Leslie M. Fischer

Association Between Composition of the Human Gastrointestinal Microbiome and Development of Fatty Liver With Choline Deficiency

BACKGROUND & AIMS Nonalcoholic fatty liver disease affects up to 30% of the US population, but the mechanisms underlying this condition are incompletely understood. We investigated how diet standardization and choline deficiency influence the composition of the microbial community in the human gastrointestinal tract and the development of fatty liver under conditions of choline deficiency. METHODS We performed a 2-month inpatient study of 15 female subjects who were placed on well-controlled diets in which choline levels were manipulated. We used 454-FLX pyrosequencing of 16S ribosomal RNA bacterial genes to characterize microbiota in stool samples collected over the course of the study. RESULTS The compositions of the gastrointestinal microbial communities changed with choline levels of diets; each individuals microbiome remained distinct for the duration of the experiment, even though all subjects were fed identical diets. Variations between subjects in levels of Gammaproteobacteria and Erysipelotrichi were directly associated with changes in liver fat in each subject during choline depletion. Levels of these bacteria, change in amount of liver fat, and a single nucleotide polymorphism that affects choline were combined into a model that accurately predicted the degree to which subjects developed fatty liver on a choline-deficient diet. CONCLUSIONS Host factors and gastrointestinal bacteria each respond to dietary choline deficiency, although the gut microbiota remains distinct in each individual. We identified bacterial biomarkers of fatty liver that result from choline deficiency, adding to the accumulating evidence that gastrointestinal microbes have a role in metabolic disorders.

Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liver disease (NAFLD)

Phosphatidylethanolamine N‐methyltransferase (PEMT) catalyzes phosphatidylcholine synthesis. PEMT knockout mice have fatty livers, and it is possible that, in humans, nonalcoholic fatty liver disease (NAFLD) might be associated with PEMT gene polymorphisms. DNA samples from 59 humans without fatty liver and from 28 humans with NAFLD were genotyped for a single nucleotide polymorphism in exon 8 of PEMT, which leads to a V175M substitution. V175M is a loss of function mutation, as determined by transiently transfecting McArdle‐RH7777 cells with constructs of wild‐type PEMT open reading frame or the V175M mutant. Met/Met at residue 175 (loss of function SNP) occurred in 67.9% of the NAFLD subjects and in only 40.7% of control subjects (P<0.03). For the first time we report that a polymorphism of the human PEMT gene (V175M) is associated with diminished activity and may confer susceptibility to NAFLD. Song, J., da Costa, K. A., Fischer, L. M., Kohlmeier, M., Kwock, L., Wang, S., Zeisel, S. H. Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liver disease (NAFLD). FASEB J. 19, 1266–1271 (2005)

Guiding Stars: the effect of a nutrition navigation program on consumer purchases at the supermarket

BACKGROUND To improve diet quality and overall population health, the need to develop nutritional rating systems that are comprehensive in scope and easy for the consumer to understand and use at the point-of-purchase has emerged. OBJECTIVE Our aim was to examine the effect of a comprehensive storewide supermarket point-of-purchase nutrition navigation intervention by using a shelf-label 3-tiered star icon on consumer food and beverage choices and their associated nutritional quality. DESIGN By using a natural experiment design, purchasing data from 2006 to 2008 were obtained from a Northeast supermarket chain with 168 stores located in northern New England and New York and examined at preimplementation and at 1- and 2-y follow-up periods. RESULTS The nutrition navigation system studied showed significant changes in food purchasing immediately after implementation, and these changes continued to be significant 1 and 2 y later. When the same 8-mo period (January-August) each year was compared, in 2006, 24.50% of items purchased earned a star rating; this proportion increased to 24.98% (P < 0.001) and 25.89% (P < 0.0001) at the 1- and 2-y follow-up periods, respectively. For a 4-wk period, 1 y after program implementation, consumers purchased significantly more ready-to-eat cereals with stars (eg, less added sugars and more dietary fiber) and fewer no-star, high-sugar, low-fiber cereals. CONCLUSION Increasing rates of obesity and declining diet quality for Americans strongly support the need for effective supermarket point-of-purchase programs, such as the Guiding Stars nutrition navigation program, that provide clear, concise, and simplified nutrition information to guide consumer food and beverage choices.

Clinical characteristics and pharmacokinetics of purified soy isoflavones: Multiple-dose administration to men with prostate neoplasia

A phase I clinical trial was conducted to determine the safety, pharmacokinetic parameters, and efficacy of orally administered isoflavones (genistein and daidzein, potential cancer chemotherapeutic agents) over a 3-mo period in men with prostate neoplasia. Twenty men, ages 40 and above, with stage B, C, or D adenocarcinoma of the prostate were treated with a multiple-dose regimen of a soy isoflavone formulation (delivering approximately 300 or 600 mg/day genistein and half this much daidzein) for 84 days. The delivered dose of isoflavones was more than 10-fold higher than that typically taken by prostate cancer patients. In men with prostate cancer, relatively minor side effects of chronic isoflavone treatment were observed including some estrogenic effects (breast changes, increased frequency of hot flashes). Serum dehydroepiandrosterone was decreased by 31.7% (P = 0.0004) at the end of treatment. Except for those subjects whose prostate-specific antigen (PSA) values were below 0.4 ng/ml, subjects had a history of increasing PSA levels prior to the trial. This increase continued during the trial both while on soy isoflavones and after treatment was discontinued. On average the rate of rise accelerated after soy isoflavones were discontinued, but that difference did not attain statistical significance. Genistein and daidzein were rapidly cleared from plasma and excreted in urine. Pharmacokinetic data for chronic dose administration were similar to single-dose administration for the isoflavones investigated except that we observed slightly longer circulation time for daidzein.

Metabolomic profiling can predict which humans will develop liver dysfunction when deprived of dietary choline

Choline is an essential nutrient, and deficiency causes liver and muscle dysfunction. Common genetic variations alter the risk of developing organ dysfunction when choline deficient, probably by causing metabolic inefficiencies that should be detectable even while ingesting a normal choline‐adequate diet. We determined whether metabolomic profiling of plasma at baseline could predict whether humans will develop liver dysfunction when deprived of dietary choline. Fifty‐three participants were fed a diet containing 550 mg choline/70 kg/d for 10 d and then fed <50 mg choline/70 kg/d for up to 42 d. Participants who developed organ dysfunction on this diet were repleted with a choline‐adequate diet for ≥3 d. Plasma samples, obtained at baseline, end of depletion, and end of repletion, were used for targeted and nontargeted metabolomic profiling. Liver fat was assessed using magnetic resonance spectroscopy. Metabolomic profiling and targeted biochemical analyses were highly correlated for the analytes assessed by both procedures. In addition, we report relative concentration changes of other small molecules detected by the nontargeted metabolomic analysis after choline depletion. Finally, we show that metabolomic profiles of participants when they were consuming a control baseline diet could predict whether they would develop liver dysfunction when deprived of dietary choline.—Sha, W., da Costa, K., Fischer, L. M., Milburn, M. V., Lawton, K. A., Berger, A., Jia, W., Zeisel, S. H. Metabolomic profiling can predict which humans will develop liver dysfunction when deprived of dietary choline. FASEB J. 24, 2962–2975 (2010). www.fasebj.org

Choline intake and genetic polymorphisms influence choline metabolite concentrations in human breast milk and plasma

BACKGROUND Choline is essential for infant nutrition, and breast milk is a rich source of this nutrient. Common single nucleotide polymorphisms (SNPs) change dietary requirements for choline intake. OBJECTIVE The aim of this study was to determine whether total choline intake and/or SNPs influence concentrations of choline and its metabolites in human breast milk and plasma. DESIGN We gave a total of 103 pregnant women supplemental choline or a placebo from 18 wk gestation to 45 d postpartum and genotyped the women for 370 common SNPs. At 45 d postpartum, we measured choline metabolite concentrations in breast milk and plasma and assessed the dietary intake of choline by using a 3-d food record. RESULTS On average, lactating women in our study ate two-thirds of the recommended intake for choline (Adequate Intake = 550 mg choline/d). Dietary choline intake (no supplement) correlated with breast-milk phosphatidylcholine and plasma choline concentrations. A supplement further increased breast-milk choline, betaine, and phosphocholine concentrations and increased plasma choline and betaine concentrations. We identified 5 SNPs in MTHFR that altered the slope of the intake-metabolite concentration relations, and we identified 2 SNPs in PEMT that shifted these curves upward. Individuals who shared sets of common SNPs were outliers in plots of intake-metabolite concentration curves; we suggest that these SNPs should be further investigated to determine how they alter choline metabolism. CONCLUSION Total intake of choline and genotype can influence the concentrations of choline and its metabolites in the breast milk and blood of lactating women and thereby affect the amount of choline available to the developing infant. This study was registered at clinicaltrials.gov as NCT00678925.

Dietary choline requirements of women: effects of estrogen and genetic variation

BACKGROUND Choline is obtained from the diet and from the biosynthesis of phosphatidylcholine. Phosphatidylcholine is catalyzed by the enzyme phosphatidylethanolamine-N-methyltransferase (PEMT), which is induced by estrogen. Because they have lower estrogen concentrations, postmenopausal women are more susceptible to the risk of organ dysfunction in response to a low-choline diet. A common genetic polymorphism (rs12325817) in the PEMT gene can also increase this risk. OBJECTIVE The objective was to determine whether the risk of low choline-related organ dysfunction increases with the number of alleles of rs12325817 in premenopausal women and whether postmenopausal women (with or without rs12325817) treated with estrogen are more resistant to developing such symptoms. DESIGN Premenopausal women (n = 27) consumed a choline-sufficient diet followed by a very-low-choline diet until they developed organ dysfunction (or for 42 d), which was followed by a high-choline diet. Postmenopausal women (n = 22) were placed on the same diets but were first randomly assigned to receive estrogen or a placebo. The women were monitored for organ dysfunction and plasma choline metabolites and were genotyped for rs12325817. RESULTS A dose-response effect of rs12325817 on the risk of choline-related organ dysfunction was observed in premenopausal women: 80%, 43%, and 13% of women with 2, 1, or 0 alleles, respectively, developed organ dysfunction. Among postmenopausal women, 73% who received placebo but only 18% who received estrogen developed organ dysfunction during the low-choline diet. CONCLUSIONS Because of their lower estrogen concentrations, postmenopausal women have a higher dietary requirement for choline than do premenopausal women. Choline requirements for both groups of women are further increased by rs12325817. This trial was registered at clinicaltrials.gov as NCT00065546.

Development and implementation of the guiding stars nutrition guidance program.

Purpose. To describe the collaborative process between a grocery retailer and a panel of nutrition experts used to develop a nutrition guidance system (Guiding Stars) that evaluates the nutrient profile of all edible products in the supermarket, and to report the results of the food and beverage ratings. Design. A collaboration between a private retailer and members of the scientific community that led to the development of a scoring algorithm used to evaluate the nutritional quality of foods and beverages. Setting/Subjects. Northeast supermarkets (n = 160). Measures. Food and beverage nutrition ratings and distribution of stars across different grocery categories. Analysis. Descriptive statistics for rating distributions were computed. T-tests were conducted to assess differences in mean nutrient values between foods with zero versus three stars or a dichotomized variable representing all foods with one to three stars. Results. All edible grocery items (n = 27,466) were evaluated, with 23.6% earning at least one star. Items receiving at least one star had lower mean levels of sodium, saturated fat, and sugars and higher amounts of fiber than products not earning stars. Conclusion. The Guiding Stars system rates edible products without regard to brand or manufacturer, and provides consumers with a simple tool to quickly identify more nutritious choices while shopping. The low percentage of products qualifying for stars reflects poorly on the food choices available to Americans.

Docosahexaenoic acid in plasma phosphatidylcholine may be a potential marker for in vivo phosphatidylethanolamine N-methyltransferase activity in humans

BACKGROUND Choline is an essential nutrient for humans, and part of this requirement is met by endogenous synthesis catalyzed by hepatic phosphatidylethanolamine N-methyltransferase (PEMT). PEMT activity is difficult to estimate in humans because it requires a liver biopsy. Previously, we showed that mice that lack functional PEMT have dramatically reduced concentrations of docosahexaenoic acid (DHA; 22:6n-3) in plasma and of liver phosphatidylcholine (PtdCho)-a phospholipid formed by PEMT. OBJECTIVE The objective was to evaluate plasma PtdCho-DHA concentrations as a noninvasive marker of liver PEMT activity in humans. DESIGN Plasma PtdCho-DHA concentrations were measured in 72 humans before and after they consumed a low-choline diet, and correlations were analyzed in relation to estrogen status, PEMT polymorphism rs12325817, the ratio of plasma S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy), and dietary choline intake; all of these factors are associated with changes in liver PEMT activity. PtdCho-DHA and PEMT activity were also measured in human liver specimens. RESULTS At baseline, the portion of PtdCho species containing DHA (pmol PtdCho-DHA/nmol PtdCho) was higher in premenopausal women than in men and postmenopausal women (P < 0.01). This ratio was lower in premenopausal women with the rs12325817 polymorphism in the PEMT gene (P < 0.05), and PtdCho-DHA concentration and PEMT activity were lower in human liver samples from women who were homozygous for PEMT rs12325817 (P < 0.05). The ratio of DHA-PtdCho to PtdCho in plasma was directly correlated with the ratio of AdoMet to AdoHcy (P = 0.0001). The portion of PtdCho species containing DHA in plasma was altered in subjects who consumed a low-choline diet. CONCLUSION PtdCho-DHA may be useful as a surrogate marker for in vivo hepatic PEMT activity in humans. This trial was registered at clinicaltrials.gov as NCT00065546.

Effects of a high daily dose of soy isoflavones on DNA damage, apoptosis and estrogenic outcomes in healthy, postmenopausal women - a Phase I clinical trial

Objective:A phase I double-blind clinical trial was conducted to evaluate the effects of a high oral dose of soy isoflavones administered daily for 84 days to healthy postmenopausal women. Principal outcome measures included DNA damage, apoptosis, and changes indicative of estrogenic stimulation. Design:After eligibility and equol-producer status were determined, stratified randomization was used to assign women to the isoflavone (active) or placebo group. Of the 30 women who completed the study, 18 were in the active group. DNA damage was assessed via COMET and apurinic/apyrimidinic site assays in lymphocytes. Apoptosis was evaluated via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and activated caspase-3 assays in lymphocytes. Estrogenic/antiestrogenic effects were assessed using a self-report questionnaire and by assaying for estrogen, follicle-stimulating hormone, luteinizing hormone, and sex hormone-binding globulin in blood. Results:In treated postmenopausal women, there was no indication that high doses of soy isoflavones caused DNA strand breakage, increased apurinic/apyrimidinic sites, or increased apoptosis in peripheral lymphocytes. There were no significant changes in mean values for estrogenic effects or other laboratory measurements. Very few adverse events occurred, and the only drug-related adverse events were mild or grade 1 in severity. Conclusions:Unconjugated soy isoflavones appear to be safe and well tolerated in healthy postmenopausal women at doses of 900 mg/day.