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Methylation demand and homocysteine metabolism: effects of dietary provision of creatine and guanidinoacetate. Am J Physiol Endocrinol Metab 281: E1095-E1100, 2001.--S-adenosyl, methionine, formed by the adenylation of methionine via S-adenosylmethionine synthase, is the methyl donor in virtually all known biological methylations. These methylation reactions produce a methylated substrate and S-adenosylhomocysteine, which is subsequently metabolized to homocysteine. The methylation of guanidinoacetate to form creatine consumes more methyl groups than all other methylation reactions combined. Therefore, we examined the effects of increased or decreased methyl demand by these physiological substrates on plasma homocysteine by feeding rats guanidinoacetate- or creatine-supplemented diets for 2 wk. Plasma homocysteine was significantly increased (~50%) in rats maintained on guanidinoacetate-supplemented diets, whereas rats maintained on creatine-supplemented diets exhibited a significantly lower (~25%) plasma homocysteine level. Plasma creatine and muscle total creatine were significantly increased in rats fed the creatine-supplemented or guanidinoacetate-supplemented diets. The activity of kidney L-arginine:glycine amidinotransferase, the enzyme catalyzing the synthesis of guanidinoacetate, was significantly decreased in both supplementation groups. To examine the role of the liver in mediating these changes in plasma homocysteine, isolated rat hepatocytes were incubated with methionine in the presence and absence of guanidinoacetate and creatine, and homocysteine export was measured. Homocysteine export was significantly increased in the presence of guanidinoacetate. Creatine, however, was without effect. These results suggest that homocysteine metabolism is sensitive to methylation demand imposed by physiological substrates. methionine; methyl balance; methyltransferases; hepatocytes; liver; kidney

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. [ABSTRACT FROM PUBLISHER]

Plasma and urinary formate concentrations were recently found to be elevated during vitamin B12 and folate deficiencies. It was proposed that formate may be a valuable biomarker of impaired one-carbon metabolism. Formate is an essential intermediary metabolite in folate-mediated one-carbon metabolism and, despite its importance, our knowledge of its metabolism is limited. Formate can be produced from several substrates (e.g., methanol, branched chain fatty acids, amino acids), some reactions being folate-dependent while others are not. Formate removal proceeds via two pathways; the major one being folate-dependent. Formate is a potentially toxic molecule and we suggest that formate may play a role in some of the pathologies associated with defective one-carbon metabolism. [ABSTRACT FROM AUTHOR]

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 14C-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 In = 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. [ABSTRACT FROM AUTHOR]

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 (BCHI is a nonmetabolizable analog of the natural GDH allosteric activator leucine. A dose of 100 ~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, However. 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 two-fold 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 n31e of GDH in regulating renal ammoniagenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 15N from 15NH4CI 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. [ABSTRACT FROM AUTHOR]

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 AlN-93G Ibetaine-free) or an AlN-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. [ABSTRACT FROM AUTHOR]

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 μM in arterial plasma and 10.9 μM in renal venous plasma for a renal arteriovenous (A-V) difference of -5.0 μM. In the rat, infusion of arginine or citrulline 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 μM, and a decreased renal A-V difference for GAA of —0.9 μM. In humans, [GAA] was 2.4 μM in arterial plasma, with a renal A-V difference of —1.1 μM. These studies show, for the first time, that GAA is produced by both rat and human kidneys in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Methionine is a dietary essential amino acid that plays unique roles, both in protein structure and in metabolism. Methionine serves as the initiating amino acid in eukaryotic protein synthesis. In globular proteins, most methionine residues are buried inside the hydrophobic core. Some methionine residues, located on the surfaces of proteins are susceptible to oxidation to methionine sulfoxide. These may be reduced back to methionine by methionine sulfoxide reductase. Methionine''s principal metabolic function lies in its conversion to S-adenosylmethionine which is the principal biological methylating agent. Methionine metabolism may be divided into transmethylation, remethylation and transsulfuration. S-adenosylmethionine, via allosteric mechanisms, exerts control over these processes. Creatine synthesis is a major user of S-adenosylmethionine-derived methyl groups. Piglets acquire considerable quantities of creatine during growth. About one third of this is provided in the milk; two thirds is synthesized within the piglet. This requires very high rates of creatine synthesis and has the potential to be a significant drain on dietary methionine. [Copyright &y& Elsevier]

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 orotic 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. [ABSTRACT FROM AUTHOR]