Alpha-aminocyclic and bicyclic alkane carboxylic acids: differential effects on selected amino acids of rat brain cortex

Abstract— The intraperitoneal administration of 1-aminocyclopentane carboxylic acid, 1-aminocyclohex-ane carboxylic acid, l-aminocycloheptane carboxylic acid, 1-aminocyclooctane carboxylic acid, exo-2-aminobicyclo(2,2. l)heptane-2-carboxylic acid. endo-2-aminobicyclo(2,2.1)heptane-2-carboxylic acid. 2-aminobicyclo(2.2.2)octane-2-carboxylic acid and 2-aminobicyclo(3,2.l)octane-2-carboxylic acid to 18-day-old male rats selectively perturbed the levels of neutral amino acids in the cerebral cortex. While the effect of the above compounds was rather diversified and usually resulted in a reduction of amino acid levels. marked elevations of the levels of valine and isoleucine were also noted. 1-Aminocycloheptane and cyclooctane carboxylic acids were particularly noteworthy, in that they elicited a marked reduction of the levels of cortical phenylalanine.     

Microbiological Asymmetric Hydroxylation of 7-Carboxybicyclo[2.2.1]heptane and hept-2-ene and Their Methyl Esters Giving Potentially Useful Chiral Synthons for Cyclopentanoids

One hundred and nineteen strains of microorganisms (yeasts, bacteria, actinomycetes and fungi) were screened as to the hydroxylation of bicyclo[2.2.1]heptane-7-carboxylic acid, bicyclo[2.2.1]hept-2-ene-7-syn-carboxylic acid, and their methyl esters. Several species belonging to the genera, Bacillus, Streptomyces, Penicillium, Aspergillus, Absidia, Beauveria, Cunninghamella, Drechslera, Mucor and Chaetomium, were found to asymmetrically hydroxylate some or all of the substrates. Bacillus thuringiensis and Aspergillus awamori were the most effective microorganisms for obtaining the chiral products, (lR)-2-hydroxy acids or esters, with enantiomeric purities of 75~90% e.e., which are potential intermediates for (?)-methyl jasmonate or natural prostaglandins.     

SYNTHETIC AMINO ACIDS AND THE NATURE OF l-DOPA TRANSPORT AT THE BLOOD-BRAIN BARRIER

—The blood-brain barrier transport of amino acids has been measured using the carotid injection technique in the rat. The synthetic amino acids, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and α-(methylamino)isobutyric acid (MeAIB), were model substrates in the Ehrlich cell for the leucine (L) and alanine (A) neutral amino acid transport mechanisms, respectively. The uptake (±)b-[carboxyl-¹⁴C]BCH at the same rate for the five brain regions tested suggested a similarity between regions for the L transport mechanism. At injectant concentrations of 0·1 mm (similar to naturally occurring aromatic neutral amino acids), BCH was mainly taken up by a saturable mediated transport mechanism (K₁, 0·16 mm and V_max, 0·03/μmol/g per min). At higher concentrations, uptake by a nonsaturable or diffusional mechanism could be demonstrated. When BCH was added as a second amino acid to l -[3-¹⁴C]DOPA, the saturable component of l -DOPA transport was significantly inhibited. MeAIB had no measurable effect on the rate of l -DOPA transport. These results suggested that the mediated transport mechanism for l -DOPA at the cerebral capillaries is similar to the l -neutral amino acid transport system.     

Glucose oxidation and contents of free amino acids in pancreatic β-cells stimulated by a non-metabolizable leucine analogue

Oxidation of glucose and the contents of free amino acids were measured in β-cell-rich pancreatic islets exposed to stereoisomers of the non-metabolizable leucine analogue 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid. The insulin-releasing isomer b(−) appeared to stimulate glucose oxidation, whereas the inactive b(+) form was without effect. 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid reduced the islet contents of aspartic acid, γ-aminobutyric acid and glutamic acid and increased that of phenylalanine. Since the two isomers of 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid did not differ in their ability to alter the intracellular contents of free amino acids, these alterations are probably not significant for the regulation of insulin release. It could not be excluded that increased glucose metabolism may contribute to the stimulation of insulin release by the b(−) isomer of 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid.     

Azetidine-2-carboxylic acid derivatives from seeds of Fagus silvatica L. and a revised structure for nicotianamine

2(S),3′(S)-N-(3-Amino-3-carboxypropyl)azetidine-2-carboxylic acid and 2(S),3′(S),3″(S)-N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid have been isolated from seeds of Fagus silvatica L. (beechnuts). The structures have been established by PMR- and ¹³C-NMR-spectroscopy and by synthesis from l-azetidine-2-carboxylic acid. The second of the new amino acids is identical with nicotianamine. previously isolated from Nicotiana tabacum but assigned a different formula. The ring opening reactions of azetidine-2-carboxylic acid in neutral solution have been studied and the chemical and possibly biochemical precursor role of this amino acid for various amino acids including the two new ones described here, nicotianine [N-(3-amino-3-carboxypropyl)nicotinic acid] and methionine is discussed.     

Neutral amino acid transport at the human blood-brain barrier

The kinetics of human blood-brain barrier neutral amino acid transport sites are described using isolated human brain capillaries as an in vitro model of the human blood-brain barrier. Kinetic parameters of transport (Km, Vmax, and KD) were determined for eight large neutral amino acids. Km values ranged from 0.30 +/- 0.08 microM for phenylalanine to 8.8 +/- 4.6 microM for valine. The amino acid analogs N-methylaminoisobutyric acid and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid were used as model substrates of the alanine- and leucine-preferring transport systems, respectively. Phenylalanine is transported solely by the L-system (which is sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and leucine is transported equally by the L- and ASC-system (which is sodium-dependent and N-methylaminoisobutyric acid-independent). Dose-dependent inhibition of the high affinity transport system by p-chloromercuribenzenesulfonic acid is demonstrated for phenylalanine, similar to the known sensitivity of blood-brain barrier transport in vivo. The Km values for the human brain capillary in vitro correlate significantly (r = 0.83, p less than 0.01) with the Km values for the rat brain capillary in vivo. The results show that the affinity of human blood-brain barrier neutral amino acid transport is very high, i.e. very low Km compared to plasma amino acid concentrations. This provides a physical basis for the selective vulnerability of the human brain to derangements in amino acid availability caused by a selective hyperaminoacidemia, e.g. hyperphenylalaninemia.     

INHIBITION OF GABA UPTAKE IN RAT BRAIN SLICES BY NIPECOTIC ACID, VARIOUS ISOXAZOLES AND RELATED COMPOUNDS

—A variety of isoxazoles structurally related to muscimol (3-hydroxy-5-aminomethylisoxazole) were tested as inhibitors of the uptake of GABA and some other amino acids in rat brain slices, and of the activity of the GABA-metabolizing enzymes l -glutamate 1-carboxylyase and GABA:2-oxo-glutarate aminotransferase. A bicyclic derivative, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol, proved to be a more potent inhibitor of GABA uptake than muscimol. Structure-activity studies on this derivative, which appeared to be a competitive inhibitor of GABA uptake, led to the findings that nipecotic acid (piperidine-3-carboxylic acid) is a powerful non-competitive inhibitor of GABA uptake, and that perhydro-1,2-oxazine-6-carboxylic acid is a relatively weak competitive inhibitor of GABA uptake.     

Further studies on amino acid transport in murine P388 leukemia cells in vitro. Presence of system y+

The transport of glycine and L-lysine into murine P388 leukemia cells has been examined. Glycine transport appears to be shared by both systems A and ASC in P388 cells. Glycine transport is Na+-dependent and is effectively blocked by alpha-(methylamino)isobutyric acid, threonine and alanine but only a marginal reduction in transport is seen with 100-fold excess cold 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. System gly is not expressed in P388 cells. Lysine is largely transported by a Na+-independent, pH-insensitive system with a Km of 0.079 mM. Lysine transport is relatively unaffected by the addition of 100-fold excess cold alpha-(methylamino)isobutyric acid, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and the anionic amino acids, L-glutamate and L-aspartate. A partial inhibition of lysine transport was observed with L-threonine and L-leucine while L-arginine and L-histidine radically decreased lysine transport. Lysine appears to be transported by a system similar to the system y+ seen in cultured human fibroblasts, Ehrlich ascites cells, and hepatoma cell lines.     

Constitutional Amino Acids of the Antibiotic YA–56

Acid hydrolysis of the antibiotic YA-56 X (Zorbamycin) and Y belonging to the phleomycin-bleomycin group was carried out and the following constitutional amino acids were isolated from the hydrolyzate of YA–56 X: β-Amino-β-(4-amino-6-carboxy-5-methylpyrimidine-2-yl)- propionic acid, β-aminoalanine, L-erythro-β-hydroxyhistidine and 3 unidentified amino acids. Though the former 3 amino acids were known to be constituents of phleomycins and bleomycins, the latter three were not found in phleomycins and bleomycins. YA–56 Y gave one more unidentified amino acid.

Furthermore, isolation of β-alanine and 2-acetylthiazole-4-carboxylic acid from the hydrolyzate indicated the presence of 2-(2-(2-aminoethyl)-Δ²-thiazoline-4-yl)-thiazole-4-carboxylic acid in YA–56 X and Y as in phleomycins.     

Na+-dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts

Mouse blastocysts which had been activated from diapause in utero appeared to take up amino acids via a Na+-dependent transport system with novel characteristics. In contrast to other cell types, uptake of 3-aminoendobicyclo [3,2,1]octane-3-carboxylic acid (BCO) by blastocysts was largely Na+ dependent. Moreover, L-alanine and BCO met standard criteria for mutual competitive inhibition of the Na+-dependent transport of each other. The Ki for each of these amino acids as an inhibitor of transport of the other had a value similar to the value of its Km for transport. In addition, both 2-aminoendobicyclo [2,2,1]heptane-2-carboxylic acid (Ki approximately 1.0 mM) and L-valine (Ki approximately 0.10 mM) appeared to inhibit Na+-dependent transport of alanine and BCO competitively. Finally, alanine and L-lysine appeared to compete for the same Na+-dependent transport sites in blastocysts. For these reasons, we conclude that lysine, alanine, and BCO are transported by a common Na+-dependent system in blastocysts. In addition, the apparent interaction of the system with other basic amino acids, such as 1-dimethylpiperidine-4-amino-4-carboxylic acid, which has a nondissociable positive charge on its side chain, and L-arginine and L-homoarginine, whose cationic forms are highly predominant at neutral pH, suggests that the cationic forms of basic amino acids are transported by the wide-scope system.     

L-threonine transport in pig jejunal brush border membrane vesicles. Functional characterization of the unique system B in the intestinal epithelium.

Uptake and inhibitory kinetics of [3H]L-threonine were evaluated in preparations of pig jejunal brush border membrane vesicles. Uptake of [3H]L-threonine under O-trans, Na+ gradient, and O-trans, Na(+)-free conditions was best described by high affinity transport (Km < 0.01 mM) plus a nonsaturable component. The maximal velocity of transport was 3-fold greater under Na+ gradient conditions. 100 mM concentrations of all of the dipolar amino acids and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid caused complete inhibition of [3H]L-threonine transport under Na+ gradient and Na(+)-free conditions. Imino acids, anionic amino acids, cationic amino acids, and methylamino-isobutyric acid caused significant partial inhibition of L-threonine uptake. Inhibitor concentration profiles for proline and lysine were consistent with low affinity competitive inhibition. The Ki values of alanine and phenylalanine approximated 0.2 and 0.5 mM, respectively, under both Na+ gradient and Na(+)-free conditions. These data indicate that the transport system available for L-threonine in the intestinal brush border membrane (system B) is functionally distinct from other amino acid transport systems. Comparison of kinetics parameters in the presence and absence of a Na+ gradient suggests that both partially and fully loaded forms of the carrier can function to translocate substrate and that Na+ serves to accelerate L-threonine transport by a mechanism that does not involve enhanced substrate binding.     

Interference of a nonmetabolized analogue of L-leucine with lipid metabolism in tumoral pancreatic islet cells

The nonmetabolized analogue of L-leucine, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), was recently found to inhibit O2 uptake and insulin release from tumoral islet cells of the RINm5F line. BCH inhibited lipogenesis, stimulated lipolysis, and severely decreased the oxidation of endogenous [U-14C]palmitate in prelabelled RINm5F cells. D-Glucose exerted metabolic effects which were sometimes opposite to those caused by BCH and, within limits, protected the islet cells against the inhibitor action of BCH. Since BCH augments NH4+ production and facilitated the catabolism of 14C-labelled amino acids in the prelabelled cells, it is proposed that the unexpected inhibition of O2 uptake by BCH is mainly attributable to a decrease in the oxidation of endogenous fatty acids.     

Reversed-phase liquid chromatographic resolution of diastereomers of protein and non-protein amino acids prepared with newly synthesized chiral derivatizing reagents based on cyanuric chloride

Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative was replaced with amino acid amide (viz l-Leu–NH₂) and amino acid (l-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and were separated on a reversed-phase C₁₈ column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery.     

Synthesis and antibacterial properties of new N4-acetylated hexahydro-2,7-dioxopyrido[2,3-f]quinoxaline-8-carboxylic acids

Direct interaction between 7-chloro-1-cyclopropyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid and primary α-amino acids (exemplified by glycine, alanine, and l-valine) in aqueous ethanolic NaHCO₃ at 70–80°C for 24–72?h produced the respective N-(4-oxoquinolin-7-yl)-α-amino acids (6a–c). The latter derivatives underwent reductive lactamization upon treatment with Na₂S₂O₄ in aqueous ethanol to afford moderate yields of the corresponding pyrido[2,3-f]quinoxaline-8-carboxylic acids (8a–c). Acetylation of 8a–c using acetyl chloride afforded N₄-acetylated hexahydro-2,7-dioxopyrido[2,3-f]quinoxaline-8-carboxylic acids (9a–c). The structures, assigned to these new heterocyclic products, are supported by analytical and spectral data. The synthesized compounds (6a–c/9a–c) showed appreciable antibacterial activity as compared with ciprofloxacin.     

Cytotoxicity as an indicator for transport mechanism. Evidence that melphalan is transported by two leucine-preferring carrier systems in the L1210 murine leukemia cell

Melphalan, l-phenylalanine mustard, is transported by the L1210 cell through carriers of the leucine (L) type. Its initial rate of transport is inhibited by both l-leucine, a naturally occurring L system amino acid and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), a synthetic amino acid which is transported by the L system in the Ehrlich ascites tumor cell. Both amino acids inhibited melphalan transport comparably in sodium-free medium. However, BCH, in medium containing sodium, was unable to reduce a component of melphalan transport which was readily inhibited by leucine but not by α-aminoisobutyric acid. Inhibition analysis indicated that leucine competes with BCH for transport but that a portion of leucine transport is not readily inhibited by BCH. These results suggest that in the L1210 cell melphalan is transported equally by a BCH-sensitive, sodium-independent L system and a BCH-insensitive, sodium-dependent L system.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

Blood-Brain Barrier Transport of 1-Aminocyclohexanecarboxylic Acid, a Nonmetabolizable Amino Acid for In Vivo Studies of Brain Transport

Regional transport of 1-aminocyclohexanecarboxylic acid (ACHC), a nonmetabolizable amino acid, across the blood-brain barrier was studied in pentobarbital-anesthetized rats using an in situ brain perfusion technique. The concentration dependence of influx was best described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants of the frontal cortex equaled 9.7 X 10(-4) mumol/s/g for Vmax, 0.054 mumol/ml for Km, and 1.0 X 10(-4) ml/s/g for KD in the absence of competing amino acids. Saturable influx could be reduced by greater than 85% by either L-phenylalanine or 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with transport by the cerebrovascular neutral amino acid transport system. The transport Km for ACHC was one-fifth that for the more commonly used homologue, 1-aminocyclopentanecarboxylic acid, and was similar to values for several natural amino acids, such as L-methionine, L-isoleucine, and L-tyrosine. The results indicate that ACHC may be a useful probe for in vivo studies of amino acid transport into brain.     

METABOLISM OF MALONIC ACID IN RAT BRAIN AFTER INTRACEREBRAL INJECTION

Labeled malonic acid ([1-¹⁴C] and [2-¹⁴C]) was injected into the left cerebral hemisphere of anesthetized adult rats in order to determine the metabolic fate of this dicarboxylic acid in central nervous tissue. The animals were allowed to survive for 2, 5, 10. 15 or 30min. Blood was sampled from the torcular during the experimental period and labeled metabolites were extracted from the brain after intracardiac perfusion. There was a very rapid efflux of unreacted malonate in the cerebral venous blood. Labeled CO₂ was recovered from the venous blood and the respired air after the injection of [1-¹⁴C]malonate but not after [2-¹⁴C]malonate. The tissue extracts prepared from the brain showed only minimal labeling of fatty acids and sterols. Much higher radioactivity was present in glutamate, glutamine, aspartate, and GABA. The relative specific activities (RSA) of glutamine never rose above 1.00. Aspartate was labeled very rapidly and revealed evidence of ¹⁴CO₂ fixation in addition to labeling through the Krebs cycle. GABA revealed higher RSA after [1-¹⁴C]malonate than after [2-¹⁴C]malonate. Sequential degradations of glutamate and aspartate proved that labeling of these amino acids occurred from [1-¹⁴C] acetyl-CoA and [2-¹⁴C] acetyl-CoA, respectively, via the Krebs cycle. Malonate activation and malonyl-CoA decarboxylation in vivo were similar to experiments with isolated mitochondria. However, labeled malonate was not incorporated into the amino acids of free mitochondria. The results were compared to data obtained after intracerebral injection of [1-¹⁴C]acetate and [2-¹⁴C]acetate.