Autotrophic synthesis of activated acetic acid from two CO2 in Methanobacterium thermoautotrophicum

In a previous study with Methanobacterium thermoautotrophicum evidence was presented that methanogenesis and autotrophic synthesis of activated acetic acid from CO₂ are linked processes. In this study one-carbon metabolism was investigated with growing cultures and in vitro.Serine was shown to be converted into glycine and activated formaldehyde, but only traces of label from [¹⁴C-3] of serine appeared in biosynthetic one-carbon positions. This seeming discrepancy could be explained if the same activated formaldehyde is an intermediate in biosynthesis and in methanogenesis from CO₂. This hypothesis was supported by demonstrating that [¹⁴C-3] of serine and [¹⁴C] formaldehyde were rapidly converted into methane, but a small portion of the label was also specifically incorporated into the methyl group of acetate. Methane and acetate synthesis in vitro were similarly stimulated by various compounds. These experiments indicate that the methyl of acetate and methane share common one-carbon precursor(s), i.e. methylene tetrahydromethanopterin, which can also be formed enzymatically from C-3 of serine or chemically from formaldehyde.Propyl iodide 20–40 M) and methyl iodide (1–3 M) completely inhibited growth in the dark. This effect was abolished by light. Methane formation was hardly affected. When ¹⁴CH₃I was applied at an only slightly inhibitory concentration, ¹⁴C was incorporated into the methyl of acetate. In vitro, similar effects on [¹⁴C] acetate formation from ¹⁴CO₂ or from [¹⁴C-3] of serine were observed, except that methyl iodide did not inhibit, but even stimulated acetate synthesis. These experiments indicate that a corrinoid is involved in acetate synthesis and probably not in methanogenesis from CO₂; the metal is light-reversibly alkylated and functions in methyl transfer to the acetate methyl.     

Influence of corrinoid antagonists on methanogen metabolism.

Iodopropane inhibited cell growth and methane production when Methanobacterium thermoautotrophicum, Methanobacterium formicicum, and Methanosarcina barkeri were cultured on H2-CO2. Iodopropane (40 microM) inhibited methanogenesis (30%) and growth (80%) when M. barkeri was cultured mixotrophically on H2-CO2-methanol. The addition of acetate to the medium prevented the observed iodopropane-dependent inhibition of growth. The concentrations of iodopropane that caused 50% inhibition of growth of M. barkeri on either H2-CO2, H2-CO2-methanol, methanol, and acetate were 112 +/- 6, 24 +/- 2, 63 +/- 11, and 4 +/- 1 microM, respectively. Acetate prevented the iodopropane-dependent inhibition of one-carbon metabolism. Cultivation of M. barkeri on H2-CO2-methanol in bright light also inhibited growth and methanogenesis to a greater extent in the absence than in the presence of acetate in the medium. Acetate was the only organic compound examined that prevented iodopropane-dependent inhibition of one-carbon metabolism in M. barkeri. The effect of iodopropane and acetate on the metabolic fates of methanol and carbon dioxide was determined with 14C tracers when M. barkeri was grown mixotrophically on H2-CO2-methanol. The addition of iodopropane decreased the contribution of methanol to methane and cell carbon while increasing the contribution of CO2 to cell carbon. Regardless of iodopropane, acetate addition decreased the contribution of methanol and CO2 to cell carbon without decreasing their contribution to methane. The corrinoid antagonists, light and iodopropane, appeared most specific for methanogen metabolic reactions involved in acetate synthesis from one-carbon compounds and acetate catabolism.     

Cholesterol and bile acids via acetate from the insect juvenile hormone analog methoprene

Metabolism of the insect growth regulator methorpene by a steer produced labeled cholesterol, cholic acid, and deoxycholic acid. Cholesterol and deoxycholic acid were degraded chemically by Kuhn-Roth and Barbier-Wieland oxidations to show catabolism of [5-¹⁴C] methoprene to [2-¹⁴C] acetate. Unless radioactive residues are rigorously characterized as primary metabolites rather than natural products, misinterpretations will occur when assessing metabolic pathways for extremely biodegradable compounds such as methoprene.     

Mitochondrial compartmentation of metabolic CO2 resulting from its site of origin in relation to urea synthesis

In isolated hepatocytes the entry into urea of metabolic ¹⁴CO₂; derived from [¹⁴C] formate is modified by the addition of dichloroacetate and hydroxypyruvate. An explanation is that this results from changes in the cytoplasmic/mitochondrial pH gradient. ¹⁴CO₂, derived from [1-¹⁴C]alanine enters into urea more readily than ¹⁴CO₂ arising from [1-¹⁴C]glutamate. It is proposed that the difference, which is more than 4-fold, is indicative of a preferred pathway for metabolic CO₂ in liver mitochondria from pyruvate dehydrogenase to carbamoylphosphate synthetase than form oxoglutarate dehydrogenase. Acetazolamide inhibition of carbonic anhydrase is without effect on this observed incorporation into urea.     

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-¹⁴C] pyruvate, and chemically synthesized O-[methyl-¹⁴C]vanillate were used to further investigate the catabolism of one-carbon (C₁) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO₂ was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO₂ readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO₂ and pyruvate predominantly labeled the carboxyl group of acetate. No CO₂ was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.     

One-carbon metabolism in methanogens: evidence for synthesis of a two-carbon cellular intermediate and unification of catabolism and anabolism in Methanosarcina barkeri

One-carbon metabolic transformations associated with cell carbon synthesis and methanogenesis were analyzed by long- and short-term ¹⁴CH₃OH or ¹⁴CO₂ incorporation studies during growth and by cell suspensions. ¹⁴CH₃OH and ¹⁴CO₂ were equivalently incorporated into the major cellular components (i.e., lipids, proteins, and nucleic acids) during growth on H₂-CO₂-methanol. ¹⁴CH₃OH was selectively incorporated into the C-3 of alanine with decreased amounts fixed in the C-1 and C-2 positions, whereas ¹⁴CO₂ was selectively incorporated into the C₁ moiety with decreasing amounts assimilated into the C-2 and C-3 atoms. Notably, ¹⁴CH₄ and [3-¹⁴C]alanine synthesized from ¹⁴CH₃OH during growth shared a common specific activity distinct from that of CO₂ or methanol. Cell suspensions synthesized acetate and alanine from ¹⁴CO₂. The addition of iodopropane inhibited acetate synthesis but did not decrease the amount of ¹⁴CH₃OH or ¹⁴CO₂ fixed into one-carbon carriers (i.e., methyl coenzyme M or carboxydihydromethanopterin). Carboxydihydromethanopterin was only labeled from ¹⁴CH₃OH in the absence of hydrogen. Cell extracts catalyzed the synthesis of acetate from ¹⁴CO (~1 nmol/min per mg of protein) and an isotopic exchange between CO₂ or CO and the C-1 of pyruvate. Acetate synthesis from ¹⁴CO was stimulated by methyl B₁₂ but not by methyl tetrahydrofolate or methyl coenzyme M. Methyl coenzyme M and coenzyme M were inhibitory to acetate synthesis. Cell extracts contained high levels of phosphotransacetylase (>6 μmol/min per mg of protein) and acetate kinase (>0.14 μmol/min per mg of protein). It was not possible to distinguish between acetate and acetyl coenzyme A as the immediate product of two-carbon synthesis with the methods employed.     

THE INFLUENCE OF INTRAVENTRICULARLY INJECTED AMINO ACID EXCITANTS ON THE LABELLING OF ENDOGENOUS BRAIN AMINO ACIDS FROM [U-14C]ACETATE IN NEMBUTALIZED MICE

Mice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U-¹⁴C]acetate metabolism were investigated. The natural excitant amino acids, l -glutamate and l -aspartate, reduced the incorporation of ¹⁴C from [U-¹⁴C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N-methyl-d -aspartate caused a reduction in the incorporation of ¹⁴C from intraventricularly injected [U-¹⁴C]acetate into all of the brain amino acids labelled by [U-¹⁴C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl-CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine-forming pool(s) of glutamate metabolism.     

Sensitive periods for developmental toxicity of orally administered artesunate in the rat

BACKGROUND: Artesunate has been reported to cause embryolethality and malformations when administered orally to rats during organogenesis. The purpose of this study was to determine the most sensitive period(s) for the induction of these effects in order to provide clues about possible mechanisms and to identify a short treatment regimen for further studies. METHODS: Pregnant rats were orally administered artesunate (10, 17 or 30 mg/kg/day) on single or multiple days of gestation. Cesarean sections and fetal evaluations were conducted on Day 21 postcoitum (pc). RESULTS: Embryolethality, cardiovascular malformations and a syndrome of skeletal defects were observed after single doses on days 10 to 14 pc, while no developmental effects were observed before (day 9 pc) or after (days 16 or 17 pc) that period. The most sensitive day for embryo lethality was day 11 pc, where lethality occurred with a very steep dose response (postimplantation loss was ～15% at 10 mg/kg and 100% at 17 mg/kg/day). The most sensitive day for the induction of malformations was day 10 pc. Malformations tended to occur in partially resorbed litters and included cardiovascular defects and bent and misshapen long bones and scapulae. CONCLUSIONS: The sensitive window for developmental toxicity of artesunate in the rat was identified as days 10 to 14 pc. Single oral doses produced embryolethality and similar cardiovascular and skeletal malformations as previously reported in longer term dosing experiments. These single dose treatment regimens could be useful to further investigate the mechanistic basis for artesunate‐induced developmental toxicity. Birth Defects Research (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

Propionate catabolism in the housefly Musca domestica and the termite Zootermopsis nevadensis

The catabolism of propionate was examined in the housefly Musca domestica (which does not contain detectable amounts of vitamin B₁₂) and the termite Zootermopsis nevadensis (which contains large amounts of vitamin B₁₂). The products from carbon-14 labeled propionate were separated by HPLC and radioactivity was determined by liquid scintillation counting. In vivo studies as a function of time showed that, in both species, products of [2-¹⁴C]propionate were acetate and 3-hydroxypropionate. [2-¹⁴C]Propionate was not efficiently converted to methylmalonate or succinate, as would occur in mammals. Studies with sub-cellular fractions in both species showed that only the mitochondrial fraction efficiently converted propionate to acetate. Radioactivity from [1-¹⁴C]propionate incubated with housefly mitochondria was recovered only in fractions corresponding to propionate and 3-hydroxypropionate. The data obtained are consistent with a metabolic pathway in which propionate is converted to 3-hydroxypropionate and then to acetate. The results presented here demonstrate this pathway in insects which have high vitamin B₁₂ levels and undetectable vitamin B₁₂ levels, suggesting that this may be a common pathway for propionate metabolism in insects.     

Comparative study of in vivo stigmasterol biosynthesis in Nicotiana tabacum and Hordeum vulgare

Six-day-old tobacco (Nicotiana tabacum) and barley (Hordeum vulgare) seedlings rapidly incorporated and metabolized exogenously supplied [4-¹⁴C]sitosterol but neither plant was able to convert it into stigmasterol. However, a sterol metabolite was isolated from both species and the acetate derivative was slightly more polar, on AgNO₃—silica gel TLC, than stigmasteryl acetate. A similar metabolite was also obtained with [4-¹⁴C]cholesterol, indicating a general metabolic reaction of plants to exogenous sterols. Both species incorporated [2-¹⁴C]mevalonic acid into sitosterol and stigmasterol. We suggest that in vascular plants, whether monocotyledons or dicotyledons, the pathway of stigmasterol biosynthesis is not via sitosterol but through a common precursor which is derived from mevalonic acid.     

Origin of end-products from the co-metabolism of glucose and citrate by Leuconostoc mesenteroides subsp. cremoris

Summary The addition of citrate to glucose broth led to an increase in specific growth rate and glucose catabolism, but a decrease in molar growth yield from glucose, in Leuconostoc mesenteroides subsp. cremoris. Acetate and formate were produced during the stationary phase of growth. According to the fermentation balance, part of the acetate and lactate came from the pyruvate of citrate metabolism. L. mesenteroides subsp. cremoris incorporated radioactive metabolites from [1,5-¹⁴C] citrate into cell material, primarily into lipids. [U-¹⁴C] Glucose was not incorporated into cell material.     

Microbial acetate conversion to methane: kinetics,yields and pathways in a two-step digestion process

Summary Organic waste is converted in a two-stage process to methane and carbon dioxide by mixed cultures of microorganisms. Acetate, a product of acidogenic and acetogenic bacteria and the main substrate for methanogenic bacteria, is an important intermediate of the anaerobic degradation process, which results in the generation of methane. It was shown by labelling experiments using (U-¹⁴C) acetate that as much as 65%–96% of the total methane produced came from the acetate. The first order utilization rate for acetate in the methanogenic stages of a two-stage digestion process was between 0.17 h^-1 and 0.5 h^-1. The kinetics as well as the mass flow and yields of acetate and the methyl group of acetate were determined by pulse-labelling experiments with (U-¹⁴C) acetate and (2-¹⁴C) acetate without a significant rise of the total concentrations. Up to 58% of the acetate carbon was transformed to methane, and about 30% to carbon dioxide; only 4%–15% was incorporated into the biomass. There are at least two parallel degradation mechanisms in the metabolic transformation of acetate to methane: acetate is cleaved either to form methane and carbon dioxide or to form hydrogen and carbon dioxide, which can be transformed by an additional reaction to methane. Labelling experiments with (2-¹⁴C) acetate show that both mechanisms took place at similar order.     

In VivoC NMR Study of the Bidirectional Reactions of the Wood–Werkman Cycle and around the Pyruvate Node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici

This study used in vivo¹³C NMR spectroscopy to directly examine bidirectional reactions of the Wood–Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-¹³C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 μmol min⁻¹ g⁻¹ (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood–Werkman cycle was evident from the formation of intermediates such as [3-¹³C]pyruvate and [3-¹³C]malate and of products like [2-¹³C]acetate from [2-¹³C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-¹³C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.     

Microbial Activity in Organic Soils as Affected by Soil Depth and Crop

The microbial activity of Pahokee muck, a lithic medisaprist, and the effect of various environmental factors, such as position in the profile and type of plant cover, were examined. Catabolic activity for [7-¹⁴C]salicylic acid, [1,4-¹⁴C]succinate, and [1,2-¹⁴C]acetate remained reasonably constant in surface (0 to 10 cm) soil samples from a fallow (bare) field from late in the wet season (May to September) through January. Late in January, the microbial activity toward all three compounds decreased approximately 50%. The microbial activity of the soil decreased with increasing depth of soil. Salicylate catabolism was the most sensitive to increasing moisture deep in the soil profile. At the end of the wet season, a 90% decrease in activity between the surface and the 60- to 70-cm depth occurred. Catabolism of acetate and succinate decreased approximately 75% in the same samples. Little effect of crop was observed. Variation in the microbial activity, as measured by the catabolism of labeled acetate, salicylate, or succinate, was not significant between a sugarcane (Saccharum officinarum L.) field and a fallow field. The activity with acetate was insignificantly different in a St. Augustine grass [Stenotaphrum secundatum (Walt) Kuntz] field, whereas the catabolism of the remaining substrates was elevated in the grass field. These results indicate that the total carbon evolved from the different levels of the soil profile by the microbial community oxidizing the soil organic matter decreased as the depth of the soil column increased. However, correction of the amount of carbon yielded at each level for the bulk density of that level reveals that the microbial contribution to the soil subsidence is approximately equivalent throughout the soil profile above the water table.     

Inhibition of hydrocarbon biosynthesis in the housefly by 2-Octadecynoate

The elongation of [9,10-³H]oleoyl-CoA with malonyl-CoA to form 20, 22, and 24 carbon monounsaturated fatty acids was demonstrated in housefly microsomes by radio-GLC. These elongation reactions, which have been postulated to be involved in hydrocarbon biosynthesis, have not been previously demonstrated in insects. 2-Octadecynoate (18:1 Δ²⁼) inhibited the in vivo incorporation of [1-¹⁴C]acetate into both fatty acids and hydrocarbons in a dose-dependent manner. At doses of 10 μg per female housefly of the alkynoic acid, the incorporation of [1-¹⁴C]acetate into hydrocarbon was inhibited 93%, the incorporation of [9,10-³H]oleate into hydrocarbon was inhibited 64%, and the incorporation of [1-¹⁴C]acetate into total internal lipid was inhibited 65%. Partially purified FAS was inhibited 50% and 95% at 15 μM and 40 μM, respectively, of the alkynoic acid. These results show that 2-octadecynoate inhibits hydrocarbon biosynthesis in the housefly by inhibiting FAS, and the in vivo data suggest that the elongation of 18:1 to longer chain fatty acids is also inhibited.     

Metabolic fate of guanosine in higher plants

The aim of the present study was to investigate the metabolic fate of guanine nucleotides in higher plants. The rate of uptake of [8-¹⁴C]guanosine by suspension-cultured Catharanthus roseus cells was more than 20 times higher than that of [8-¹⁴C]guanine. The rate of uptake of [8-¹⁴C]guanosine increased with the age of the culture. Pulse-chase experiments with [8-¹⁴C]guanosine revealed that some of the guanosine that had been taken up by the cells was converted to guanine nucleotides and incorporated into nucleic acids. A significant amount of [8-¹⁴C]guanosine was degraded directly to xanthine, allantoin and allantoic acid, with the generation of ¹⁴CO₂ as the final product. The rate of salvage of [8-¹⁴C]guanosine for the synthesis of nucleic acids was highest in young cells, while the rate of degradation increased with the age of the cells. In segments of roots from Vigna mungo seedlings, nearly 50% of the [8-¹⁴C]guanosine that had been absorbed over the course of 15 min was recovered in guanine nucleotides. A significant amount of the radioactivity in nucleotides became associated with nucleic acids and ureides during ‘chase’ periods. In segments of young leaves of Camellia sinensis, [8-¹⁴C]guanosine was initially incorporated into guanine nucleotides, nucleic acids, theobromine and ureides, and the radioactivity in these compounds was transferred to caffeine and CO₂ during a 24-h incubation. Our results suggest that guanosine is an intermediate in the catabolism of guanine nucleotides and that it is re-utilised for nucleotide synthesis by ‘salvage’ reactions. Guanosine was catabolised by the conventional degradation pathway via xanthine and allantoin. In some plants, guanosine is also utilised for the formation of ureide or the biosynthesis of caffeine.     

Participation of propionate in cholesterol biosynthesis by rat liver

Incorporation of (2¹⁴C) propionate into cholesterol was demonstrated using rat liver slices and homogenates. The incorporation of (2¹⁴C) propionate was greater than that of (2¹⁴C) acetate. Using the same liver homogenate preparation (2 ¹⁴C) succinate and (2¹⁴C) pyruvate were incorporated into cholesterol to a lesser extent than (2¹⁴C) acetate and (2 ¹⁴C) propionate. Addition of unlabeled acetate failed to dilute the incorporation of (2 ¹⁴C) propionate. Incorporation of the 2 and 3 carbon atoms pf propionate were equal; little incorporation of the 1 carbon atom was demonstrable. These results indicate that propionate is an excellent source of 2 carbon units for isoprenoid biosynthesis; the intermediary pathway does not involve a common acetate pool nor can these results be satisfactorily explained by citric acid intermediary metabolism.     

Ureide Catabolism of Soybeans : II. Pathway of Catabolism in Intact Leaf Tissue

Allantoin catabolism studies have been extended to intact leaf tissue of soybean (Glycine max L. Merr.). Phenyl phosphordiamidate, one of the most potent urease inhibitors known, does not inhibit ¹⁴CO₂ release from [2,7-¹⁴C]allantoin (urea labeled), but inhibits urea dependent CO₂ release ≥99.9% under similar conditions. Furthermore, ¹⁴CO₂ and [¹⁴C] allantoate are the only detectable products of [2,7-¹⁴C]allantoin catabolism. Neither urea nor any other product were detected by analysis on HPLC organic acid or organic base columns although urea and all commercially available metabolites that have been implicated in allantoin and glyoxylate metabolism can be resolved by a combination of these two columns. In contrast, when allantoin was labeled in the two central, nonureido carbons ([4,5-¹⁴C]allantoin), its catabolism to [¹⁴C]allantoate, ¹⁴CO₂, [¹⁴C]glyoxylate, [¹⁴C]glycine, and [¹⁴C]serine in leaf discs could be detected. These data are fully consistent with the metabolism of allantoate by two amidohydrolase reactions (neither of which is urease) that occur at similar rates to release glyoxylate, which in turn is metabolized via the photorespiratory pathway. This is the first evidence that allantoate is metabolized without urease action to NH₄⁺ and CO₂ and that carbons 4 and 5 enter the photorespiratory pathway.     

Teratogenicity of dimethoxyethyl phthalate and its metabolites methoxyethanol and methoxyacetic acid in the rat

It is hypothesized that the known teratogen di(2-methoxyethyl) phthalate (DMEP) acts by in vivo hydrolysis to 2-methoxyethanol (2-ME), also a known teratogen, which in turn is metabolized to methoxyacetic acid (MAA), the proximate teratogen. Teratological studies were conducted with Wistar rats, with the administration of these three agents on day 12 of gestation. On an equimolar dosage basis, DMEP, 2-ME, and MAA were equally potent, which is consistent with the hypothesis. There was a striking similarity in the defects produced by these agents, mainly hydronephrosis, heart defects, and short limbs and tails. In particular all three agents produced unusual heart defects (dilated ductus arteriosus and dilated aortic arch) not seen with other agents, as well as ventral polydactyly, a rarely seen defect, suggesting teratogenic action by a common mechanism or component; 4-methylpyrazole, an alcohol dehydrogenase inhibitor, provided significant protection against 2-ME. This combination of effects strongly suggests that following the administration of DMEP, 2-ME, or MAA, MAA is the proximate teratogen.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.