Metabolism of cerebrosides and sulfatides in subcellular fractions of developing rat brain

Previous studies on myelinating rat brain indicated that microsomes, Golgi-enriched and cytosol fractions may process galactolipids destined for myelin. To extend these findings we labeled brain galactolipids in vivo and determined the specific radioactivity of cerebrosides and sulfatides in several subcellular fractions. 17-day-old rats were treated by intracranial injection with [14C]galactose 60 min prior to and [3H]galactose 15 min prior to killing. Subcellular fractions were prepared from brain stem, and concentrations of cerebrosides and sulfatides were determined, their radioactivity measured and the 3H/14C ratio compared. Our results showed that the heavier Golgi-enriched fraction (designated Fraction 2) is unique in its low galactolipid content and high specific radioactivities of cerebrosides and sulfatides. The low ratio of the specific activity of cerebroside to that of sulfatide in Fraction 2 compared to other fractions indicates that it may be the site of most rapid conversion of newly synthesized cerebrosides to sulfatides. The specific radioactivities of cerebrosides and sulfatides in cytosol are intermediate between those in Golgi-enriched Fraction 2 and microsomes and those in myelin, consistent with the role postulated for cytoplasmic elements in the transport of cerebrosides and sulfatides to myelin.     

BIOSYNTHESIS AND COMPOSITION OF BRAIN GALACTOLIPIDS IN NORMAL AND HYPOTHYROID RATS

Abstract— Newborn rats were rendered hypothyroid by methimazole treatment. Incorporation of [1-¹⁴C]galactose both in vivo and in vitro into brain cerebrosides of hypothyroid rats was significantly lower than in normals. Biosynthesis of sulphatides was affected by hypothyroidism to a smaller extent than cerebrosides. Assay of cerebroside biosynthesis from [1-¹⁴C]galactose or UDP-[1-¹⁴C]galactose by brain preparations revealed that incorporation of the sugar in both cases is affected to the same extent by methimazole treatment, suggesting that the phenomenon is not due to impairment of the nucleotide biosynthesis. A radioactive galactolipid tentatively characterized as glycerogalactolipid was synthesized in vitro and its biosynthesis was reduced to a large extent in the brain preparations from hypothyroid rats. The fatty acid composition of cerebrosides and sulphatides from the brains of hypothyroid rats was found to be different from that of normal rats. The percentage of normal C₂₄ fatty acids was significantly decreased in the methimazole-treated rats. Brain sphingomyelin fatty acids did not differ between normal and hypothyroid rats.     

Decreased Metabolism of Cerebrosides and Sulfatides in Rat Sciatic Nerve After Intraneural Injection of Colchicine

To obtain an understanding of the importance of the neuronal cytoskeleton in Schwann cell metabolism, an antimicrotubular agent (colchicine) was injected into the rat sciatic nerve 24 or 48 h before incubation of the nerve with labeled precursor: [35S]sulfate, [14C]galactose, or [3H]-galactose. Colchicine inhibited the incorporation of 35S radioactivity into sulfatides and, to a lesser extent, into proteins. With galactose as the radioactive precursor, synthesis of cerebrosides was reduced by colchicine injection, whereas incorporation of radioactivity into phosphatidylserine and phosphatidylcholine increased. Intraneural injection of lumicolchicine had no effect. The effects of colchicine on the metabolism of the Schwann cell are discussed in relation to its action on microtubules.     

Effects of desmethylimipramine and diphenylpropene derivatives on synthesis of rat brain lipids in vivo

The effects of antidepressant compounds on the synthesis of brain lipids from [1-¹⁴C] acetate $\text{in vivo}$ in 15 day old rats have been investigated. Compounds used included the drug desmethylimipramine (DMI), the tetrabenazine antagonist 3-methylamino-1:1-diphenylprop-1-ene (II) and the primary (I) and tertiary (III) amine analogues of (II). Compound (II), the most potent tetrabenazine antagonist in the diphenylpropene series, significantly increased lipogenesis, whereas the remaining compounds did not. The results from fractionation of the lipid extract from rats treated with (II) indicated that the incorporation of radioactivity from [1-¹⁴C] acetate increased proportionally in all neutral lipids and phospholipids. Tetrabenazine also increased brain lipogenesis $\text{in vivo}$ and altered the distribution within lipid classes of radioactivity from [1-¹⁴C] acetate. Using [¹⁴C] labelled compound, the concentration of (II) in the brain under the present experimental conditions has been determined.     

A study on the precursors of the acetyl moiety of acetylcholine in brain slices. Observations on the compartmentalization of the acetyl-coenzyme A pool

1. A method was devised for the determination of the specific radioactivity of the acetyl moiety of acetylcholine synthesized from various (14)C-labelled substrates. 2. The precursor for the acetyl moiety of acetylcholine was studied in slices of striatum and cerebral cortex from rat and guinea-pig brain. Incorporation of radioactivity into acetylcholine was determined after incubating the slices in the presence of [2-(14)C]acetate, [(14)C]bicarbonate, [1,5-(14)C]citrate, dl-[1- or 5-(14)C]glutamate or [1- or 2-(14)C]pyruvate. 3. After incubation for 1h, acetylcholine was accumulated significantly in both striatum slices (4.1nmol/mg of protein) and cerebral-cortex slices (0.57nmol/mg of protein) from the rat. Final concentrations were about 11 and 5 times respectively the initial values. 4. With slices from rat striatum, rat cerebral cortex and guinea-pig cerebral cortex, the specific radioactivity of acetylcholine derived from [2-(14)C]pyruvate was very high, reaching approx. 30, 20 and 6% respectively of the initial specific radioactivity of added pyruvate in the medium. With the striatum slices this high value was reached after incubation for 15min. Incorporation of radioactivity from [2-(14)C]acetate was only 1.25, 5.3 and 19.7% of that from [2-(14)C]pyruvate in rat striatum, rat cerebral-cortex and guinea-pig cerebral-cortex slices respectively. A small but definite incorporation was found from [5-(14)C]glutamate. No incorporation was found from the other substrates. The findings suggest that pyruvate is the most important precursor for the synthesis of the acetyl moiety of acetylcholine in brain slices. 5. The specific radioactivity of acetylcholine relative to that of citrate when [2-(14)C]pyruvate was used compared with that obtained when [2-(14)C]acetate was used. A marked difference was found in all slices, suggesting metabolic compartmentation of the acetyl-CoA pool.     

FLOW OF GLUCOSE CARBON INTO BRAIN LIPIDS IN ADULT RATS FOLLOWING FOOD DEPRIVATION

Abstract— Rates of flow of glucose carbon in vivo into brain cholesterol, phospholipids, cerebrosides and gangliosides and concentrations of these lipids in the brain, were determined in adult rats after various periods of food deprivation. The rates were calculated from two measurements, the curve representing the decrease of plasma [¹⁴C]glucose specific activity with time and the specific activity of the brain lipid 180 min after intravenous injection of a tracer dose of d -[U-¹⁴C]glucose. Specific activities of brain lipids in rats deprived of food for 72h were significantly higher than in postabsorptive rats which were treated with the same dose of [¹⁴C]glucose. These higher specific activities were interpreted as a result of more labelled glucose available to lipid synthesis in the brain of fasted rats due to the substantial decrease in the rate of irreversible disposal of glucose by the whole body, commonly observed in fasted animals. The possibility that the higher specific activity values resulted from enhanced synthesis of brain lipids from glucose was ruled out since no changes were observed in the rate of flow of glucose carbon into brain lipids after food deprivation. The rate of flow of glucose carbon into gangliosides (15.4 ng C/min/mg C) was more than twice as fast as into either phospholipids or cerebrosides and about 4 times as fast as into cholesterol. The rates of carbon flow were used to calculate half lives of glucose carbon in the different classes of brain lipids. These half life values were 31 days for gangliosides, 72 days for phospholipids, 82 days for cerebrosides and 133 days for cholesterol. The results suggest that the synthesis of brain lipids from glucose is not affected by prolonged starvation in the adult rat.     

Metabolism of cerebrosides and sulfatides in the nervous system ofXenopus tadpole during metamorphosis

Xenopus laevis tadpoles undergoing metamorphosis were used to study the turnover of cerebrosides and sulfatides in the nervous system of the frog. Tadpoles at the beginning of metamorphosis were treated by intraperitoneal injection with [U-¹⁴C]glucose and radioactivity incorporated into galactosphingolipids of brain and tail was measured after various times. The specific activity of brain cerebrosides increased rapidly for the first 24 hr after injection, reached a plateau after 48hr, and then declined 40% by 7 days. The specific activity of sulfatides changed somewhat more slowly. Hydroxy fatty acid-containing galactosphingolipids had nearly twice the specific activity compared with their nonhydroxy counterparts in brain. Despite the complete regression of tail nerve cord, metabolism of glycosphingolipids in this tissue also indicated active synthesis as well as degradation during this period. The specific activities of these lipids were similar and all reached a peak 24 hr after injection. Examination of the components of these galactosphingolipids disclosed that only a small fraction (7–25%) of the radioactivity was in the galactose moiety in both brain and tail. The ratios of the radioactivity in fatty acid to that in the sphingoid base were much higher for hydroxycerebroside and hydroxysulfatide than for the nonhydroxy isomers.Abbreviations used: Cerebroside is N-acyl, 1-0--galactosyl derivative of sphingoid base (D-erythro-2-amino-alkyl-1,3-diol) Sulfatide is the galactose-3-sulfated derivative of cerebroside. The prefixes hydroxy and nonhydroxy indicate cerebroside or sulfatide containing -hydroxy and nonhydroxy fatty acids, respectively     

METABOLISM IN VIVO OF BRAIN GALACTOLIPIDS: THE JIMPY MUTANT

Abstract— The incorporation in vivo of [U-¹⁴C]glucose into the galactolipids of the brain of control and Jimpy mutant mice was examined. Over a 24-h period of incorporation there was no indication of an increased rate of turnover of brain galactolipids in the mutant. The Jimpy mutant was identified at ages prior to and at the inception of myelination (7–10 days post partum) with a coat marker (Tabby). There was similar total radioactivity in galactolipids of the Jimpy at these ages but a reduction to 13 per Cent of control at 13 days and to 6 per cent at 16 days of postnatal age. This devetopmental pattern of galactolipid synthesis in Jimpy brain is not in accord with a primary defect in the biosynthesis of cerebrosides and sulphatides.     

Long-term metabolism of [U-14C]glucose in the whole rat brain

The experiment was performed on rats to which a single injection of [U-14C]glucose had been administered. Results were observed from the 7th to the 281st day following contamination. At 280 days only the lipids in the brain contained radioactivity, the highest degree of specific activity being found in the cerebrosides.     

Levels and syntheses of cerebrosides and sulfatides in subcellular fractions of jimpy mutants

During the period of brain development, the levels of nonhydroxy- and hydroxy-cerebrosides in the cytosol from brains of jimpy mutants were determined by high-performance liquid chromatography and compared to those in the rest of the particulates from the same brains as well as those in the littermate controls. The concentrations of cerebrosides in jimpy brain preparations were much lower than in controls at all ages. In another experiment, [U-¹⁴C]glucose was injected intraperitoneally into jimpy mutants and their littermate controls. The amounts of radioactivity incorporated into cerebrosides and sulfatides in brain cytosol, the microsome-rich fraction, and the rest of the heavier particles were determined. Although the total radioactivity incorporated into these lipids was much lower in jimpy, the specific activities were 2–3 times the control value in all subcellular fractions.     

Lipid content in brain and spinal cord during experimental allergic encephalomyelitis in rats

Abstract— The content of cerebrosides, sulphatides, gangliosides, cholesterol and phospholipids was evaluated in the brain and spinal cord of rats during the acute and recovery stages of experimental allergic encephalomyelitis (EAE). During the acute stage there was a significant decrease of sulphatides and gangliosides in spinal cord; in brain, only sulphatides were diminished. In the recovery stage, cerebrosides and gangliosides were decreased in the brain, whereas the lipid content of the spinal cord was similar to that in control animals. Cholesterol esters were detected in the brain and spinal cord during both periods. The results show that the changes are not the same for brain and spinal cord during the acute and recovery stages and that glycosphingolipids from either white or grey matter seem to be preferentially altered.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

A comparative study of brain and kidney glycolipids in metachromatic leucodystrophy

Sulphatides and cerebrosides from white matter of brains of patients with metachromatic leucodystrophy (MLD) have been isolated and compared in fatty acid composition to those glycolipids found in MLD kidney tissue. A marked difference in glycolipid composition was found between the brain and kidney tissues. The sulphatides accumulated in MLD kidney have the same fatty acid profile as those found in normal kidney tissue and are typical‘kidney sulphatides.’The neutral glycolipids of MLD kidney retain larger amounts of the longer chain acids than do the cerebrosides of MLD brain white matter and thus resemble more closely in fatty acid composition, glycolipids of normal tissue. Structurally, the sulfate group is located at the C-3 position of the galactose molecule in sulphatides from normal and MLD tissue. As in the brain white matter, the sulphatides which accumulate in the kidney tissue of patients with MLD are normal in structure and composition.     

Malonate as a precursor in the biosynthesis of aflatoxins.

Incorporation of [I-14C]acetate and [2-14C]malonate into aflatoxins by resting mycelia of Aspergillus parasiticus resuspended in different buffers was studied. A decrease in pH from 5-8 to 2-8, as well as addition of EDTA, markedly stimulated the incorporation of malonate but the effect on acetate incorporation was less pronounced. Mycelia took up comparatively more acetate than malonate, but more malonate (4-3%) entering mycelia was incorporated into aflatoxins than was acetate (1-6%). Furthermore, the addition of unlabelled acetate reduced the incorporation of label from [I-14C]acetate by 75% but from [2-14C]malonate by only 25%. These results suggest that malonate is an intermediate in aflatoxin synthesis and that is can be incorporated without prior conversion to acetate.     

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.     

Metabolic compartmentation of pyruvate in the isolated perfused rat heart.

1. Prompted by the finding of markedly differing specific radioactivities of tissue alanine and lactate in isolated rat hearts perfused with [1-14C]pyruvate, a more detailed study on the cytosolic subcompartmentalization of pyruvate was undertaken. Isolated rat hearts were perfused by the once-through Langendorff technique under metabolic and isotopic steady-state conditions but with various routes of radioactive label influx, and the specific radioactivities of pyruvate, lactate and alanine were determined. An enzymic method was devised to determine the specific radioactivity of C-1 of pyruvate. 2. Label introduction as [1-14C]pyruvate resulted in a higher specific radioactivity of tissue alanine and mitochondrial pyruvate than of lactate, and a higher specific radioactivity of perfusate lactate than of tissue lactate. Label introduction as [1-14C]lactate resulted in a roughly similar isotope dilution into the tissue and perfusate pyruvate and the tissue alanine. Label introduction as [3,4-14C]glucose resulted in the same specific radioactivity of tissue lactate and alanine and a roughly similar specific radioactivity of mitochondrial pyruvate. 3. The results can be reconciled with a metabolic model containing two cytosolic functional pyruvate pools. One pool (I) communicates more closely with the glycolytic system, whereas the other (II) communicates with extracellular pyruvate and intracellular alanine. Pool II is in close connection with intramitochondrial pyruvate. The physical identity of the cytosolic subcompartments of pyruvate is discussed.     

Lipid metabolism in various regions of squid giant nerve fiber

The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region.     

Conversion of [U-14C]threonine into 14C-labelled amino acids in the brain of thiamin-deficient rats.

In confirmation of the findings of Gaitonde et al. (1974), a decrease in the brain concentration of threonine and serine, and an increase in glycine, were observed in rats maintained on a thiamin-deficient diet. Similar changes were found in the blood, and the concentration of several other amino acids in the blood decreased significantly. There was a correlation between the concentrations of threonine, serine, aspartate and asparagine in the brain and blood. In experiments in which [U-14C]threonine was injected into rats most of the radioactivity in the brain and blood of control rats was, as expected, in threonine in the acid soluble metabolites. In contrast, a considerable proportion of radioactivity was also found in other amino acids, namely glutamate, glutamine, aspartate, gamma-aminobutyrate and alanine, in the brain of thiamin-deficient rats. [U-14C]Threonine was also converted into 14C-labelled lactate and glucose, but the extent of this conversion was severalfold higher in thiamin-deficient than in control rats. This finding gave evidence of the stimulation in thiamin-deficient rats of the catabolism of [U-14C]threonine to [14C]lactate by the aminoacetone pathway catalysed by threonine dehydrogenase, and into succinate via propionate by the alpha-oxobutyrate pathway catalysed by threonine dehydratase (deaminase). The measurement of specific radioactivities of glutamate, aspartate and glutamine after injection of [U-14C]threonine, indicated a stimulation of the activities of threonine dehydrogenase and threonine dehydratase (deaminase) in the brain of thiamin-deficient rats. The specific radioactivities of glutamate, asparatate and glutamine int he brain were consistent with an alteration in the metabolism of threonine, mainly in the 'large' compartment of the brain of thiamin-deficient rats. The measurement of relative specific radioactivity of proteins after injection of [U-14C]threonine indicated a marked decrease in the synthesis of proteins, mainly in the liver of thiamin-deficient rats.     

CHANGES WITHIN METABOLIC COMPARTMENTS IN THE BRAINS OF YOUNG RATS INGESTING LEAD

—[2-¹⁴C]Glucose and [³H]acetate were injected simultaneously into 19-day-old rats suckling from mothers fed either a normal diet or a diet containing 4·5% lead acetate. Changes in the rate of conversion of both precursors into amino acids associated with the tricarboxylic acid cycle were observed. [^I4C]Glucose. In the brain of young rats ingesting lead, the specific radioactivity of glutamate, aspartate, γ-aminobutyrate and glutamine were all significantly lowered relative to that of glucose. Glutamine labelling was the most affected. [³H]Acetate. In comparison with controls, the total amount of ³H in either water or acid-soluble constituents of the brain was the same, but the ³H content of the amino acids was significantly reduced in the lead-treated rats. In both groups, glutamine had the highest specific radioactivity but the time courses of the labelling of glutamine were different. In the control the peak incorporation was reached during the first 5 min, whereas in the experimental animals this occurred at about 10 min after the injection of the precursor, and the specific radioactivity even at that time was less than in controls. When compared with controls, the depression in the labelling of glutamine was accompanied at 5 min by an increase in the specific radioactivity of aspartate. In the lead-treated rats the labelling of GABA was also slowed and the time course seemed to follow that of glutamine rather than glutamate. In spite of the differences in the metabolism of [³H]acetate, metabolic compartmentation of glutamate, assessed by a glutamine : glutamate specific radioactivity ratio higher than 1, was evident even in the brain of the lead-treated animals, although the values of the ratio at 5 and 10 min were less than in controls. There was no evidence of a diminished supply of substrates to the brain in lead intoxication. The overall changes would be consistent with a retardation in the biochemical maturation of the brain in terms of development of glucose metabolism and metabolic compartmentation.     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.