Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

Lipids and lipid metabolism in the brown alga, Fucus serratus

The lipids of the brown alga Fucus serratus were isolated, identified and quantified. The major acyl lipids were the three glycosylglycerides, diacylgalactosylglycerol, diacyldigalactosylglycerol and diacylsulphoquinovosylglycerol. These represent over 70% of the total acyl lipids. The fatty acid compositions of the major lipids were examined and most showed rather distinctive fatty acid contents. For example, diacylgalactosylglycerol was enriched in n-3 polyunsaturated fatty acids while phosphatidylcholine and phosphatidylethanolamine had very high levels of arachidonate. Phosphatidylglycerol contained the unusual trans-Δ3-hexadecenoic acid. The labelling of lipids and fatty acids from [¹⁴C]acetate was examined and the distribution of label between individual components as a function of the incubation period and in algae collected at different times of the year is reported. Algae collected in the winter incorporated much more radioactivity into non-esterified fatty acids when compared to algae collected in the summer. All algae could label myristate, palmitate, stearate and oleate at high rates. Longer incubation times allowed the labelling of polyunsaturated fatty acids such as linoleic acid.     

Effects of acute hypoxia on incorporation of [1-14C]arachidonic acid into glycerolipids of rat brain

The effects of 1 min of acute hypoxic treatment (1% O₂ in N₂) on incorporation of [1-¹⁴C]arachidonic acid into brain lipids of 16-day-old rats were investigated at 3, 6, and 12 min after intracerebral injection of the labeled fatty acid. The hypoxic-hypoxia condition associated with convulsive seizures caused a decrease in the conversion of labeled arachidonate to its acyl-CoA as well as incorporation of the label into the brain phospholipids. Among the phospholipids, there was a specific decrease in the labeling of diacylglycerophosphoinositol (GPI), and this change was accompanied by an increase in labeling of the diacylglycerols. These results indicate that metabolism of the long-chain fatty acids and some glycero-lipids in brain are vulnerable to acute hypoxic treatment.     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

Changes in Amino Acid Metabolism in vivo with Time after Feeding

Changes in the metabolism in vivo of amino acids with the lapse of time after feeding a diet were investigated by measuring the incorporation of ¹⁴C into some body components one hour after injection with ¹⁴C-amino acid mixture.

The incorporation of ¹⁴C into protein in the liver and carcass was rather constant, but that into blood sugar, liver glycogen, and lipids in the liver and carcass showed a change with the lapse of time after feeding a 25% casein diet or a protein-free diet. The incorporation of ¹⁴C into liver glycogen was stimulated shortly after feeding, but it was reduced at 7 hr, when a large amount of glycogen was still in the liver. On the contrary, the specific activity of blood sugar increased with the lapse of time after feeding. The conversion of ¹⁴C-amino acids into lipids in the liver and carcass was stimulated shortly after feeding.

The incorporation of ¹⁴C into protein was higher in the rats fed the protein-free diet than in those fed the 25% casein diet, and the higher incorporation was partly counterbalanced by the lower incorporation of ¹⁴C into lipids and glycogen in the rats fed the protein-free diet.     

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U-¹⁴C]glucose and [1-¹⁴C]acetate, with lipid synthesis from exogenous albumin-bound [1-¹⁴C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain-length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non-esterified fatty acid pool in the two situations. At the period of their most active formation, the very long-chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C₁₈ acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

Ketone-body utilization and lipid synthesis by developing rat brain—a comparison between in vivo and in vitro experiments

The distribution of ketone bodies between oxidation and lipid synthesis was analysed in homogenates of developing rat brain. The capacity for lipid synthesis of homogenized or minced brain preparations was compared with rates of lipid synthesis in vivo, assessed by incorporation of ³H from ³H₂O into fatty acids and cholesterol. Brain homogenates of suckling rats (but not those of adults) incorporated label from [3-¹⁴C]ketone bodies into lipids, but this process was slow as compared to ¹⁴CO₂ production (< 5%) and much slower than the total rate of ketone-body utilization (< 0.5%). Study of ³H₂O incorporation demonstrated that the rates of lipogenesis and cholesterogenesis are at least one order of magnitude higher in vivo than in vitro. Maximal rates of ³H incorporation into fatty acids (3 μmol/g brain . h) and into cholesterol (0.6 μmol/g brain . h) were found during the third postnatal week. Adult rats still incorporated ³H into brain fatty acids at an appreciable rate (1 μmol/g brain . h), whereas cholesterogenesis was very low. It is concluded that in vitro measurements of lipid synthesis severely underestimate the rates that occur in developing rat brain in vivo. The high rate of ³H incorporation into lipids by developing and adult rat brain as compared to the amounts of these lipids present in the brain suggests an important contribution of endogenous lipid synthesis during brain development and an appreciable rate of fatty acid turnover during brain growth, but also in the adult brain.     

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5–8-day old rats. Cultures containing 60–80% oligodendrocytes were incubated for 16h with [3-¹⁴C]acetoacetate, d-[3-¹⁴C]3-hydroxybutyrate, [U-¹⁴C]glucose, l-[U-¹⁴C]glutamine and [1-¹⁴C]pyruvate or [2-¹⁴C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO₂ was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.     

Fatty Acid Incorporation in Normal and Degenerating Rat Sciatic Nerve In Vivo

Abstract: Labeled palmitic acid ([16-¹⁴C]palmitate) (0).5 μCi) was injected into rat sciatic nerves in vivo to characterize thc incorporation of this fatty acid into complex peripheral nerve lipids after time lapses of 1 min to 2 weeks. For the first 30 min after intraneural injection, the label was concentrated in nerve diglycerides. Thereafter, the relative diglyccride label declined rapidly, and phospholipid radioactivity rose steadily. After 120 min, phospholipids contained over 70% of the total lipid radioactivity. Among the phospholipids, phosphatidylcholine had the largest percentage of total phospholipid label, and acylation of lysophosphatidylcholine accounted for approximately 75% of this label. With time, there was conversion of [16-¹⁴C]palmitate to other long-chain fatty acids by elongation and desaturation. Phosphatidic acid was labeled also, suggesting the operation of the de novo biosynthetic mechanism. However, the specific radioactivity of 1,2-diacylglycerol was much higher than that of phosphatidic acid, suggesting phosphorylation of diglycerides by diglyceride kinase. After nerve section and survival of 2 h to 50 days, the injection of [16-¹⁴C]palmitate into the degenerating distal segment revealed an overall decline of phospholipid labeling and a commensurate increase of triglyceride radioactivity. Phosphatidylcholine in degenerating nerve contained a larger percentage of the fatty acid label than that in normal nerve. Almost all of the labeling was due to acylation of lysophosphatidylcholine, implying a much smaller contribution of the de novo pathway. Phosphatidylethanolamine and phosphatidylserine showed a relative loss of radioactivity. The changes were apparent at 1 day, but not at 2 h, suggesting loss of homeostatic control, presumably by interruption of axonal flow. An incidental observation was the stimulation of phosphatidylcholine biosynthesis by acylation of lysophosphatidylcholine in the contralateral unoperated sciatic nerve.     

Lipogenesis in Fatty Liver by Amino Acid Imbalance

Lipogenesis in the fatty liver of rat which was induced by feeding an amino acid-irnbalanced diet containing 8% casein supplemented with 0.3% dl-methionine has been investigated by measuring the incorporation of acetate-1-¹⁴C into various lipid fractions during in vitro incubation of liver slices.

In the incorporation of acetate-1-¹⁴C into the total lipid per one g of the slices, no significant difference for the imbalance group was observed. However, the total radioactivity of liver lipid per 100 g of the body and the incorporation into triglyceride in the lipids were significantly higher in the imbalance group than in the control group. Conversion of acetate-1-¹⁴C to CO₂ was not impaired in the imbalance group.

It is evident from these results that the induction of this type of fatty liver is due mainly to the synthesis of triglyceride.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

Phospholipid metabolism in boar spermatozoa and role of diacylglycerol species in the De Novo formation of phosphatidylcholine

We have investigated pathways of lipid metabolism in boar spermatozoa sperm cells incubated for up to 3 days with [¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid or incorporated these precursors into diglycerides and/or phospholipids. When spermatozoa were incubated with [¹⁴C]palmitic acid or [¹⁴C]glycerol, there was first an incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidyl-choline (PC). This indicates that the de novo pathway of phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of di-saturated DAG, which represented a considerable proportion of the high basal levels of DAG, accumulated the majority of label. Another DAG pool, containing saturated fatty acids in position 1 and unsaturated fatty acids in position 2 and representing the remaining basal DAG, was in equilibrium with PC. When spermatozoa were incubated with [¹⁴C]arachidonic acid, there was a considerable incorporation of label into PC, which indicates the presence of an active deacylation/reacylation cycle. The behaviour of certain lipid pools varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of [¹⁴C]palmitic acid or [¹⁴C]arachidonic acid, there was more incorporation of label into PC when spermatozoa were incubated at 25°C than when incubated at 17°C. Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may therefore be possible to design methods to evaluate the metabolic activity of boar spermatozoa based on the incorporation of lipid precursors under standardized conditions. Mol. Reprod. Dev. 47:105–112, 1997. © 1997 Wiley-Liss, Inc.     

Biosynthesis of lipids in chloroplasts isolated from jack pine needles

Suspensions of isolated pine needle chloroplasts were shown to incorporate galactose from UDP galactose-[¹⁴C] into galactolipids. The incorporation of the label among galactolipids was always considerably higher in the monogalactosyl diglycerides than in the digalactosyl diglycerides. The galactosyl incorporation into both galactolipid fractions was optimal at pH 8.0 and was inhibited by sulphydryl reagents (p-chloromercuribenzoate, N-ethyl maleimide and CdCl₂). The chloroplast preparations were also able to biosynthesize various phospholipids and galactolipids from palmitoyl-[1-¹⁴C]-CoA; the major portion of the label appeared in phosphatidyl choline. The incorporation of palmitic-[1-¹⁴C] acid into various lipids was very poor compared to that of palmitoyl-[1-¹⁴C]-CoA. However, addition of ATP and CoA markedly stimulated lipid biosynthesis from palmitic-[1-¹⁴C] acid, suggesting the presence of activating enzymes. These chloroplast suspensions did not show any de novo fatty acid synthesis.     

Inhibition of fatty acid synthesis by RMI 14,514 (5-tetradecyloxy-2-furoic acid)

RMI 14,514 strongly inhibited the incorporation of label from [1-¹⁴C]acetyl-CoA into fatty acids by rat liver homogenates. No inhibition was observed when [2-¹⁴C]malonyl-CoA was used as the labeled fatty acid precursor. These results suggest that the drug inhibits $\text{de novo}$ fatty acid biosynthesis at the step mediated by acetyl-CoA carboxylase. The data presented in this communication support earlier reports that RMI 14,514 probablyexerts its hypolipidemic effects by inhibition of fatty acid biosynthesis.     

Nature of DNA and RNA Degradation in Escherichia coli Induced by Colicin E2

Lipogenesis in the fatty liver of rat, which was induced by feeding an amino acid unbalanced diet containing 8% casein supplemented with 0.3% dl-methionine, has been studied by measuring the incorporation of glycerol-1-¹⁴C, palmitate-1-¹⁴C, citrate-1,5-¹⁴C, pyruvate-1-¹⁴C and pyruvate-2-¹⁴C into various lipid fractions and ¹⁴CO₂ during in vitro incubation of liver slices.

The total radioactivity of liver lipid per 100 g of the body and the incorporation of each substrate into triglyceride in the lipid were significantly higher in the imbalance group than the control group. Conversion of each substrate to ¹⁴CO₂ was not imparied in the imbalance group.

It is evident from these results that the induction of this type of fatty liver is due mainly to the triglyceride synthesis by both the fatty acid synthesis and the transesterification of fatty acid.

These results are considered to support the previous assumption in which acetate-1-¹⁴C was used as a precursor.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

Lipid metabolism in brain tissue explants

Abstract— Tissue explants from frontal lobes of rat brain were used for the study of cerebral fatty acid metabolism. After tissues had been maintained in serum-supplemented medium, a lipid-free medium was substituted and metabolic studies were carried out. Under these conditions explants continued to take up lipid precursors for at least 48 h, as judged by incorporation of dl -[2-¹⁴C]mevalonic acid into cellular lipids. [l-¹⁴C]Stearic acid and [l-¹⁴C]palmitic acid were bound to cells as the free fatty acids, or incorporated into neutral lipids (particularly triglycerides), glycolipids and phospholipids. In the galactolipid fraction, cerebrosides were the principal radioactive lipids. Choline phosphoglycerides, ethanolamine phosphoglycerides, inositol phosphoglycerides and serine phosphoglycerides were the principal radioactive phospholipids. Fatty acids were incorporated into cellular lipids either unchanged or after desaturation, chain elongation, or both. Maximum incorporation of stearate occurred in tissues derived from 3-day-old animals. With increasing age the uptake of fatty acid dropped sharply. When the labelling of lipids as a function of time was followed in 3-day-old animals, triglycerides and choline phosphoglycerides were the first fractions to take up labelled stearate. Labelling of cerebrosides occurred slowly, only becoming evident after 24 h. These studies exemplify the usefulness of tissue explants for prolonged metabolic studies in normal and pathological specimens of brain.     

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'¹⁴C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [¹⁴C]succinate was rapid, as shown by the incorporation of ¹⁴C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [¹⁴C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-¹⁴C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of ¹⁴C into GABA was very low (< 1 per cent of the amino acid -¹⁴C) after the injection of [1.4-¹⁴C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-¹⁴C]succinate 2 min beforehand. Since the oxidation of [l,4-¹⁴C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce ¹⁴C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD⁺ ratios and [¹⁴C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.     

Turnover of palmitic and arachidonic acids in the phospholipids from different brain areas of adult and aged rats

[³H]Palmitic acid and [¹⁴C]arachidonic acid were injected together into the cerebral ventricle of 4-month and 24-month-old rats. At different time intervals from the injection, the distribution of these fatty acids in the lipids from different brain areas was examined. The fatty acids were rapidly incorporated into the lipids through different mechanisms. The time-specific activity relationship indicate that the utilization of the fatty acid differs according to the different areas and aging decreases the utilization of both the fatty acids. The decline of arachidonic acid incorporation into phospholipids is particularly evident, indicating that aging affects mainly the utilization of polyunsaturated fatty acids.