Targeting liver aldehyde dehydrogenase-2 prevents heavy but not moderate alcohol drinking.

Aldehyde dehydrogenase 2 (ALDH2), a key enzyme for detoxification the ethanol metabolite acetaldehyde, is recognized as a promising therapeutic target to treat alcohol use disorders (AUDs). Disulfiram, a potent ALDH2 inhibitor, is an approved drug for the treatment of AUD but has clinical limitations due to its side effects. This study aims to elucidate the relative contribution of different organs in acetaldehyde clearance through ALDH2 by using global- (Aldh2-/-) and tissue-specific Aldh2-deficient mice, and to examine whether liver-specific ALDH2 inhibition can prevent alcohol-seeking behavior. Aldh2-/- mice showed markedly higher acetaldehyde concentrations than wild-type (WT) mice after acute ethanol gavage. Acetaldehyde levels in hepatocyte-specific Aldh2 knockout (Aldh2Hep-/-) mice were significantly higher than those in WT mice post gavage, but did not reach the levels observed in Aldh2-/- mice. Energy expenditure and motility were dramatically dampened in Aldh2-/- mice, but moderately decreased in Aldh2Hep-/- mice compared to controls. In the 2-bottle paradigm and the drinking-in-the-dark model, Aldh2-/- mice drank negligible volumes from ethanol-containing bottles, whereas Aldh2Hep-/- mice showed reduced alcohol preference at high but not low alcohol concentrations. Glial cell- or neuron-specific Aldh2 deficiency did not affect voluntary alcohol consumption. Finally, specific liver Aldh2 knockdown via injection of shAldh2 markedly decreased alcohol preference. In conclusion, although the liver is the major organ responsible for acetaldehyde metabolism, a cumulative effect of ALDH2 from other organs likely also contributes to systemic acetaldehyde clearance. Liver-targeted ALDH2 inhibition can decrease heavy drinking without affecting moderate drinking, providing molecular basis for hepatic ALDH2 targeting/editing for the treatment of AUD.

Relationship between inorganic ion distribution, resting membrane potential, and the ΔG' of ATP hydrolysis: a new paradigm.

Cell membrane potential and inorganic ion distributions are currently viewed from a kinetic electric paradigm, which ignores thermodynamics. The resting membrane potential is viewed as a diffusion potential. The 9 major inorganic ions found in blood plasma (Ca2+, Na+, Mg2+, K+, H+, Cl-, HCO3-, H2PO4-, and HPO42-) are distributed unequally across the plasma membrane. This unequal distribution requires the energy of ATP hydrolysis through the action of the Na+-K+ ATPase. The cell resting membrane potential in each of 3 different tissues with widely different resting membrane potentials has been shown to be equal to the Nernst equilibrium potential of the most permeant inorganic ion. The energy of the measured distribution of the 9 major inorganic ions between extra- and intracellular phases was essentially equal to the independently measured energy of ATP hydrolysis, showing that the distribution of these 9 major ions was in near-equilibrium with the ΔG' of ATP. Therefore, thermodynamics does appear to play an essential role in the determination of the cell resting membrane potential and the inorganic ion distribution across the plasma membrane.-Veech, R. L., King, M. T., Pawlosky, R., Bradshaw, P. C., Curtis, W. Relationship between inorganic ion distribution, resting membrane potential, and the ΔG' of ATP hydrolysis: a new paradigm.

A Dietary Ketone Ester Normalizes Abnormal Behavior in a Mouse Model of Alzheimer's Disease.

Because of a decreased sensitivity toward insulin, a key regulator of pyruvate dehydrogenase (PDH), Alzheimer's patients have lower brain glucose utilization with reductions in Tricarboxylic Acid (TCA) cycle metabolites such as citrate, a precursor to n-acetyl-aspartate. In the 3xTgAd mouse model of Alzheimer's disease (AD), aging mice also demonstrate low brain glucose metabolism. Ketone metabolism can overcome PDH inhibition and restore TCA cycle metabolites, thereby enhancing amino acid biosynthesis. A ketone ester of d-ß-hydroxybutyrate was incorporated into a diet (Ket) and fed to 3xTgAd mice. A control group was fed a calorically matched diet (Cho). At 15 months of age, the exploratory and avoidance-related behavior patterns of the mice were evaluated. At 16.5 months of age, the animals were euthanized, and their hippocampi were analyzed for citrate, α-ketoglutarate, and amino acids. In the hippocampi of the Ket-fed mice, there were higher concentrations of citrate and α-ketoglutarate as well as higher concentrations of glutamate, aspartate and n-acetyl-aspartate compared with controls. There were positive associations between (1) concentrations of aspartate and n-acetyl-aspartate (n = 14, R = 0.9327), and (2) α-ketoglutarate and glutamate (n = 14, R = 0.8521) in animals maintained on either diet. Hippocampal n-acetyl-aspartate predicted the outcome of several exploratory and avoidance-related behaviors. Ketosis restored citrate and α-ketoglutarate in the hippocampi of aging mice. Higher concentrations of n-acetyl-aspartate corresponded with greater exploratory activity and reduced avoidance-related behavior.

The "great" controlling nucleotide coenzymes.

Nucleotide coenzymes dot the map of metabolic pathways providing energy to drive the reactions of the pathway and play an important role in regulating and controlling energy metabolism through their shared potential energy, which is widely unobserved due to the paradox that the energy in the coenzyme pools cannot be determined from the concentration of the coenzyme couples. The potential energy of the nucleotide couples in the mitochondria or the cytoplasm is expressed in the enzyme reactions in which they take part. The energy in these couples, [NAD+]/[NADH], [NADP+]/[NADPH], [acetyl CoA]/[CoA], and [ATP]/[ADP]x[Pi], regulates energy metabolism. The energy contained in the couples can be altered by suppling energy equivalents in the form of ketones, such as, D-ß-hydroxybutyrate to overcome insulin resistance, to restore antioxidants capacity, to form potential treatments for Alzheimer's and Parkinson's diseases, to enhance life span, and to increase physiological performance. © 2019 IUBMB Life, 71(5):565-579, 2019.

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.

Effects of a dietary ketone ester on hippocampal glycolytic and tricarboxylic acid cycle intermediates and amino acids in a 3xTgAD mouse model of Alzheimer's disease.

In patients with Alzheimer's disease (AD) and in a triple transgenic (3xTgAD) mouse model of AD low glucose metabolism in the brain precedes loss of memory and cognitive decline. The metabolism of ketones in the brain by-passes glycolysis and therefore may correct several deficiencies that are associated with glucose hypometabolism. A dietary supplement composed of an ester of D-ß-hydroxybutyrate and R-1,3 butane diol referred to as ketone ester (KE) was incorporated into a rodent diet and fed to 3xTgAD mice for 8 months. At 16.5 months of age animals were killed and brains dissected. Analyses were carried out on the hippocampus and frontal cortex for glycolytic and TCA (Tricarboxylic Acid) cycle intermediates, amino acids, oxidized lipids and proteins, and enzymes. There were higher concentrations of d-ß-hydroxybutyrate in the hippocampus of KE-fed mice where there were also higher concentrations of TCA cycle and glycolytic intermediates and the energy-linked biomarker, N-acetyl aspartate compared to controls. In the hippocampi of control-fed animals the free mitochondrial [NAD+ ]/[NADH] ratio were highly oxidized, whereas, in KE-fed animals the mitochondria were reduced. Also, the levels of oxidized protein and lipids were lower and the energy of ATP hydrolysis was greater compared to controls. 3xTgAD mice maintained on a KE-supplemented diet had higher concentrations of glycolytic and TCA cycle metabolites, a more reduced mitochondrial redox potential, and lower amounts of oxidized lipids and proteins in their hippocampi compared to controls. The KE offers a potential therapy to counter fundamental metabolic deficits common to patients and transgenic models. Read the Editorial Highlight for this article on page 162.

Ketone bodies mimic the life span extending properties of caloric restriction.

The extension of life span by caloric restriction has been studied across species from yeast and Caenorhabditis elegans to primates. No generally accepted theory has been proposed to explain these observations. Here, we propose that the life span extension produced by caloric restriction can be duplicated by the metabolic changes induced by ketosis. From nematodes to mice, extension of life span results from decreased signaling through the insulin/insulin-like growth factor receptor signaling (IIS) pathway. Decreased IIS diminishes phosphatidylinositol (3,4,5) triphosphate (PIP3 ) production, leading to reduced PI3K and AKT kinase activity and decreased forkhead box O transcription factor (FOXO) phosphorylation, allowing FOXO proteins to remain in the nucleus. In the nucleus, FOXO proteins increase the transcription of genes encoding antioxidant enzymes, including superoxide dismutase 2, catalase, glutathione peroxidase, and hundreds of other genes. An effective method for combating free radical damage occurs through the metabolism of ketone bodies, ketosis being the characteristic physiological change brought about by caloric restriction from fruit flies to primates. A dietary ketone ester also decreases circulating glucose and insulin leading to decreased IIS. The ketone body, d-ß-hydroxybutyrate (d-ßHB), is a natural inhibitor of class I and IIa histone deacetylases that repress transcription of the FOXO3a gene. Therefore, ketosis results in transcription of the enzymes of the antioxidant pathways. In addition, the metabolism of ketone bodies results in a more negative redox potential of the NADP antioxidant system, which is a terminal destructor of oxygen free radicals. Addition of d-ßHB to cultures of C. elegans extends life span. We hypothesize that increasing the levels of ketone bodies will also extend the life span of humans and that calorie restriction extends life span at least in part through increasing the levels of ketone bodies. An exogenous ketone ester provides a new tool for mimicking the effects of caloric restriction that can be used in future research. The ability to power mitochondria in aged individuals that have limited ability to oxidize glucose metabolites due to pyruvate dehydrogenase inhibition suggests new lines of research for preventative measures and treatments for aging and aging-related disorders. © 2017 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 69(5):305-314, 2017.

Novel ketone diet enhances physical and cognitive performance.

Ketone bodies are the most energy-efficient fuel and yield more ATP per mole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high-fat, low-carbohydrate diets has been used for the treatment of drug-refractory epilepsy and for neurodegenerative diseases, such as Parkinson's disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)-3-hydroxybutyl (R)-3-hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a treadmill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palm oil (P < 0.05). Ketone-fed rats completed an 8-arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts from rats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [31P]-NMR spectroscopy. The novel ketone diet, therefore, improved physical performance and cognitive function in rats, and its energy-sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.-Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M. K., Atherton, H. J., Schroeder, M. A., Deacon, R. M. J., Kashiwaya, Y., King, M. T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance.

Metabolite regulation of nucleo-cytosolic trafficking of carbohydrate response element-binding protein (ChREBP): role of ketone bodies.

The carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in converting excess carbohydrate to storage fat in liver. In response to changing glucose levels, ChREBP activity is regulated by nucleo-cytoplasmic shuttling of ChREBP via interactions with 14-3-3 proteins and importins. The nuclear/cytosol trafficking is regulated partly by phosphorylation/dephosphorylation of serine 196 mediated by cAMP-dependent protein kinase and protein phosphatase. We show here that protein-free extracts of starved and high fat-fed livers contain metabolites that activate interaction of ChREBP·14-3-3 and inhibit the ChREBP/importin α interaction, resulting in cytosolic localization. These metabolites were identified as ß-hydroxybutyrate and acetoacetate. Nuclear localization of GFP-ChREBP is rapidly inhibited in hepatocytes incubated in ß-hydroxybutyrate or fatty acids, and the observed inhibition is closely correlated with the production of ketone bodies. These observations show that ketone bodies play an important role in the regulation of ChREBP activity by restricting ChREBP localization to the cytoplasm, thus inhibiting fat synthesis during periods of ketosis.

Microwave irradiation decreases ATP, increases free [Mg²âº], and alters in vivo intracellular reactions in rat brain.

Rapid inactivation of metabolism is essential for accurately determining the concentrations of metabolic intermediates in the in vivo state. We compared a broad spectrum of energetic intermediate metabolites and neurotransmitters in brains obtained by microwave irradiation to those obtained by freeze blowing, the most rapid method of extracting and freezing rat brain. The concentrations of many intermediates, cytosolic free NAD(P)(+) /NAD(P)H ratios, as well as neurotransmitters were not affected by the microwave procedure. However, the brain concentrations of ATP were about 30% lower, whereas those of ADP, AMP, and GDP were higher in the microwave-irradiated compared with the freeze-blown brains. In addition, the hydrolysis of approximately 1 µmol/g of ATP, a major in vivo Mg(2+) -binding site, was related to approximately five-fold increase in free [Mg(2+) ] (0.53 ± 0.07 mM in freeze blown vs. 2.91 mM ± 0.48 mM in microwaved brains), as determined from the ratio [citrate]/[isocitrate]. Consequently, many intracellular properties, such as the phosphorylation potential and the ∆G' of ATP hydrolysis were significantly altered in microwaved tissue. The determinations of some glycolytic and TCA cycle metabolites, the phosphorylation potential, and the ∆G' of ATP hydrolysis do not represent the in vivo state when using microwave-fixed brain tissue.

Oral 28-day and developmental toxicity studies of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate.

(R)-3-Hydroxybutyl (R)-3-hydroxybutyrate (ketone monoester) has been developed as an oral source of ketones, which may be utilized for energy. In a 28-day toxicity study, Crl:WI (Wistar) rats received diets containing, as 30% of the calories, ketone monoester (12 and 15 g/kg body weight/day for male and female rats, respectively). Control groups received either carbohydrate- or fat-based diets. Rats in the test group consumed less feed and gained less weight than control animals; similar findings have been documented in studies of ketogenic diets. Between-group differences were noted in selected hematology, coagulation, and serum chemistry parameters; however, values were within normal physiological ranges and/or were not accompanied by other changes indicative of toxicity. Upon gross and microscopic evaluation, there were no findings associated with the ketone monoester. In a developmental toxicity study, pregnant Crl:WI (Han) rats were administered 2g/kg body weight/day ketone monoester or water (control) via gavage on days 6 through 20 of gestation. No Caesarean-sectioning or litter parameters were affected by the test article. The overall incidence of fetal alterations was higher in the test group; however, there were no specific alterations attributable to the test substance. The results of these studies support the safety of ketone monoester.

Kinetics, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate in healthy adult subjects.

Induction of mild states of hyperketonemia may improve physical and cognitive performance. In this study, we determined the kinetic parameters, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, a ketone monoester administered in the form of a meal replacement drink to healthy human volunteers. Plasma levels of ß-hydroxybutyrate and acetoacetate were elevated following administration of a single dose of the ketone monoester, whether at 140, 357, or 714 mg/kg body weight, while the intact ester was not detected. Maximum plasma levels of ketones were attained within 1-2h, reaching 3.30 mM and 1.19 mM for ß-hydroxybutyrate and acetoacetate, respectively, at the highest dose tested. The elimination half-life ranged from 0.8-3.1h for ß-hydroxybutyrate and 8-14 h for acetoacetate. The ketone monoester was also administered at 140, 357, and 714 mg/kg body weight, three times daily, over 5 days (equivalent to 0.42, 1.07, and 2.14 g/kg/d). The ketone ester was generally well-tolerated, although some gastrointestinal effects were reported, when large volumes of milk-based drink were consumed, at the highest ketone monoester dose. Together, these results suggest ingestion of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate is a safe and simple method to elevate blood ketone levels, compared with the inconvenience of preparing and consuming a ketogenic diet.

A ketone ester diet increases brain malonyl-CoA and Uncoupling proteins 4 and 5 while decreasing food intake in the normal Wistar Rat.

Three groups of male Wistar rats were pair fed NIH-31 diets for 14 days to which were added 30% of calories as corn starch, palm oil, or R-3-hydroxybutyrate-R-1,3-butanediol monoester (3HB-BD ester). On the 14th day, animal brains were removed by freeze-blowing, and brain metabolites measured. Animals fed the ketone ester diet had elevated mean blood ketone bodies of 3.5 mm and lowered plasma glucose, insulin, and leptin. Despite the decreased plasma leptin, feeding the ketone ester diet ad lib decreased voluntary food intake 2-fold for 6 days while brain malonyl-CoA was increased by about 25% in ketone-fed group but not in the palm oil fed group. Unlike the acute effects of ketone body metabolism in the perfused working heart, there was no increased reduction in brain free mitochondrial [NAD(+)]/[NADH] ratio nor in the free energy of ATP hydrolysis, which was compatible with the observed 1.5-fold increase in brain uncoupling proteins 4 and 5. Feeding ketone ester or palm oil supplemented diets decreased brain L-glutamate by 15-20% and GABA by about 34% supporting the view that fatty acids as well as ketone bodies can be metabolized by the brain.

Brain ethanol metabolism by astrocytic ALDH2 drives the behavioural effects of ethanol intoxication.

Alcohol is among the most widely used psychoactive substances worldwide. Ethanol metabolites such as acetate, thought to be primarily the result of ethanol breakdown by hepatic aldehyde dehydrogenase 2 (ALDH2), contribute to alcohol's behavioural effects and alcoholism. Here, we show that ALDH2 is expressed in astrocytes in the mouse cerebellum and that ethanol metabolism by astrocytic ALDH2 mediates behavioural effects associated with ethanol intoxication. We show that ALDH2 is expressed in astrocytes in specific brain regions and that astrocytic, but not hepatocytic, ALDH2 is required to produce ethanol-derived acetate in the mouse cerebellum. Cerebellar astrocytic ALDH2 mediates low-dose ethanol-induced elevation of GABA levels, enhancement of tonic inhibition and impairment of balance and coordination skills. Thus, astrocytic ALDH2 controls the production, cellular and behavioural effects of alcohol metabolites in a brain-region-specific manner. Our data indicate that astrocytic ALDH2 is an important, but previously under-recognized, target in the brain to alter alcohol pharmacokinetics and potentially treat alcohol use disorder.

Beta-carotene conversion to vitamin A decreases as the dietary dose increases in humans.

It has been suggested that high doses of beta-carotene limit its conversion to vitamin A, yet this effect has not been well established in humans. A feeding study was conducted in a randomized crossover design in which volunteers consumed 2 doses of deuterium-labeled beta-carotene on 2 occasions, with beta-carotene and vitamin A response assessed by plasma area under the concentration time curve (AUC). Seven volunteers (4 men, 3 women) consumed each of 2 doses of beta-carotene-d8 and provided serial blood samples for 37 d after each dose. beta-Carotene doses were 20 and 40 mg. Plasma beta-carotene-d8 was assessed by HPLC-MS. Plasma retinol (ROH)-d4, which was derived from the beta-carotene-d8, was evaluated by GC-MS after saponification to convert retinyl esters to ROH prior to the formation of the trimethylsilylether. The plasma AUC for beta-carotene-d8 increased 2-fold from the 20-mg dose to the 40-mg dose. The plasma AUC for ROH-d4 increased 36% from the 20-mg dose to the 40-mg dose. These results establish that, in humans, beta-carotene conversion to vitamin A decreases as the dietary dose increases.

Alterations in brain glucose utilization accompanying elevations in blood ethanol and acetate concentrations in the rat.

BACKGROUND: Previous studies in humans have shown that alcohol consumption decreased the rate of brain glucose utilization. We investigated whether the major metabolite of ethanol, acetate, could account for this observation by providing an alternate to glucose as an energy substrate for brain and the metabolic consequences of that shift. METHODS: Rats were infused with solutions of sodium acetate, ethanol, or saline containing (13)C-2-glucose as a tracer elevating the blood ethanol (BEC) and blood acetate (BAcC) concentrations. After an hour, blood was sampled and the brains of animals were removed by freeze blowing. Tissue samples were analyzed for the intermediates of glucose metabolism, Krebs' cycle, acyl-coenzyme A (CoA) compounds, and amino acids. RESULTS: Mean peak BEC and BAcC were approximately 25 and 0.8 mM, respectively, in ethanol-infused animals. Peak blood BAcC increased to 12 mM in acetate-infused animals. Both ethanol and acetate infused animals had a lower uptake of (13)C-glucose into the brain compared to controls and the concentration of brain (13)C-glucose-6-phosphate varied inversely with the BAcC. There were higher concentrations of brain malonyl-CoA and somewhat lower levels of free Mg(2+) in ethanol-treated animals compared to saline controls. In acetate-infused animals the concentrations of brain lactate, alpha-ketoglutarate, and fumarate were higher. Moreover, the free cytosolic [NAD(+)]/[NADH] was lower, the free mitochondrial [NAD(+)]/[NADH] and [CoQ]/[CoQH(2)] were oxidized and the DeltaG' of ATP lowered by acetate infusion from -61.4 kJ to -59.9 kJ/mol. CONCLUSIONS: Animals with elevated levels of blood ethanol or acetate had decreased (13)C-glucose uptake into the brain. In acetate-infused animals elevated BAcC were associated with a decrease in (13)C-glucose phosphorylation. The co-ordinate decrease in free cytosolic NAD, oxidation of mitochondrial NAD and Q couples and the decrease in DeltaG' of ATP was similar to administration of uncoupling agents indicating that the metabolism of acetate in brain caused the mitochondrial voltage dependent pore to form.

Maternal smoking is associated with decreased 5-methyltetrahydrofolate in cord plasma.

BACKGROUND: Maternal-fetal folate transport via the placenta has been shown to be concentrative. Exposure to cigarette smoke is associated with decreased maternal folate status through altered dietary intakes and possibly through nondietary mechanisms such as increased folate turnover. The effect of maternal smoking on fetal folate status has not been documented. OBJECTIVE: The objective was to determine the effect of maternal smoking on plasma 5-methyltetrahydrofolic acid (5-MTHFA) concentrations in umbilical cord blood. DESIGN: African American women were recruited from an antenatal clinic in Detroit, MI. Plasma 5-MTHFA concentrations were measured in maternal-umbilical cord pairings (n = 58). The participants completed a structured interview to determine demographic characteristics, including smoking. RESULTS: Concentrations of 5-MTHFA were significantly higher in venous cord plasma (16.8 +/- 7.5 ng/mL) than in maternal plasma (13.0 +/- 7.5 ng/mL) but remained associated (r = 0.60, P < 0.001) with each other. Cigarettes smoked by the mothers was negatively associated (r = -0.31, P = 0.019) with venous cord 5-MTHFA concentrations and remained so after control for maternal plasma 5-MTHFA and other variables. Venous cord plasma 5-MTHFA was significantly lower in smoking (15.1 +/- 7.6 ng/mL; n = 32) than in nonsmoking (19.0 +/- 7.0 ng/mL; n = 26) mothers. CONCLUSIONS: Cord plasma 5-MTHFA concentrations were elevated relative to maternal blood, as expected, because the placenta is capable of concentrative folate transport to the fetus. The negative effect of maternal smoking on infant, but not on maternal, 5-MTHFA status indicates that maternal smoking may impair folate transport to the fetus.

[6S]-5-Methyltetrahydrofolate is at least as effective as folic acid in preventing a decline in blood folate concentrations during lactation.

BACKGROUND: Studies in nonpregnant, nonlactating women suggest that folate supplementation in the form of 5-methyltetrahydrofolate ([6S]-5-methylTHF) is at least as effective as folic acid in increasing blood folate indexes. No data, however, are available on the effect of supplemental [6S]-5-methylTHF on blood folate concentrations during lactation. OBJECTIVE: We assessed the relative effectiveness of [6S]-5-methylTHF, a placebo, and folic acid in maintaining blood folate indexes during lactation in a sample of healthy Canadian women consuming folic acid-fortified foods. DESIGN: This study was designed as a 16-wk, randomized, placebo-controlled intervention. Pregnant women (n = 72) advised to consume a folic acid-containing prenatal supplement (1000 microg/d) during pregnancy were enrolled at 36 wk gestation. After delivery, the women were randomly assigned to receive [6S]-5-methylTHF (416 microg/d, 906 nmol/d) or a placebo or were assigned to a folic acid (400 microg/d, 906 nmol/d) reference group. RESULTS: At 16 wk of lactation, the mean red blood cell (RBC) folate concentration in women in the [6S]-5-methylTHF group (2178; 95% CI: 1854, 2559 nmol/L) was greater than that in the folic acid (1967; 1628, 2377 nmol/L; P < 0.05) and placebo (1390; 1198, 1613 nmol/L; P < 0.002) groups after adjustment for baseline concentrations (36 wk gestation). The distribution of folate forms in RBCs did not differ significantly between the [6S]-5-methylTHF and placebo groups. However, the folic acid group had greater amounts of 5-formylTHF (P < 0.03). CONCLUSION: [6S]-5-MethylTHF appeared to be as effective as, and perhaps more effective than, folic acid in preserving RBC folate concentrations during lactation.

Status of plasma folate after folic acid fortification of the food supply in pregnant African American women and the influences of diet, smoking, and alcohol consumption.

BACKGROUND: African American women and socioeconomically challenged women are at risk of compromised folate status and, thus, of folate-related birth defects. Data are limited on circulating folate concentrations in pregnant African American women after folic acid fortification of the food supply was implemented. OBJECTIVE: The objective was to determine the influence of smoking and alcohol consumption on plasma 5-methyltetrahydrofolic acid (5-MTHFA) concentrations in pregnant African American women. DESIGN: Alcohol consumption, smoking exposure, and other characteristics of pregnant African American women reporting to an inner-city antenatal clinic were assessed. At 24 wk of gestation, blood samples and food-frequency intake data were collected. Plasma 5-MTHFA concentrations were determined by liquid chromatography-mass spectrometry for 116 subjects and examined in a correlational study design. RESULTS: Dietary folate and markers of alcohol consumption were positively associated, whereas exposure to smoke was negatively associated with plasma 5-MTHFA. More than one-half of the participants in this population failed to meet the recommended dietary allowance for dietary folate equivalents of 600 microg/d during pregnancy. CONCLUSIONS: Most inner-city African American women are not meeting the recommended dietary allowance for dietary folate during pregnancy, and smoking may further compromise their folate status. Programs to reduce smoking and raise awareness about the importance of folate and multivitamin supplementation during pregnancy need to target this population.

An Ester of ß-Hydroxybutyrate Regulates Cholesterol Biosynthesis in Rats and a Cholesterol Biomarker in Humans.

In response to carbohydrate deprivation or prolonged fasting the ketone bodies, ß-hydroxybutyrate (ßHB) and acetoacetate (AcAc), are produced from the incomplete ß-oxidation of fatty acids in the liver. Neither ßHB nor AcAc are well utilized for synthesis of sterols or fatty acids in human or rat liver. To study the effects of ketones on cholesterol homeostasis a novel ßHB ester (KE) ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate) was synthesized and given orally to rats and humans as a partial dietary carbohydrate replacement. Rats maintained on a diet containing 30-energy % as KE with a concomitant reduction in carbohydrate had lower plasma cholesterol and mevalonate (-40 and -27 %, respectively) and in the liver had lower levels of the mevalonate precursors acetoacetyl-CoA and HMG-CoA (-33 and -54 %) compared to controls. Whole liver and membrane LDL-R as well as SREBP-2 protein levels were higher (+24, +67, and +91 %, respectively). When formulated into a beverage for human consumption subjects consuming a KE drink (30-energy %) had elevated plasma ßHB which correlated with decreased mevalonate, a liver cholesterol synthesis biomarker. Partial replacement of dietary carbohydrate with KE induced ketosis and altered cholesterol homeostasis in rats. In healthy individuals an elevated plasma ßHB correlated with lower plasma mevalonate.