-Luc mice, and the luciferase activity was measured in ex vivo and in vivo. To evaluate ER stress in the NASH model, ERAI-Luc mice were fed a modified choline-deficient l-amino acid-defined (mCDAA) diet for 14 weeks. After measurement of ER stress by luminescence imaging, levels of liver lipids and pro-fibrotic and pro-inflammatory gene expression were measured as NASH-related indexes. In non-invasive whole-body imaging, TM elevated luciferase activity in the liver, induced by activation of ER stress. The highest luminescence in the liver was confirmed by ex vivo imaging of isolated tissues. In parallel with progression of NASH, elevated luminescence induced by ER stress in liver was observed in mCDAA diet-fed ERAI-Luc mice. Luciferase activity was significantly and positively correlated to levels of triglyceride and free cholesterol in the liver, as well as to the mRNA expression of type 1 collagen α1 chain and tumor necrosis factor α. These data indicated that the use of ERAI-Luc mice was effective in the detection of ER stress in the liver. Moreover, the NASH model using ERAI-Luc mice can be a useful tool to clarify the role of ER stress in pathogenesis of NASH and to evaluate effects of drugs targeted against ER stress.

Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts.

Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2-3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired.

Choline Supplementation With a Structured Lipid in Children With Cystic Fibrosis: A Randomized Placebo-Controlled Trial.

BACKGROUND: Choline depletion is seen in cystic fibrosis (CF) and pancreatic insufficiency in spite of enzyme treatment and may result in liver, fatty acid, and muscle abnormalities. This study evaluated the efficacy and safety of an easily absorbed choline-rich structured lipid (LYM-X-SORB™ [LXS]) to improve choline status. METHODS: Children with CF and pancreatic insufficiency were randomized to LXS or placebo in a 12-month double blind trial. Dietary choline intake, plasma cholines, plasma and fecal phospholipids, coefficient of fat absorption, pulmonary function, growth status, body composition, and safety measures were assessed. Magnetic resonance spectroscopy for calf muscle choline and liver fat were assessed in a subgroup and compared with a healthy comparison group matched for age, sex, and body size. RESULTS: A total of 110 subjects were enrolled (age 10.4 ±â€Š3.0 years). Baseline dietary choline, 88% recommended, increased 3-fold in the LXS group. Plasma choline, betaine, and dimethylglycine increased in the LXS but not placebo (P = 0.007). Plasma lysophosphatidylcholine and phosphatidylcholine increased, and fecal phosphatidylcholine/phosphatidylethanolamine ratio decreased (P ≤ 0.05) in LXS only, accompanied by a 6% coefficient of fat absorption increase (P = 0.001). Children with CF had higher liver fat than healthy children and depleted calf muscle choline at baseline. Muscle choline concentration increased in LXS and was associated with improvement in plasma choline status. No relevant changes in safety measures were evident. CONCLUSIONS: LXS had improved choline intake, plasma choline status, and muscle choline stores compared with placebo group. The choline-rich supplement was safe, accepted by participants, and improved choline status in children with CF.

Plasma free choline, betaine and cognitive performance: the Hordaland Health Study.

Choline and betaine are nutrients involved in one-carbon metabolism. Choline is essential for neurodevelopment and brain function. We studied the associations between cognitive function and plasma concentrations of free choline and betaine. In a cross-sectional study, 2195 subjects (55 % women), aged 70-74 years, underwent extensive cognitive testing including the Kendrick Object Learning Test (KOLT), Trail Making Test (part A, TMT-A), modified versions of the Digit Symbol Test (m-DST), Block Design (m-BD), Mini-Mental State Examination (m-MMSE) and Controlled Oral Word Association Test (COWAT). Compared with low concentrations, high choline (>8·4 µmol/l) was associated with better test scores in the TMT-A (56·0 v. 61·5, P=0·004), m-DST (10·5 v. 9·8, P=0·005) and m-MMSE (11·5 v. 11·4, P=0·01). A generalised additive regression model showed a positive dose-response relationship between the m-MMSE and choline (P=0·012 from a corresponding linear regression model). Betaine was associated with the KOLT, TMT-A and COWAT, but after adjustments for potential confounders, the associations lost significance. Risk ratios (RR) for poor test performance roughly tripled when low choline was combined with either low plasma vitamin B12 (≤257 pmol/l) concentrations (RR(KOLT)=2·6, 95 % CI 1·1, 6·1; RR(m-MMSE)=2·7, 95 % CI 1·1, 6·6; RR(COWAT)=3·1, 95 % CI 1·4, 7·2) or high methylmalonic acid (MMA) (≥3·95 µmol/l) concentrations (RR(m-BD)=2·8, 95 % CI 1·3, 6·1). Low betaine (≤31·1 µmol/l) combined with high MMA was associated with elevated RR on KOLT (RR(KOLT)=2·5, 95 % CI 1·0, 6·2). Low plasma free choline concentrations are associated with poor cognitive performance. There were significant interactions between low choline or betaine and low vitamin B12 or high MMA on cognitive performance.

Choline supply of preterm infants: assessment of dietary intake and pathophysiological considerations.

BACKGROUND: Choline forms the head group of phosphatidylcholines, comprising 40-50 % of cellular membranes and 70-95 % of phospholipids in surfactant, bile, and lipoproteins. Moreover, choline serves as the precursor of acetylcholine and is important for brain differentiation and function. While accepted as essential for fetal and neonatal development, its role in preterm infant nutrition has not yet gained much attention. METHODS: The adequate intake of choline of preterm infants was estimated from international recommendations for infants, children, and adults. Choline intake relative to other nutrients was determined retrospectively in all inborn infants below 1,000 g (extremely low birth weight) or below 28 weeks gestational age, admitted to our department in 2006 and 2007 (N = 93). RESULTS: Estimation of adequate intake showed that children with 290 g body weight need more choline than those with 1,200 g (31.4 and 25.2 mg/kg/day, respectively). Day-by-day variability was high for all nutrient intakes including choline. In contrast to the continuous intrauterine choline delivery, median supply reached a plateau at d11 (21.7 mg/kg/day; 25th/75th percentile: 19.6; 23.9). Individual choline supply at d0-d1 and d2-d3 was <10 mg/kg/day in 100 and 69 % of infants, respectively. Furthermore, intakes <10 mg/kg/day were frequently observed beyond day 11. Median adequate intakes (27.4 mg/kg/day at 735 g body weight) were achieved in <2 %. CONCLUSIONS: Nutritional intake of choline in this cohort of preterm infants was frequently less than the estimated adequate intake, with particular shortage until postnatal d10. Because choline is important for brain development, future studies are needed to investigate the effects of adequate nutritional choline intake on long-term neurodevelopment in VLBW infants.

Choline and betaine in health and disease.

Choline is an essential nutrient, but is also formed by de novo synthesis. Choline and its derivatives serve as components of structural lipoproteins, blood and membrane lipids, and as a precursor of the neurotransmitter acetylcholine. Pre-and postnatal choline availability is important for neurodevelopment in rodents. Choline is oxidized to betaine that serves as an osmoregulator and is a substrate in the betaine-homocysteine methyltransferase reaction, which links choline and betaine to the folate-dependent one-carbon metabolism. Choline and betaine are important sources of one-carbon units, in particular, during folate deficiency. Choline or betaine supplementation in humans reduces concentration of total homocysteine (tHcy), and plasma betaine is a strong predictor of plasma tHcy in individuals with low plasma concentration of folate and other B vitamins (B2, B6, and B12) in combination TT genotype of the methylenetetrahydrofolate reductase 677 C->T polymorphism. The link to one-carbon metabolism and the recent availability of food composition data have motivated studies on choline and betaine as risk factors of chronic diseases previously studied in relation to folate and homocysteine status. High intake and plasma level of choline in the mother seems to afford reduced risk of neural tube defects. Intake of choline and betaine shows no consistent relation to cancer or cardiovascular risk or risk factors, whereas an unfavorable cardiovascular risk factor profile was associated with high choline and low betaine concentrations in plasma. Thus, choline and betaine showed opposite relations with key components of metabolic syndrome, suggesting a disruption of mitochondrial choline oxidation to betaine as part of the mitochondrial dysfunction in metabolic syndrome.

Oral choline supplementation in children with intestinal failure.

Choline deficiency leads to steatohepatitis, elevated transaminases, susceptibility to septic shock, and an increased risk of central catheter thrombosis. Children with intestinal failure (IF) are at risk for choline deficiency. In an unblinded, open-label study, we studied 7 children with IF on parenteral nutrition, measured their plasma free choline level, and, if low, supplemented enterally with adequate intake (AI) doses of choline. Four to 6 weeks later we remeasured their plasma free choline. Unlike adults, infants did not respond to oral choline supplementation at AI doses. Additionally, we have calculated plasma free choline percentiles versus age for normal children.

Choline-deprivation alters crucial brain enzyme activities in a rat model of diabetic encephalopathy.

Diabetic encephalopathy describes the moderate cognitive deficits, neurophysiological and structural central nervous system changes associated with untreated diabetes. It involves neurotoxic effects such as the generation of oxidative stress, the enhanced formation of advanced glycation end-products, as well as the disturbance of calcium homeostasis. Due to the direct connection of choline (Ch) with acetylcholine availability and signal transduction, a background of Ch-deficiency might be unfavorable for the pathology and subsequently for the treatment of several metabolic brain diseases, including that of diabetic encephalopathy. The aim of this study was to shed more light on the effects of adult-onset streptozotocin (STZ)-induced diabetes and/or Ch-deprivation on the activities of acetylcholinesterase (AChE) and two important adenosine triphosphatases, namely Na(+),K(+)-ATPase and Mg(2+)-ATPase. Male adult Wistar rats were divided into four main groups, as follows: control (C), diabetic (D), Ch-deprived (CD), and Ch-deprived diabetic (D+CD). Deprivation of Ch was provoked through the administration of Ch-deficient diet. Both the induction of diabetes and the beginning of dietary-mediated provoking of Ch-deprivation occurred at the same day, and rats were killed by decapitation after 30 days (1 month; groups C1, D1, CD1 and D1+CD1) and 60 days (2 months; groups C2, D2, CD2 and D2+CD2, respectively). The adult rat brain AChE activity was found to be significantly increased by both diabetes (+10%, p < 0.001 and +11%, p < 0.01) and Ch-deprivation (+19%, p < 0.001 and +14%, p < 0.001) when compared to the control group by the end of the first (C1) and the second month (C2), respectively. However, the Ch-deprived diabetic rats' brain AChE activity was significantly altered only after a 60-day period of exposure, resulting in a +27% increase (D2+CD2 vs. C2, p < 0.001). Although the only significant change recorded in the brain Na(+),K(+)-ATPase activity after the end of the first month is attributed to Ch-deprivation (+21%, p < 0.05, CD1 vs. C1), all groups of the second month exhibited a statistically significant decrease in brain Na(+),K(+)-ATPase activity (-24%, p < 0.01, D2 vs. C2; -21%, p < 0.01, CD2 vs. C2; -22%, p < 0.01, D2+CD2 vs. C2). As concerns Mg(2+)-ATPase, the enzyme's activity demonstrates no significant changes, with the sole exception of the D2+CD2 group (+21%, p < 0.05, D2+CD2 vs. C2). In addition, statistically significant time-dependent changes concerning the brain Mg(2+)-ATPase activity were recorded within the diabetic (p < 0.05, D2 vs. D1) and the Ch-deprived (p < 0.05, CD2 vs. CD1) rat groups. Our data indicate that Ch-deprivation seems to be an undesirable background for the above-mentioned enzymatic activities under untreated diabetes, in a time-evolving way. Further studies on the issue should focus on a region-specific reevaluation of these crucial enzymes' activities as well as on the possible oxidative mechanisms involved.

Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta?

BACKGROUND: Hepatic oval cells, progenitor cells in the liver, can differentiate into hepatocytes and bile duct cells both in vitro and in vivo. Although hepatic stellate cells are another important cell component in the liver, less attention has been focused on the relationship between hepatic oval cells and hepatic stellate cells. METHODS: Hepatic oval cells were isolated from rats fed a choline-deficient diet supplemented with 0.1% ethionine for 6 weeks and characterized by electron microscopy, flow cytometry, reverse transcription polymerase chain reaction, Western blot and bi-direction differentiation. After treatment with transforming growth factor-beta1 (TGF-beta1), changes in cell viability, morphology, extracellular matrix (ECM) expression and immune phenotype were analysed in these cultured and adherent hepatic oval cells. RESULTS: The primary cultured hepatic oval cells were positive for the oval cell-specific markers OV-6, BD-1/BD-2 and M2PK as well as the hepatocyte markers albumin and alpha-foetoprotein. These hepatic oval cells differentiated bipotentially into hepatocytes or bile duct-like cells under appropriate conditions. It is noteworthy that these bipotential hepatic oval cells expressed ECM genes stably, including collagens, matrix metalloproteinases and tissue inhibitor of mellatoproteinase. Furthermore, except for growth inhibition and morphological changes in the hepatic oval cells after exposure to TGF-beta1, there was an increased expression of ECM genes, the onset expression of snail and loss expression of E-cadherin. During this process, TGF-beta1 treatment induced an upregulation of marker genes for hepatic stellate cells in hepatic oval cells, such as desmin and GFAP. CONCLUSION: Except for the expression of ECM, the cultured hepatic oval cells could induce an increased expression of hepatic stellate cell markers by TGF-beta1 through an epithelial-mesenchymal transition process, which might indicate the contribution of hepatic oval cells to liver fibrosis.

Butein protects the nonalcoholic fatty liver through mitochondrial reactive oxygen species attenuation in rats.

One of the worldwide metabolic health dilemma is nonalcoholic fatty liver diseases (NAFLD). Researchers are searching effective drug to manage NAFLD patients. One of the best way to manage the metabolic imperfection is through natural principal isolated from different sources. Butein, a natural compound known to have numerous pharmacological application. In the current study we assessed the therapeutic effect of butein administration on liver function tests, oxidative stress, antioxidants, lipid abnormalities, serum inflammatory cytokines, and mitochondrial reactive oxygen species levels, in rats with methionine-choline deficient (MCD) diet induced NAFLD. Male Wistar rats were treated with MCD diet with/without butein (200 mg/kg body wt. orally) for 6 weeks. The protective effect of butein, were evident from decreased transaminase activities, restoration of albumin, globulin, albumin/globulin ratio, and oxidants in serum (P < 0.01), further it improved liver antioxidant status (P < 0.01). Butein significantly lowered lipid profile parameters (P < 0.01), suppressed inflammatory cytokines (P < 0.01), and improved liver histology. Further to understand the possible mechanism behind the hepatoprotective and lipid lowering effect of butein, the activities of heme oxygenase (HO1), myeloperoxidase (MPO), and mitochondrial reactive oxygen species (ROS) were measured. We found that butein supplementation significantly decreased the activity of HO1 (P < 0.001), and increased the activity of MPO (P < 0.001). Furthermore butein attenuated mitochondrial ROS produced in NAFLD condition. Present study shows that butein supplementation restore liver function by altering liver oxidative stress, inflammatory markers, vital defensive enzyme activities, and mitochondrial ROS. In summary, butein has remarkable potential to develop effective hepato-protective drug. © 2018 BioFactors, 44(3):289-298, 2018.

Pro-thrombotic and pro-oxidant effects of diet-induced hyperhomocysteinemia.

Elevated plasma homocysteine levels are associated with the risk of atherosclerosis and arterial and venous thrombosis. We have previously demonstrated that rabbits rendered hyperhomocysteinemic by parenteral administration of homocysteine develop a dysfibrinogenemia that is associated with the formation of fibrin clots that are abnormally resistant to fibrinolysis. We suggested that this acquired dysfibrinogenemia contributes to the thrombotic tendency in hyperhomocysteinemia. However, it was possible that the homocysteine-associated dysfibrinogenemia was an artifact of the parenteral administration model. Therefore, the goals of the current study were to develop a diet-induced model of homocysteinemia in rabbits and determine whether a dysfibrinogenemia and evidence of oxidative stress develop in this model as they do when homocysteine is injected. We found that rabbits fed a diet severely deficient in folate and mildly deficient in choline develop mild hyperhomocysteinemia: 14.8+/-4.0 microM in deficient rabbits compared to 9.0+/-1.7 microM in controls. The deficient rabbits also develop evidence of oxidant stress: increased lipid peroxidation in liver, impaired mitochondrial enzyme activities in liver and elevated caspase-3 levels in plasma. Most importantly, the deficient rabbits also develop a dysfibrinogenemia characterized by increased resistance to fibrinolysis. We believe that this dietary model of homocysteinemia is clinically relevant and reproduces many features associated with hyperhomocysteinemia in previous work using in vitro and in vivo models. Our findings suggest that an acquired dysfibrinogenemia could play a role in the increased risk of atherothrombotic disease in mildly hyperhomocysteinemic human subjects.

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The necroptosis-inducing kinase RIPK3 dampens adipose tissue inflammation and glucose intolerance.

Receptor-interacting protein kinase 3 (RIPK3) mediates necroptosis, a form of programmed cell death that promotes inflammation in various pathological conditions, suggesting that it might be a privileged pharmacological target. However, its function in glucose homeostasis and obesity has been unknown. Here we show that RIPK3 is over expressed in the white adipose tissue (WAT) of obese mice fed with a choline-deficient high-fat diet. Genetic inactivation of Ripk3 promotes increased Caspase-8-dependent adipocyte apoptosis and WAT inflammation, associated with impaired insulin signalling in WAT as the basis for glucose intolerance. Similarly to mice, in visceral WAT of obese humans, RIPK3 is overexpressed and correlates with the body mass index and metabolic serum markers. Together, these findings provide evidence that RIPK3 in WAT maintains tissue homeostasis and suppresses inflammation and adipocyte apoptosis, suggesting that systemic targeting of necroptosis might be associated with the risk of promoting insulin resistance in obese patients.

Restriction of dietary methyl donors limits methionine availability and affects the partitioning of dietary methionine for creatine and phosphatidylcholine synthesis in the neonatal piglet.

Methionine is required for protein synthesis and provides a methyl group for >50 critical transmethylation reactions including creatine and phosphatidylcholine synthesis as well as DNA and protein methylation. However, the availability of methionine depends on dietary sources as well as remethylation of demethylated methionine (i.e., homocysteine) by the dietary methyl donors folate and choline (via betaine). By restricting dietary methyl supply, we aimed to determine the extent that dietary methyl donors contribute to methionine availability for protein synthesis and transmethylation reactions in neonatal piglets. Piglets 4-8 days of age were fed a diet deficient (MD-) (n=8) or sufficient (MS+) (n=7) in folate, choline and betaine. After 5 days, dietary methionine was reduced to 80% of requirement in both groups to elicit a response. On day 8, animals were fed [(3)H-methyl]methionine for 6h to measure methionine partitioning into hepatic protein, phosphatidylcholine, creatine and DNA. MD- feeding reduced plasma choline, betaine and folate (P<.05) and increased homocysteine ~3-fold (P<.05). With MD- feeding, hepatic phosphatidylcholine synthesis was 60% higher (P<.05) at the expense of creatine synthesis, which was 30% lower during MD- feeding (P<.05); protein synthesis as well as DNA and protein methylation were unchanged. In the liver, ~30% of dietary label was traced to phosphatidylcholine and creatine together, with ~50% traced to methylation of proteins and ~20% incorporated in synthesized protein. Dietary methyl donors are integral to neonatal methionine requirements and can affect methionine availability for transmethylation pathways.

MicroRNA-21 is associated with fibrosis in a rat model of nonalcoholic steatohepatitis and serves as a plasma biomarker for fibrotic liver disease.

Evidence indicates that hepatic fibrosis is the initial lesion of cirrhosis or hepatocellular carcinoma in diseases such as nonalcoholic steatohepatitis (NASH). To induce NASH, we fed rats a choline-deficient and iron-supplemented L-amino acid-defined (CDAA) diet. Histopathological examination revealed that fibrosis appeared from week 4 and progressed to bridging fibrosis from week 12. Using qRT-PCR assays, we detected increased expression of miR-21, Mmp-9, and Timp-1 in liver that peaked during week 4, when fibrosis was first detected. The expression pattern of miR-21 in plasma paralleled that in liver. Fibrosis tended to be resolved within 12 weeks of a recovery period after 12 weeks of feeding, and the expression of miR-21, Timp-1, and Mmp-9 decreased in liver. Comprehensive analyses of miRNA and mRNA expression in the liver using samples acquired at week 4 detected 16 miRNAs and 11 mRNAs that are mutually-interacting fibrosis-related factors. We therefore conclude that miR-21 was closely associated with fibrosis in a rat model of NASH and has potential as a plasma biomarker for hepatic fibrosis.

Free choline and phospholipid-bound choline concentrations in serum and dialysate during peritoneal dialysis in children and adults.

OBJECTIVES: This study tested whether continuous ambulatory peritoneal dialysis (CAPD) changes free or phospholipid-bound choline concentrations in serum or peritoneal dialysis fluid of patients with end stage renal disease (ESRD). DESIGN AND METHODS: Serum and dialysate choline and phospholipid-bound choline were measured before, during and after 6 h CAPD. RESULTS: Serum choline concentrations were higher in patients with ESRD compared with age-matched controls. CAPD lowered serum choline concentrations significantly although it did not influence phospholipid-bound choline. Choline accumulated in the dialysate, reaching 28.4 +/- 2.7 microM in children and 18.2 +/- 1.4 microM in adults, during six hours CAPD; phospholipid-bound choline increased to 22.9 +/- 2.5 microM and 10.8 +/- 1.4 microM in children and adults, respectively. The total daily loss of choline into the dialysate was 181 +/- 20 micromoles in children and 260 +/- 18 micromoles in adults. CONCLUSION: CAPD causes a substantial loss of choline into peritoneal dialysates and reduces serum choline concentrations significantly.

Verbal and visual memory improve after choline supplementation in long-term total parenteral nutrition: a pilot study.

BACKGROUND: Previous investigations have demonstrated that choline deficiency, manifested in low plasma-free choline concentration and hepatic injury, may develop in patients who require long-term total parenteral nutrition (TPN). Preliminary studies have suggested lecithin or choline supplementation might lead to improved visual memory in the elderly and reverse abnormal neuropsychological development in children. We sought to determine if choline-supplemented TPN would lead to improvement in neuropsychological test scores in a group of adult, choline-deficient outpatients receiving TPN. METHODS: Eleven subjects (8 males, 3 females) who received nightly TPN for more than 80% of their nutritional needs for at least 12 weeks before entry in the study were enrolled. Exclusion criteria included active drug abuse, mental retardation, cerebral vascular accident, head trauma, hemodialysis or peritoneal dialysis, (prothrombin time [PT] >2x control), or acquired immune deficiency syndrome (AIDS). Patients were randomly assigned to receive their usual TPN regimen (n = 6, aged 34.0 +/- 12.6 years) over a 12-hour nightly infusion or their usual TPN regimen plus choline chloride (2 g) (n = 5, aged 37.3 +/- 7.3 years). The following neuropsychological tests were administered at baseline and after 24 weeks of choline supplementation (or placebo): Weschler Adult Intelligence Scale-Revised (WAIS-R, intellectual functioning), Weschler Memory Scale-Revised (WMS-R, two subtests, verbal and visual memory), Rey-Osterrieth Complex Figure Test (visuospatial functioning and perceptual organization), Controlled Oral Word Association Test (verbal fluency), Grooved Pegboard (manual dexterity and motor speed), California Verbal Learning Test (CVLT, rote verbal learning ability), and Trail Making Parts A & B (visual scanning, psychomotor speed and set shifting). Scores were reported in terms of standard scores including z scores and percentile ranks. Mean absolute changes in raw scores were compared between groups using the Wilcoxon rank sum test, where p values < .05 constituted statistical significance. RESULTS: Significant improvements were found in the delayed visual recall of the WMS-R (7.0 +/- 2.7 vs -.33 +/- 5.7, p = .028), and borderline improvements in the List B subset of the CVLT (1.0 +/- 0.8 vs -2.0 +/- 2.4, p = .06) and the Trails A test (-3.8 +/- 8.1 vs 3.7 +/- 4.5 seconds, p = .067). No other statistically significant changes were seen. CONCLUSIONS: This pilot study indicates both verbal and visual memory may be impaired in patients who require long-term TPN and both may be improved with choline supplementation.

Alcohol-nutrition interaction: 1984 update.

Alcohol remains a prevailing cause of malnutrition resulting in a variety of deficiency states secondary to decreased intake of nutrients. In addition to various well described primary malnutrition syndromes, secondary malnutrition may result from the interaction of ethanol with nutrient digestion, absorption or utilization. Some of the latter alcohol-nutrient interactions have been recently defined and their pathogenesis is discussed in this review. Included are interactions with thiamine, folic acid, vitamin A and disorders secondary to amino acid imbalances. The rationale for various forms of therapy is reviewed, including the treatment aimed at correcting the "hypermetabolic state" in alcoholics ad the pitfalls of excess nutrient administration (particularly as it pertains to pyridoxine, vitamin A and amino acids). The desirability of recognizing early precirrhotic stages of alcoholic fibrosis is emphasized, in order to start therapy prior to the medical and/or social disintegration of the alcoholic.