Formate, a crucial component of one-carbon metabolism, is increasingly recognized as an important intermediate in production and transport of one-carbon units. Unlike tetrahydrofolate-linked intermediates, it is not restricted to the intracellular milieu so that circulating levels of formate can provide insight into cellular events. We report a novel isotope-dilution, GC-MS assay employing derivatization by 2,3,4,5,6-pentafluorobenzyl bromide for the determination of formate in biological samples. This assay is robust and sensitive; it may be applied to the measurement of formate in serum, plasma and urine. We demonstrate how this method may be applied by providing the first characterization of formate levels in a human population; formate levels were higher in males than in females. We also show how this procedure may be applied for the measurement of in vivo kinetics of endogenous formate production in experimental animals.

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.Keywords: folic acid; formic acid; vitamin B2; vitamin B6; vitamin B12. The importance of formate in intermediary metabolismFormic acid and its conjugate base formate are essential endogenous one-carbon metabolites in virtually all living organisms. Formic acid is sometimes called methanoic acid and with its chemical formula, HCOOH, it is the simplest carboxylic acid. The name formic acid takes its origin from formica , the latin for ants from which it was first distilled by Hulse and Fisher after they observed that the ants secreted a substance that caused blue flowers to turn red [ 1 ]. For some time this acid was believed by chemists to be acetic acid, malic acid or a mixture of both. However, after thorough examination, it was demonstrated that it differed from acetic and malic acids on the basis of its specific gravity, its reaction with alkali, its metallic salts and its chemical affinities [ 2 ]. Formic acid has a pKa of 3.77 and, as a result, most of it occurs in the body as the formate anion.The importance of formate in intermediary metabolism was recognized over six decades ago when it was shown that the carbon of formate is incorporated into nucleic acids and into the glucogenic amino acid serine [ 3 -6 ]. The incorporation of formate into nucleic acids and serine requires its activation by tetrahydrofolate (THF) [ 7 ] and therefore the metabolism of formate and folate are tightly related. Our current understanding of cellular folate metabolism involves two parallel pools of folate, one mitochondrial and the other cytosolic, connected by one-carbon donors such as glycine, serine and formate [ 8 ]. For instance, mitochondria can take up serine, oxidize two of its three carbons to formate and export this to the cytosol where it re-enters the folate pool to participate in methylation reactions or nucleotide synthesis (reviewed by Tibbetts [ 8 ]). These observations, combining the roles of formate in amino and nucleic acid metabolism, illustrate the central and essential role of formate in onecarbon metabolism.Despite the importance of formate in intermediary metabolism, our knowledge of its metabolism is limited. Annison [ 9 ] showed that formate represents 10% -30% of the total volatile fatty acids in the blood of numerous anim...

Background:The mitochondrial production of formate is critical to the generation of one-carbon groups. Results: We have shown that the in vivo production of formate is markedly reduced in folate deficiency. Conclusion: Folate deficiency reduces both the production and utilization of one-carbon groups. Significance: Folate status affects the production and utilization of one-carbon groups and the role of choline metabolites as precursors of these groups.

We carry out molecular dynamics (MD) and Monte Carlo (MC) simulations to characterize nucleation in liquid clusters of 600 Lennard-Jones particles over a broad range of temperatures. We use the formalism of mean first-passage times to determine the rate and find that Classical Nucleation Theory (CNT) predicts the rate quite well, even when employing simple modelling of crystallite shape, chemical potential, surface tension and particle attachment rate, down to the temperature where the droplet loses metastability and crystallization proceeds through growth-limited nucleation in an unequilibrated liquid. Below this crossover temperature, the nucleation rate is still predicted when MC simulations are used to directly calculate quantities required by CNT. Discrepancy in critical embryo sizes obtained from MD and MC arises when twinned structures with five-fold symmetry provide a competing free energy pathway out of the critical region. We find that crystallization begins with hcp-fcc stacked precritical nuclei and differentiation to various end structures occurs when these embryos become critical. We confirm that using the largest embryo in the system as a reaction coordinate is useful in determining the onset of growth-limited nucleation and show that it gives the same free energy barriers as the full cluster size distribution once the proper reference state is identified. We find that the bulk melting temperature controls the rate, even though the solid-liquid coexistence temperature for the droplet is significantly lower. The value of surface tension that renders close agreement between CNT and direct rate determination is significantly lower than what is expected for the bulk system.