Lifestyle, metabolite, and genetic determinants of formate concentrations in a cross-sectional study in young, healthy adults.

Background: Formate is an important metabolite that serves as a donor of one-carbon groups to the intracellular tetrahydrofolate pool. However, little is known of its circulating concentrations or of their determinants. Objective: This study aimed to define formate concentrations and their determinants in a healthy young population. Design: Serum formate was measured in 1701 participants from the Trinity Student Study. The participants were men and women, aged 18 to 28 y, enrolled at Trinity College, Dublin. Formate concentrations were compared with other one-carbon metabolites, vitamin status, potential formate precursors, genetic polymorphisms, and lifestyle factors. Results: Serum formate concentrations ranged from 8.7 to 96.5 µM, with a mean of 25.9 µM. Formate concentrations were significantly higher in women than in men; oral contraceptive use did not further affect them. There was no effect of smoking or of alcohol ingestion, but the TT genotype of the methylenetetrahydrofolate reductase (MTHFR) 677C→T (rs1801133) polymorphism was associated with a significantly decreased formate concentration. Formate was positively associated with potential metabolic precursors (serine, methionine, tryptophan, choline) but not with glycine. Formate concentrations were positively related to serum folate and negatively related to serum vitamin B-12. Conclusions: Formate concentrations were sensitive to the concentrations of metabolic precursors. In view of the increased susceptibility of women with the TT genotype of MTHFR to give birth to infants with neural tube defects as well as the effectiveness of formate supplementation in decreasing the incidence of folate-resistant neural tube defects in susceptible mice, it will be important to understand how this genotype decreases the serum formate concentration. This trial was registered at www.clinicaltrials.gov as NCT03305900.

Formate: The Neglected Member of One-Carbon Metabolism.

Formate, the only non-tetrahydrofolate (THF)-linked intermediate in one-carbon metabolism, is produced in mammals from a variety of metabolic sources. It occurs in serum of adults at a concentration of approximately 30 µM. Its principal function lies as a source of one-carbon groups for the synthesis of 10-formyl-THF and other one-carbon intermediates; these are primarily used for purine synthesis, thymidylate synthesis, and the provision of methyl groups for synthetic, regulatory, and epigenetic methylation reactions. Although formate is largely produced in mitochondria, these functions mostly occur in the cytoplasm and nucleus. Formate plays a significant role in embryonic development, as evidenced by the effectiveness of formate in the pregnant dam's drinking water on the incidence of neural tube defects in some genetic models. High formate concentrations in fetal lambs may indicate a role in fetal development and suggest that extracellular formate may play a role in the interorgan distribution of one-carbon groups.

Formate supplementation enhances folate-dependent nucleotide biosynthesis and prevents spina bifida in a mouse model of folic acid-resistant neural tube defects.

The curly tail mouse provides a model for neural tube defects (spina bifida and exencephaly) that are resistant to prevention by folic acid. The major ct gene, responsible for spina bifida, corresponds to a hypomorphic allele of grainyhead-like 3 (Grhl3) but the frequency of NTDs is strongly influenced by modifiers in the genetic background. Moreover, exencephaly in the curly tail strain is not prevented by reinstatement of Grhl3 expression. In the current study we found that expression of Mthfd1L, encoding a key component of mitochondrial folate one-carbon metabolism (FOCM), is significantly reduced in ct/ct embryos compared to a partially congenic wild-type strain. This expression change is not attributable to regulation by Grhl3 or the genetic background at the Mthfd1L locus. Mitochondrial FOCM provides one-carbon units as formate for FOCM reactions in the cytosol. We found that maternal supplementation with formate prevented NTDs in curly tail embryos and also resulted in increased litter size. Analysis of the folate profile of neurulation-stage embryos showed that formate supplementation resulted in an increased proportion of formyl-THF and THF but a reduction in proportion of 5-methyl THF. In contrast, THF decreased and 5-methyl THF was relatively more abundant in the liver of supplemented dams than in controls. In embryos cultured through the period of spinal neurulation, incorporation of labelled thymidine and adenine into genomic DNA was suppressed by supplemental formate, suggesting that de novo folate-dependent biosynthesis of nucleotides (thymidylate and purines) was enhanced. We hypothesise that reduced Mthfd1L expression may contribute to susceptibility to NTDs in the curly tail strain and that formate acts as a one-carbon donor to prevent NTDs.

Creatine supplementation as a possible new therapeutic approach for fatty liver disease: early findings.

Over the last few years, consistent data have demonstrated that creatine (Cr) supplementation prevents the accumulation of fat in rat liver as well as the progression of fatty liver disease in different situations. Studies have demonstrated that Cr is effective and prevents fatty liver in high-fat and choline-deficient diets and in hepatoma cells in vitro. Because Cr synthesis is responsible for a considerable consumption of hepatic methyl groups, studies have tested the idea that Cr supplementation could modulate phospholipid formation and VLDL secretion. Studies have also demonstrated Cr is able to modulate the expression of key genes related to fatty acid oxidation in hepatocyte cell culture and in rat liver. However, to date, the mechanism by which Cr exerts protective effects against fatty liver is poorly understood. Therefore, the present review aims to summarize the studies involving the therapeutic use of Cr supplementation on fatty liver disease and to explore the mechanisms involved in one-carbon and fatty acid metabolism for the preventive effects of Cr supplementation on fat liver accumulation. Although a small number of studies have been conducted to date, we consider Cr as a new and promising therapeutic strategy to control fat accumulation in the liver as well as the progression of fatty liver disease.

The role of dietary creatine.

The daily requirement of a 70-kg male for creatine is about 2 g; up to half of this may be obtained from a typical omnivorous diet, with the remainder being synthesized in the body Creatine is a carninutrient, which means that it is only available to adults via animal foodstuffs, principally skeletal muscle, or via supplements. Infants receive creatine in mother's milk or in milk-based formulas. Vegans and infants fed on soy-based formulas receive no dietary creatine. Plasma and muscle creatine levels are usually somewhat lower in vegetarians than in omnivores. Human intake of creatine was probably much higher in Paleolithic times than today; some groups with extreme diets, such as Greenland and Alaskan Inuit, ingest much more than is currently typical. Creatine is synthesized from three amino acids: arginine, glycine and methionine (as S-adenosylmethionine). Humans can synthesize sufficient creatine for normal function unless they have an inborn error in a creatine-synthetic enzyme or a problem with the supply of substrate amino acids. Carnivorous animals, such as lions and wolves, ingest much larger amounts of creatine than humans would. The gastrointestinal tract and the liver are exposed to dietary creatine in higher concentrations before it is assimilated by other tissues. In this regard, our observations that creatine supplementation can prevent hepatic steatosis (Deminice et al. J Nutr 141:1799-1804, 2011) in a rodent model may be a function of the route of dietary assimilation. Creatine supplementation has also been reported to improve the intestinal barrier function of the rodent suffering from inflammatory bowel disease.

Tryptophan catabolism and vitamin B-6 status are affected by gender and lifestyle factors in healthy young adults.

BACKGROUND: Abnormalities of tryptophan (Trp) metabolism through the kynurenine (Kyn) pathway have been reported in various diseases; however, nutritional and lifestyle factors that affect this pathway in healthy individuals are not well documented. OBJECTIVE: Our aim was to examine the effect of vitamin B-6 status and lifestyle factors including the use of vitamin B-6 supplements, alcohol, smoking, and oral contraceptives on Trp and its Kyn metabolites in a cohort of 2436 healthy young adults aged 18-28 y. METHODS: Anthropometric and lifestyle data were collected by questionnaire. Participants provided blood samples for analysis of Trp, Kyn, anthranilic acid, kynurenic acid (KA), 3-hydroxykynurenine (HK), 3-hydroxyanthranilic acid (HAA), and xanthurenic acid (XA). Vitamin B-6 species were also measured. RESULTS: Serum Trp metabolites were 10-15% higher among men (n = 993) compared with women (n = 1443; P < 0.0001), except for HK and XA. In all participants, serum Trp was positively associated with plasma pyridoxal 5'-phosphate (PLP; r = 0.28, P < 0.0001), reaching a plateau at PLP concentrations of ∼83 nmol/L. HK was inversely associated with PLP (r = -0.14, P < 0.01). Users of vitamin B-6 supplements (n = 671) had 6% lower concentrations of HK than nonusers (n = 1765; P = 0.0006). Oral contraceptive users (n = 385) had lower concentrations of KA (20.7%) but higher XA (24.1%) and HAA (9.0%) than did nonusers (n = 1058; P < 0.0001). After adjustment for gender and other lifestyle variables, XA concentrations were 16% higher in heavy drinkers (n = 713) than in never or occasional drinkers (n = 975; P = 0.0007). Concentrations of 2 other essential amino acids, methionine and arginine, also were positively associated with serum Trp (r = 0.65 and 0.33, respectively; P < 0.0001). CONCLUSIONS: In this population of healthy young adults, gender has the largest influence on serum Kyn metabolite concentrations. The significant covariance of Trp with unrelated amino acids suggests that protein intake may be an important consideration in evaluating Kyn metabolism.

Betaine supplementation prevents fatty liver induced by a high-fat diet: effects on one-carbon metabolism.

The purpose of this study was to examine the effects of betaine supplementation on the regulation of one-carbon metabolism and liver lipid accumulation induced by a high-fat diet in rats. Rats were fed one of three different liquid diets: control diet, high-fat diet and high-fat diet supplemented with betaine. The control and high-fat liquid diets contained, respectively, 35 and 71 % of energy derived from fat. Betaine supplementation involved the addition of 1 % (g/L) to the diet. After three weeks on the high-fat diet the rats had increased total liver fat concentration, liver triglycerides, liver TBARS and plasma TNF-α. The high-fat diet decreased the hepatic S-adenosylmethionine concentration and the S-adenosylmethionine/S-adenosylhomocysteine ratio compared to the control as well as altering the expression of genes involved in one-carbon metabolism. Betaine supplementation substantially increased the hepatic S-adenosylmethionine concentration (~fourfold) and prevented fatty liver and hepatic injury induced by the high-fat diet. It was accompanied by the normalization of the gene expression of BHMT, GNMT and MGAT, which code for key enzymes of one-carbon metabolism related to liver fat accumulation. In conclusion, the regulation of the expression of MGAT by betaine supplementation provides an additional and novel mechanism by which betaine supplementation regulates lipid metabolism and prevents accumulation of fat in the liver.

Synthesis of guanidinoacetate and creatine from amino acids by rat pancreas.

Creatine is an important molecule involved in cellular energy metabolism. Creatine is spontaneously converted to creatinine at a rate of 1·7% per d; creatinine is lost in the urine. Creatine can be obtained from the diet or synthesised from endogenous amino acids via the enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). The liver has high GAMT activity and the kidney has high AGAT activity. Although the pancreas has both AGAT and GAMT activities, its possible role in creatine synthesis has not been characterised. In the present study, we examined the enzymes involved in creatine synthesis in the pancreas as well as the synthesis of guanidinoacetate (GAA) and creatine by isolated pancreatic acini. We found significant AGAT activity and somewhat lower GAMT activity in the pancreas and that pancreatic acini had measurable activities of both AGAT and GAMT and the capacity to synthesise GAA and creatine from amino acids. Creatine supplementation led to a decrease in AGAT activity in the pancreas, though it did not affect its mRNA or protein abundance. This was in contrast with the reduction of AGAT activity and mRNA and protein abundance in the kidney, suggesting that the regulatory mechanisms that control the expression of this enzyme in the pancreas are different from those in the kidney. Dietary creatine increased the concentrations of GAA, creatine and phosphocreatine in the pancreas. Unexpectedly, creatine supplementation decreased the concentrations of S-adenosylmethionine, while those of S-adenosylhomocysteine were not altered significantly.

Creatine and guanidinoacetate content of human milk and infant formulas: implications for creatine deficiency syndromes and amino acid metabolism.

Creatine is essential for normal neural development; children with inborn errors of creatine synthesis or transport exhibit neurological symptoms such as mental retardation, speech delay and epilepsy. Creatine accretion may occur through dietary intake or de novo creatine synthesis. The objective of the present study was to determine how much creatine an infant must synthesise de novo. We have calculated how much creatine an infant needs to account for urinary creatinine excretion (creatine's breakdown product) and new muscle lay-down. To measure an infant's dietary creatine intake, we measured creatine in mother's milk and in various commercially available infant formulas. Knowing the amount of milk/formula ingested, we calculated the amount of creatine ingested. We have found that a breast-fed infant receives about 9 % of the creatine needed in the diet and that infants fed cows' milk-based formula receive up to 36 % of the creatine needed. However, infants fed a soya-based infant formula receive negligible dietary creatine and must rely solely on de novo creatine synthesis. This is the first time that it has been shown that neonatal creatine accretion is largely due to de novo synthesis and not through dietary intake of creatine. This has important implications both for infants suffering from creatine deficiency syndromes and for neonatal amino acid metabolism.

Creatine supplementation prevents the accumulation of fat in the livers of rats fed a high-fat diet.

The aim of the present study was to examine the effects of creatine supplementation on liver fat accumulation induced by a high-fat diet in rats. Rats were fed 1 of 3 different diets for 3 wk: a control liquid diet (C), a high-fat liquid diet (HF), or a high-fat liquid diet supplemented with creatine (HFC). The C and HF diets contained, respectively, 35 and 71% of energy derived from fat. Creatine supplementation involved the addition of 1% (wt:v) of creatine monohydrate to the liquid diet. The HF diet increased total liver fat concentration, liver TG, and liver TBARS and decreased the hepatic S-adenosylmethionine (SAM) concentration. Creatine supplementation normalized all of these perturbations. Creatine supplementation significantly decreased the renal activity of l-arginine:glycine amidinotransferase and plasma guanidinoacetate and prevented the decrease in hepatic SAM concentration in rats fed the HF diet. However, there was no change in either the phosphatidylcholine:phosphatidylethanolamine (PE) ratio or PE N-methyltransferase activity. The HF diet decreased mRNA for PPARα as well as 2 of its targets, carnitine palmitoyltransferase and long-chain acylCoA dehydrogenase. Creatine supplementation normalized these mRNA levels. In conclusion, creatine supplementation prevented the fatty liver induced by feeding rats a HF diet, probably by normalization of the expression of key genes of ß-oxidation.

Creatine metabolism and the urea cycle.

Because creatine and creatine phosphate are irreversibly converted to creatinine, there is a continuous need for their replacement. This occurs by means of diet and de novo synthesis. Dietary creatine is provided in animal products and can amount to about half of the required amount. Synthesis provides the remainder. Creatine synthesis is a major component of arginine metabolism, amounting to more than 20% of the dietary intake of this amino acid. Creatine metabolism is of importance to patients with urea cycle disorders in two ways, both related to arginine levels. In patients with arginase deficiency, markedly elevated arginine levels may result in higher concentrations of guanidinoacetate and higher rates of creatine synthesis. This is of concern because it is thought that elevated levels of guanidinoacetate may exert neurotoxic effects. In the case of the other urea cycle disorders, arginine levels are markedly decreased unless the patients are supplemented with this amino acid. Decreased levels of arginine may result in decreased rates of creatine synthesis. This may be compounded by the fact that such patients, maintained on low protein diets, will also have lower dietary creatine intakes. There is some evidence that this may decrease brain creatine levels which may contribute to the neurological symptoms exhibited by these patients. It is clear that patients with urea cycle disorders also have altered creatine metabolism. Whether this contributes in a significant way to their neurological symptoms remains an open question.

Creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in the rat in vitro and in vivo.

Since creatinine excretion reflects a continuous loss of creatine and creatine phosphate, there is a need for creatine replacement, from the diet and/or by de novo synthesis. Creatine synthesis requires three amino acids, methionine, glycine, and arginine, and two enzymes, l-arginine:glycine amidinotransferase (AGAT), which produces guanidinoacetate acid (GAA), and guanidinoacetate methyltransferase (GAMT), which methylates GAA to produce creatine. In the rat, high activities of AGAT are found in the kidney, whereas high activities of GAMT occur in the liver. Rat hepatocytes readily convert GAA to creatine; this synthesis is stimulated by the addition of methionine, which increases cellular S-adenosylmethionine concentrations. These same hepatocytes are unable to produce creatine from methionine, arginine, and glycine. (15)N from (15)NH(4)Cl is readily incorporated into urea but not into creatine. Hepatic uptake of GAA is evident in vivo by livers of rats fed a creatine-free diet but not when rats were fed a creatine-supplemented diet. Rats fed the creatine-supplemented diet had greatly decreased renal AGAT activity and greatly decreased plasma [GAA] but no decrease in hepatic GAMT or in the capacity of hepatocytes to produce creatine from GAA. These studies indicate that hepatocytes are incapable of the entire synthesis of creatine but are capable of producing it from GAA. They also illustrate the interplay between the dietary provision of creatine and its de novo synthesis and point to the crucial role of renal AGAT expression in regulating creatine synthesis in the rat.

Elevated tissue betaine contents in developing rats are due to dietary betaine, not to synthesis.

The time course of betaine accumulation and activities of enzymes involved in betaine metabolism were studied in developing rats. In study 1, pups weaned on a nonpurified diet had a transient increase in liver and kidney betaine content followed by a decline after approximately 42-56 d. In study 2, dams and, following weaning, pups were fed an AIN-93G (betaine-free) or an AIN-93G betaine-supplemented diet (0.3%) to determine the source of the transient increase in betaine levels previously observed. In study 2, only rats fed betaine had an increase in plasma betaine concentration. Similarly, liver and kidney betaine contents increased postweaning; however, betaine levels returned to that found in rats fed a betaine-free diet by 49 d of age. The dietary content of betaine fed to dams did not affect pup betaine. The activities of choline dehydrogenase, an enzyme of betaine synthesis, and betaine:homocysteine methyltransferase (BHMT), which is the only known betaine-consuming enzyme in mammals, were also measured in study 2. Liver BHMT activity decreased after weaning, whereas liver and kidney choline dehydrogenase activity increased with age, possibly reaching a plateau by 42 d of age. We conclude that the transient increase in betaine reflects high dietary betaine and not a change in endogenous betaine synthesis.

Creatine: endogenous metabolite, dietary, and therapeutic supplement.

Creatine and phosphocreatine serve not only as an intracellular buffer for adenosine triphosphate, but also as an energy shuttle for the movement of high-energy phosphates from mitochondrial sites of production to cytoplasmic sites of utilization. The spontaneous loss of creatine and of phosphocreatine to creatinine requires that creatine be continuously replaced; this occurs by a combination of diet and endogenous synthesis. Vegetarians obtain almost no dietary creatine. Creatine synthesis makes major demands on the metabolism of glycine, arginine, and methionine. Large doses of creatine monohydrate are widely taken, particularly by athletes, as an ergogenic supplement; creatine supplements are also taken by patients suffering from gyrate atrophy, muscular dystrophy, and neurodegenerative diseases. Children with inborn errors of creatine synthesis or transport present with severe neurological symptoms and a profound depletion of brain creatine. It is evident that creatine plays a critical, though underappreciated, role in brain function.