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BackgroundFormate 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.ObjectiveThis study aimed to define formate concentrations and their determinants in a healthy young population.DesignSerum 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.ResultsSerum 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.ConclusionsFormate 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.

During lactation, there may be a higher need for creatine replacement due to the provision of creatine to the milk. Our objectives were to: 1) quantify the creatine concentration in rat milk; 2) determine the origin of milk creatine; 3) determine the activities of the enzymes of creatine synthesis in lactating rats and pups; and 4) quantify the origin of the creatine that accumulates in rat pups. The origin of milk creatine was determined in 4 dams following the administration of [sup.14]C-creatine by measuring the isotopic enrichment of creatine in the milk and plasma. The activities of the 2 enzymes involved in creatine synthesis were compared in lactating and virgin females (n = 7). For all experiments, the litter size was standardized to 8 pups. The data indicated that the mammary gland extracts creatine from the circulation rather than synthesizing it. This was confirmed by our failure to find substantial activities of the enzymes of creatine synthesis in mammary glands. The provision of milk creatine requires an additional 35-55% of creatine above the daily requirement by lactating rat dams. However, there was no increased creatine synthesis by these dams; the additional creatine was largely provided by hyperphagia, because creatine is present in commercial rat diet. There was a substantial accumulation of creatine in the growing pups, but only ~12% was obtained from milk. The great bulk of creatine accretion was via de novo synthesis by the pups, which imposed a substantial metabolic burden on them. J. Nutr. 140:1570-1573, 2010. doi: 10.3945/jn.110.125476.

The hyperinsulism/hyperammonemia (HI/HA) syndrome is caused by glutamate dehydrogenase (GDH) gain-of-function mutations that reduce the inhibition by GTP, consequently increasing the activity of GDH in vivo. The source of the hyperammonemia in the HI/HA syndrome remains unclear. We examined the effect of systemic activation of GDH on ammonia metabolism in the rat. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) is a nonmetabolizable analog of the natural GDH allosteric activator leucine. A dose of 100 [micro]mol BCH/100 g rat resulted in a mild systemic hyperammonemia. Using arterial-venous (A-V) differences, we exclude the liver, intestine, and skeletal muscle as major contributors to this BCH-induced hyperammonemia. How ever, renal ammonia output increased, as demonstrated by an increase in A-V difference for ammonia across the kidney in BCH-treated animals. Isolated renal cortical tubules incubated with BCH increased the rate of ammoniagenesis from glutamine by 40%. The flux through GDH increased more than twofold when BCH was added to renal mitochondria respiring on glutamine. The flux through glutaminase was not affected by BCH, whereas glutamate-oxaloacetate transaminase flux decreased when normalized to glutaminase flux. These data show that increased renal ammoniagenesis due to activation of GDH can explain the BCH-induced hyperammonemia. These results are discussed in relation to the organ source of the ammonia in the HI/HA syndrome as well as the role of GDH in regulating renal ammoniagenesis. kidney; mitochondria; glutaminase; 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid doi: 10.1152/ajpendo.00028.2010.

Our objectives in this study were as follows: 1) to determine the rate of creatine accretion by the neonatal piglet; 2) identify the sources of this creatine; 3) measure the activities of the enzymes of creatine synthesis; and 4) to estimate the burden that endogenous creatine synthesis places on the metabolism of the 3 amino acids required for this synthesis: glycine, arginine, and methionine. We found that piglets acquire 12.5 mmol of total creatine (creatine plus creatine phosphate) between 4 and 11 d of age. As much as one-quarter of creatine accretion in neonatal piglets may be provided by sow milk and three-quarters by de novo synthesis by piglets. This rate of creatine synthesis makes very large demands on arginine and methionine metabolism, although the magnitude of the demand depends on the rate of remethylation of homocysteine and of reamidination of ornithine. Of the 2 enzymes of creatine synthesis, we found high activity of L-arginine:glycine amidinotransferase in piglet kidneys and pancreas and of guanidinoacetate methyltransferase in piglet livers. Piglet livers also had appreciable activities of methionine adenosyltransferase, which synthesizes S-adenosylmethionine, and of betaine:homocysteine methyltransferase, methionine synthase, and methylene tetrahydrofolate reductase, which are required for the remethylation of homocysteine to methionine. Creatine synthesis is a quantitatively major metabolic process in piglets.

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-zarginine: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 Sadenosylmethionine concentrations. These same hepatocytes are unable to produce creatine from methionine, arginine, and glycine. [sup.15]N from [sup.15]N[H.sub.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. interorgan metabolism; in vivo hepatic fluxes; S-adenosyl-homocysteine hydrolase; methylation demand

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 ~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.

A fraction of the body's creatine and creatine phosphate spontaneously degrades to creatinine, which is excreted by the kidneys. In humans, this amounts to ~1-2 g/day and demands a comparable rate of de novo creatine synthesis. This is a two-step process in which L-arginine:glycine amidinotransferase (AGAT) catalyzes the conversion of glycine and arginine to ornithine and guanidinoacetate (GAA); guanidinoacetate methyltransferase (GAMT) then catalyzes the S-adenosylmethionine-dependent methylation of GAA to creatine. AGAT is found in the kidney and GAMT in the liver, which implies an interorgan movement of GAA from the kidney to the liver. We studied the renal production of this metabolite in both rats and humans. In control rats, [GAA] was 5.9 [micro]M in arterial plasma and 10.9 [micro]M in renal venous plasma for a renal arteriovenous (A-V) difference of -5.0 [micro]M. In the rat, infusion of arginine or citndline markedly increased renal GAA production but infusion of glycine did not. Rats fed 0.4% creatine in their diet had decreased renal AGAT activity and mRNA, an arterial plasma [GAA] of 1.5 [micro]M, and a decreased renal A-V difference for GAA of -0.9 [micro]M. In humans, [GAA] was 2.4 [micro]M in arterial plasma, with a renal A-V difference of - 1.1 [micro]M. These studies show, for the first time, that GAA is produced by both rat and human kidneys in vivo. L-arginine: glycine amidinotransferase; transamidinase; amino acid metabolism

The urinary excretion of orotic acid, an intermediate in the pyrimidine biosynthetic pathway, is markedly increased in many inborn errors of the urea cycle and in a number of other disorders involving arginine metabolism. Carbamoyl phosphate, which accumulates within hepatic mitochondria in patients with ornithine transcarbamoylase deficiency, can diffuse to the cytosol and enter the pyrimidine pathway, resulting in greatly increased orotic acid production and excretion. This orotie aciduria also occurs in inborn errors of the mitochondrial ornithine/citrulline transporter, arginase, argininosuccinate synthetase, and argininosuccinate lyase. Increased orotic acid excretion is also found in a number of hypoargininemic states, such as lysinuric protein intolerance. However, orotic aciduria should not be used uncritically as an index of arginine deficiency because it is found in patients with arginase deficiency who exhibit hyperargininemia. Increased orotic acid excretion can also arise as a result of impairments of pyrimidine synthesis, whether brought about by a genetic defect (e.g., in UMP synthase) or by drugs that inhibit the terminal part of the pathway (e.g., allopurinol or 6-azauridine). When used appropriately, measurement of urinary orotic acid is a valuable tool for the study of many derangements of arginine metabolism, including arginine depletion, and to assess the efficacy of therapies used to replete this amino acid.

Methionine, cysteine, homocysteine, and taurine are the 4 common sulfur-containing amino acids, but only the first 2 are incorporated into proteins. Sulfur belongs to the same group in the periodic table as oxygen but is much less electronegative. This difference accounts for some of the distinctive properties of the sulfur-containing amino acids. Methionine is the initiating amino acid in the synthesis of virtually all eukaryotic proteins; N-formylmethionine serves the same function in prokaryotes. Within proteins, many of the methionine residues are buried in the hydrophobic core, but some, which are exposed, are susceptible to oxidative damage. Cysteine, by virtue of its ability to form disulfide bonds, plays a crucial role in protein structure and in protein-folding pathways. Methionine metabolism begins with its activation to S-adenosylmethionine. This is a cofactor of extraordinary versatility, playing roles in methyl group transfer, 5'-deoxyadenosyl group transfer, polyamine synthesis, ethylene synthesis in plants, and many others. In animals, the great bulk of S-adenosylmethionine is used in methylation reactions. S-Adenosylhomocysteine, which is a product of these methyltransferases, gives rise to homocysteine. Homocysteine may be remethylated to methionine or converted to cysteine by the transsulfuration pathway. Methionine may also be metabolized by a transamination pathway. This pathway, which is significant only at high methionine concentrations, produces a number of toxic endproducts. Cysteine may be converted to such important products as glutathione and taurine. Taurine is present in many tissues at higher concentrations than any of the other amino acids. It is an essential nutrient for cats. KEY WORDS: * methionine * cysteine * taurine * homocysteine * S-adenosylmethionine

Elevation of plasma homocysteine levels has been recognized as an independent risk factor for the development of cardiovascular disease, a major complication of diabetes. Plasma homocysteine reflects a balance between its synthesis via S-adenosyl-L-methionine-dependent methylation reactions and its removal through the transmethylation and the transsulfuration pathways. Betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5) is one of the enzymes involved in the remethylation pathway. BHMT, a major zinc metalloenzyme in the liver, catalyzes the transfer of methyl groups from betaine to homocysteine to form dimethylglycine and methionine. We have previously shown that plasma homocysteine levels and the transsulfuration pathway are affected by diabetes. In the present study, we found increased BHMT activity and mRNA levels in livers from streptozotocin-diabetic rats. In the rat hepatoma cell line (H4IIE cells), glucocorticoids (triamcinolone) increased the level and rate of BHMT mRNA synthesis. In the same cell line, insulin decreased the abundance of BHMT mRNA and the rate of de now) mRNA transcription of the gene. Thus the decreased plasma homocysteine in various models of diabetes could be due to enhanced homocysteine removal brought about by a combination of increased transsulfuration of homocysteine to cysteine and increased remethylation of homocysteine to methionine by BHMT. homocysteine; diabetes; insulin; glucocorticoids; remethylation

The three branched-chain amino acids (BCAAs) are the most hydrophobic of the amino acids and play crucial roles in determining the structures of globular proteins as well as the interaction of the transmembrane domains of membranous proteins with phospholipid bilayers. However, the three BCAAs do not behave identically. In terms of protein secondary structure, valine and isoleucine exhibit a definite preference for the [beta]-structure, whereas leucine has a higher preference for the [alpha]-helix. Although mutation of one BCAA to another is commonly regarded as conservative, there are well-documented examples of such substitutions that have a significant effect on protein function. The occurrence of BCAA in nature is, therefore, attributable to their primary role in protein structure, not to their secondary metabolic roles. These functions are important for almost all proteins; therefore, BCAA commonly account for ~20-25% of most dietary proteins. Dietary BCAA largely escape first-pass splanchnic metabolism. The first steps in their catabolism are common to all three, involving the BCAA aminotransferase (BCAT) and branched-chain [alpha]-keto acid dehydrogenase (BCKD). Their further metabolism employs distinct pathways to different end-products (glucose and/or ketone bodies). However, the fact that the flux-generating step for the catabolism of the three BCAAs occurs at one of the common steps indicates that the production of these downstream products are not individually regulated and, hence, may not play important individual roles. The catabolism of the BCAAs is highly regulated by both allosteric and covalent mechanisms. BCKD is inhibited by phosphorylation and activated by dephosphorylation. Allosteric inhibition of the kinase by the branched-chain keto acids (BCKA) (particularly by [alpha]-ketoisocaproate) serves both as a mechanism for promoting the catabolism of excess quantities of these amino acids as well as for conserving low concentrations of these dietary essential amino acids. Cytosolic and mitochondrial isoenzymes of BCAT have been identified. They are thought to play an important role in brain neurotransmitter metabolism. KEY WORDS: * leucine * isoleucine * valine * protein structure * muscle metabolism

The kidney plays a major role in arginine metabolism in 3 principal ways: arginine synthesis, creatine synthesis, and arginine reabsorption. Appreciable quantities of arginine are synthesized in the kidney from citrulline produced by the intestine. The renal enzymes of arginine synthesis, argininosuccinate synthetase and argininosuccinate lyase, occur in the cells of the proximal tubule. The rate of arginine synthesis depends on citrulline delivery and does not appear to be regulated by dietary arginine availability. Renal arginine synthesis in humans produces ~2 g arginine/d, which may be compared to an intake, from a Western diet, of ~4 to 5 g/d. Spontaneous, nonenzymatic breakdown of creatine and creatine phosphate to creatinine causes the excretion of 1 to 2 g creatinine/d and requires the replacement of an equivalent amount of creatine from the diet and by endogenous synthesis. The first enzyme of creatine biosynthesis, L-arginine:glycine amidinotransferase, occurs in the kidney and produces guanidinoacetate, which is released into the renal vein. The renal output of guanidinoacetate, however, is rather low, and we propose that the entire pathway of creatine synthesis may also occur in the liver. Renal arginine reabsorption salvages ~3 g arginine/d. At the apical membrane of proximal tubular cells, arginine shares a transporter with lysine, ornithine, and cystine. Defects in this heteromeric transporter cause cystinuria, which is also characterized by urinary loss of arginine, lysine, and ornithine. Arginine is transported out of the proximal tubular cells at the basolateral membrane by another heteromeric transporter, which also transports lysine and ornithine. Defects in this transporter cause lysinuric protein intolerance. J. Nutr. 134: 2791S-2795S, 2004. KEY WORDS: * arginine synthesis * citrulline * kidney * creatine synthesis * arginine reabsorption

There is growing awareness that costs associated with egg production play a significant role in shaping avian life histories. The life-history strategy of the Thick-billed Murre (Uria lomvia), a colonial, cliff-breeding seabird of Arctic waters, is characterized by a high annual adult survival rate, deferred breeding, and laying of a single-egg clutch. The single-egg clutch is a widespread phenomenon among seabirds and is generally thought to reflect demands of chick provisioning, rather than egg production. We compared composition of eggs laid by Thick-billed Murres most likely to be physiologically constrained in their capacity to produce eggs (young females with no prior experience, and females forming replacement eggs) to that of first eggs laid by early laying females (typically older, more experienced members of the population). Young, inexperienced females laid 4-18 days past the populationwide median laying date, and their eggs averaged 13% lighter in mass than those laid by early layers. Compared to early laid eggs, shell mass on young females' eggs was similar to that predicted from egg mass, but their eggs had a lower yolk-to-albumen ratio. There was little difference between the two groups in relative protein content of albumen, relative protein or lipid content of yolk, or amino acid makeup of protein in yolk or albumen. Replacement eggs averaged 6% lighter in mass than first eggs laid by the same females earlier that season. As with young females' eggs, replacement eggs had shells similar in mass to that predicted from egg mass, but lower yolk-to-albumen ratios, when compared to early laid eggs. Both protein and lipid concentrations in yolk were similar in first and replacement eggs, but replacements were deficient in albumen protein. Amino acid makeup of protein in yolk and albumen was similar in the two groups. Those results suggest that any limitations on egg production acting on young, inexperienced females are manifested in delayed laying and reductions in overall egg mass and proportional yolk content, but not in variation in biochemical composition of their eggs. Limitations on females forming replacement eggs are manifested not only in reduced egg mass and yolk content (as with young females), but also in changes in the biochemical composition of eggs; in particular, evidence suggests that relaying females may face a deficit of endogenous protein. The existence of such limitations suggests that demands of egg production can be significant even in a species that lays a single-egg clutch.

Interorgan amino acid transport is a highly active and regulated process that provides amino acids to all tissues of the body, both for protein synthesis and to enable amino acids to be used for specific metabolic functions. It is also an important component of plasma amino acid homeostasis. Net movement of amino acids depends on the physiological and nutritional state. For example, in the fed state the dominant flux is from the intestine to the other tissues. In starvation the dominant flux is from muscle to the liver and kidney. A number of general principles underlie many amino acid fluxes: i) The body does not have a store for amino acids. This means that dietary amino acids, in excess of those required for protein synthesis, are rapidly catabolized; ii) Amino acid catabolism must occur in a manner that does not elevate blood ammonia. Thus, extrasplanchnic amino acid metabolism often involves an innocuous means of transporting nitrogen to the liver; iii) Because most amino acids are glucogenic, there will be a considerable flux of amino acids to the gluconeogenic organs when there is a need to produce glucose. In addition to these bulk flows, fluxes of many specific amino acids underlie specific organ function. These include intestinal oxidation of enteral amino acids, the intestinal/renal axis for arginine production, the brain uptake of neurotransmitter precursors and renal glutamine metabolism. There is no single means of regulating amino acid fluxes; rather, such varied mechanisms as substrate supply, enzyme activity, transporter activity and competitive inhibition of transport are all found. J. Nutr. 133: 2068S-2072S, 2003. KEY WORDS: * arteriovenous differences * intestinal metabolism * perivenous and periportal hepatocytes * glutamine * arginine

House, James D., Beatrice N. Hall, and John T. Brosnan. Threonine metabolism in isolated rat hepatocytes. Am J Physiol Endocrinol Metab 281: E1300-E1307, 2001.-- The removal of the 1-carbon of threonine can occur via threonine dehydrogenase or threonine aldolase, this carbon ending up in glycine to be liberated by the mitochondrial glycine cleavage system and producing C[O.sub.2]. Alternatively, in the threonine dehydratase pathway, the 1-carbon ends up in [alpha]-ketobutyrate, which is oxidized in the mitochondria to C[O.sub.2]. Rat hepatocytes, incubated in Krebs-Henseleit medium, were incubated with 0.5 mM L-[1-.sup.14 C] threonine, and [sup.14C][O.sub.2] production was measured. Added glycine (0.3 mM) marginally suppressed threonine oxidation. Cysteamine (0.5 mM), a potent inhibitor of the glycine cleavage system, reduced threonine oxidation to 65% of controls. However, [alpha] cyanocinnamate (0.5 mM), a competitive inhibitor of mitochondrial [alpha] keto acid uptake, reduced threonine oxidation to 35% of controls. These data provided strong evidence that ~65% of threonine oxidation occurs through the glycine-independent threonine dehydratase pathway. Glucagon ([10.sup.-7] M) increased threonine oxidation and stimulated threonine uptake by these cells. In summary, the majority of threonine oxidation occurs through the threonine dehydratase pathway in rat hepatocytes, and threonine oxidation is increased by glucagon, which also increases threonine's transport. glucagon; threonine dehydratase; glycine; transport Received 23 May 2001; accepted in final form 31 July 2001

Studies were made on the effects of dietary arginine and protein on the renal arginine synthesis of adult rats. The rats had diets with graded amounts of arginine and protein for one week. Results suggest that renal arginine synthesis goes on independently of arginine or protein intake. Citrulline availability in the intestines seems to be the necessary factor in keeping up renal arginine synthesis. A mole of citrulline in the kidneys produces a mole of arginine, an essential element in protein synthesis. However, dietary arginine deficiency causes less food intake and body weight loss.

An analysis of the metabolic processes occurring in the liver revealed that amino acids constitute its major energy fuel. The degradation of amino acids to carbon dioxide (CO2) exceeds the energy needs of the liver, so CO2 is converted to glucose by oxidation. The adequate supply of adenosine triphosphate (ATP) prevents excessive formation of excess ATP which is a rate-limiting step in glucose formation. This results in excess energy for utilization by peripheral tissues, thus sparing its own energy supplies.