THE ACCUMULATION OF ARACHIDONATE AND DOCOSAHEXAENOATE IN THE DEVELOPING RAT BRAIN

—Fatty acids typical of grey matter lipids (C20:4 and C22:6) and of myelin lipids (C20:1 and C24:1) were estimated in developing rat brains. The polyenoic fatty acids (C20:4 and C22:6) are synthesized from the essential fatty acids (C18:2 and C18:3). The results showed that more than 50 per cent of the adult content of the brain polyenoic acids were laid down by day 15. In contrast, the fatty acids characteristic of myelin lipids did not appear in significant quantities until after this time. These findings distinguish biochemically the different periods of brain development associated firstly with cell division (formation of neurons and glial cells) and secondly with myelination. It is of special interest that the period of cell proliferation is accompanied by the appearance in brain lipids of long-chain polyenoic acids derived from the essential fatty acids.     

COLD RESISTANCE OF THE BRAIN DURING HIBERNATION: THE ROLE OF STEARYL CoA DESATURASE IN BRAIN AND LIVER AS THE SOURCE FOR MONOENES

Brain and liver stearyl CoA desaturase activity and its associated microsomal electron transport chain was investigated in both the warm-adapted and hibernating hamster. It was shown that the activity of this enzyme in brain was essentially the same in both the warm-adapted and hibernating hamster. In liver an 8-fold increase in desaturase activity was observed for the hibernator without corresponding increases in the activity of the microsomal electron transport chain. It is concluded that the increase of monoenes in brain that contributes to the lipid adaptation probably results from peripheral production of these fatty acids.     

THE FATTY ACID COMPOSITION OF PHOSPHOLIPIDS AND GLYCOLIPIDS IN JIMPY MOUSE BRAIN

Abstract— Phospholipids and sphingolipids from brains of normal and Jimpy mice were isolated in a pure form by thin-layer chromatographic procedures. The fatty acid composition of the major phospholipids, i.e. ethanolamine glycerophospholipids, serine glycerophospholipids, choline glycerophospholipids and inositol glycerophospholipids, as well as sphingomyelin, cerebrosides and sulphatides was determined by gas-liquid chromatography. A specific fatty acid pattern for each of the four glycerophospholipids was found. The fatty acid composition of inositol glycerophospholipid, which has not previously been studied in mouse brain, was characterized by a high concentration of arachidonic acid. After 16 days of age, fatty acid analysis showed definite differences between the phospholipids from normal and mutant brains. A small increase of polyunsaturated fatty acids in glycerophospholipids of ethanolamine, serine and choline from the Jimpy central nervous system was found, which has been explained by the myelin deficiency. Sphingomyelin, cerebrosides and sulphatide analyses showed a wide distribution of saturated and mono-unsaturated fatty acids in both normal and mutant mice. A reduction in the amount of long-chain fatty acids was demonstrated in mutant brain sphingolipids; in sulphatides and cerebrosides, the amount of non-hydroxy fatty acids was reduced to a greater extent than in sphingomyelin. The distribution of fatty acids in sphingolipids from the myelin and microsomal fractions was also investigated in both types of mice. Cerebrosides were characterized by a high content of long-chain fatty acids in myelin as well as in microsomes. Sulphatides and sphingomyelin, on the other hand, showed a higher content of medium-chain fatty acids in microsomes than in myelin. In the mutant brain, the amount of long-chain fatty acids was reduced in both subcellular fractions. The deviation from normal in the pattern of fatty acid distribution in Jimpy brain is discussed in relation to the current concepts of glycolipid biosynthesis.     

THE INCORPORATION OF LABELLED ACETATE INTO CEREBROSIDE AND OTHER LIPIDS OF THE DEVELOPING MOUSE BRAIN

—Cerebroside in the brain is highly localized in myelin and has a relatively slow turnover rate. The aim of this study was to evaluate the true cerebroside biosynthetic activity under conditions in which the degradation and reutilization of brain lipids were as small as possible. The 3-week-old mice were decapitated at 0·5, 1, 2·5, 5 and 15 min after the intraperitoneal injection of labelled acetate and the incorporation of radioactivity into each lipid class was examined. Even at 0·5 min, a considerable amount of radioactivity was found in simple lipids, especially in the free fatty acid fraction, and in the course of time the radioactivity of complex lipids increased. On the other hand, the incorporation of radioactivity into cerebrosides was extremely small throughout the experimental period. Results indicated that the low radioactivity of cerebroside might be due to its high content of long-chain fatty acids which were weakly labelled. The radioactivity of the sphingosine moiety was also low. In short, one of the rate-limiting steps of cerebroside synthesis in brain might exist in long-chain fatty acid and sphingosine synthesis. In addition, the incorporation curves of each component of cerebroside were compared with each other and the difference of the incorporation pattern of non-hydroxy fatty acids of cerebroside was noted.     

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.     

EFFECT OF FATTY ACIDS ON RAT BRAIN PARTICULATE HEXOKINASE

Abstract— The effect of free fatty acids on rat brain particulate hexokinase was studied in vitro. Hexokinase bound with brain mitochondrial fraction was found to be sensitive to the action of free fatty acids, resulting in the solubilization of at least part of bound enzyme activity into the supernatant. The decrease of total enzyme activity observed at the highest free fatty acid concentration was probably due to the inhibition of hexokinase. The physiological consequence of hexokinase solubilization by low concentrations of free fatty acids, similar to that observed in vivo , is discussed in relation to activity changes of soluble and particulate enzyme forms demonstrated previously under hypoxic conditions.     

OBSERVATIONS ON THE BIOSYNTHESIS OF RAT BRAIN CERAMIDE AND CEREBROSIDE IN VIVO

Abstract— The incorporation in vivo of l -[¹⁴C]serine into ceramide and cerebroside of young rat brain has been studied. Acid hydrolysis of labelled ceramide and galactosyl-ceramide followed by selective partitioning of the resulting components indicated that 88 per cent of the radioactivity was present in the long-chain base portion. At early time points (10 min, 20 min) the precursor was incorporated into ceramide and to a lesser degree into glucosyl-ceramide. During time intervals of 5 and 10 h, the specific activity values (d.p.m./μmol) for ceramide and glucosyl-ceramide decreased, while values for galactosyl-ceramide, containing either unsubstituted fatty acids (NFA) or α-hydroxy fatty acids (HFA), increased 50 and 30 per cent, respectively. Analysis of labelled ceramide at all time points studied (10 min-10 h) indicated that l -[¹⁴C]serine was incorporated onto the NFA type. This observation suggests that HFA-ceramide may not be the physiological precursor of HFA-galactosyl-ceramide. In this context, the postulated precursor roles of both ceramide and psychosine in the biosynthesis of brain cerebrosides are discussed.     

FATTY ACID COMPOSITION OF CEREBROSIDES IN MICROSOMES AND MYELIN OF MOUSE BRAIN

Abstract— The fatty acid composition of cerebrosides isolated from myelin and from light and heavy microsomes of adult mouse brain was determined. 2-Hydroxy fatty acids represented 80 per cent of the fatty acids in myelin cerebrosides and approximately 55 per cent of the fatty acids in both light and heavy microsomes. In myelin, the majority of the fatty acids, both normal and hydroxy, were of chain length > C-20; in microsomes, shorter chain acids (C-16 to C-20) predominated.     

METABOLISM OF BRAIN SPHINGOMYELINS: HALF-LIVES OF SPHINGOSINE, FATTY ACIDS AND PHOSPHATE FROM TWO TYPES OF RAT BRAIN SPHINGOMYELIN

Abstract— The metabolism of rat brain sphingomyelins containing short-chain (C₁₆-C₁₈) and long-chain (C₂₀- C₂₄) fatty acids has been studied by determination of the content of radioactivity in the sphingo-sinc. fatty acids and phosphate of the sphingomyelins over a period of 60 days following the intracisternal injection of [¹⁴C]acetate and [³²P]phosphate. From the rate of decrease of the specific radioactivities of the different constituents of short-chain fatty acid sphingomyelins, we have calculated a half-life of 65 days for sphingosine. 41 days for fatty acids and 62 days for phosphate. For the long-chain fatty acid sphingomyelins the half-life of sphingosine was approximately 465 days. The fatty acids and phosphate from these sphingomyelins had fast and slow turnover pools. The half-life for the fast pool was 7 days for the two constituents and the estimated half-lives for the slow pool were 220 days for fatty acids and 480 days for phosphate. These results suggest that one can distinguish at least three metabolic pools of brain sphingomyelins: (a) sphingomyelins with long-chain fatty acids situated in myelin whose half-lives are 465 days for sphingosine, 220 days for fatty acids and 480 days for phosphate; (b) sphingomyelins with long-chain fatty acids located mainly in non-myelin structures having half-lives of 465 days for sphingosine. 7 days for fatty acids and 7 days for phosphate; (c) sphingomyeiins with short-chain fatty acids with half-lives of 65 days for sphingosine. 41 days for fatty acids and 62 days for phosphate. The differences between the half-lives of the three metabolic pools of sphingomyelin, together with the subcellular localizations of the two molecular species of these compounds, suggest that the metabolism of the different molecular species of sphingomyelin are independent and that in various subcellular fractions the long-chain fatty acid and short-chain fatty acid sphingomyelins have different turnover rates.     

ISOLATION AND CHARACTERIZATION OF GLUCOCEREBROSIDES FROM ADULT RAT BRAIN1

Abstract— By chromatography on borate-coated silicic acid, glucocerebrosides, galactocerebrosides, sulfatides and sphingomyelins from brain tissue could be efficiently separated. Adult rat brain was found to contain 54.1 ± 1.5 nmol of glucocerebrosides per gram fresh weight. Ninety percent of the glucocere-broside fatty acids were palmitate, stearate and oleate; fatty acids with chain lengths above C₂₀ were virtually absent. No hydroxy fatty acids were found. The long chain bases of adult rat brain glucocerebrosides consisted of 74.6% C₁₈-sphingosine, 24.4% C₁₈-sphinganine and 1.1% C₂₀-sphingosine. These results are compared to those obtained from glucocerebrosides from immature rat brains (Abe & Norton , 1974) and discussed in respect to changes occurring during brain development.     

THE RELEASE OF BRAIN FREE FATTY ACIDS DURING ISCHAEMIA IN ESSENTIAL FATTY ACID-DEFICIENT RATS

—The release of free fatty acids and especially of free arachidonic acid occurring in rat brain during ischaemia has been studied in essential fatty acid-deficient animals. Free fatty acid levels are lower in essential fatty acid-deficient brains before and especially after 5 min of ischaemia. The percentage of arachidonic acid, in respect of total free fatty acids, is similar in both control and essential fatty acid-deficient brains before ischaemia (0 min), but is greatly reduced in deficient brains in respect of controls after ischaemia (5 min). Total levels of free arachidonic acid are thus greatly reduced after ischaemia in the brain of essential fatty acid-deficient rats. Focussed microwave irradiation of the brain, a technique which instantaneously kills the animals, and which was used for the determination of brain basal levels of free fatty acids and free arachidonic acid, does not per se modify brain lipid and fatty acid composition.     

PHOSPHOFRUCTOKINASE AND FUMARATE HYDRATASE IN DEVELOPING RAT BRAIN

The developmental patterns of phosphofructokinase, fumarate hydratase and lactate dehydrogenase were determined and compared using homogenates of rat brain. Phosphofructokinase activity, expressed in terms of tissue wet wt., was relatively constant from 5 days before birth to 8 days postnatal; a 110 per cent increase in activity occurred between 12 and 21 days of age, when adult levels were achieved. The degree of inhibition of phosphofructokinase by 1-0 mM-ATP changed little during development; inhibition by 2-5 mM-citrate was about 50 per cent in both newborn and adult brain. Phosphofructokinase development more closely resembled that of lactate dehydrogenase than that of fumarate hydratase.     

THE CHARACTERIZATION OF SPHINGOLIPIDS OF OLIGODENDROGLIA FROM CALF BRAIN

Abstract— Purified oligodendroglia isolated from bovine brain white matter were found to contain, in addition to galactosylceramide, sulfatide and sphingomyelin, significant quantities of glucosylcerai-mide, dihexosylceramide and esterified galactosylceramide. These sphingolipids were isolated and quan-titated and their fatty acid and long chain base patterns compared with those from sphingolipids isolated from bovine myelin, white matter and gray matter.
The minor glycosphingolipids, glucosylceramide, dihexosylceramide and esterified galactosylceramide, constituted a higher percentage of glial lipids than of myelin lipids. Glucosylceramide accounted for 12% of the total glial monohexosylceramide fraction and 0.8% of total lipids; dihexosylceramide was 0.9% of total glial lipids. Both of these lipids had small quantities of α-hydroxy fatty acids. The unsubstituted fatty acids of glucosylceramide were mostly short chain (16 and 18 carbons) and were different from those of the dihexosylceramides which were a mixture of short and long chain. The hydroxy acids of each of these lipids were, however, similar and resembled those of galactosylceramide.
The fatty acid patterns of galactosylceramide, sulfatide and sphingomyelin from glial cells resembled those of the corresponding lipids from myelin and white matter. The amide-linked acids of esterified galactosylceramide contained both unsubstituted and α-hydroxy chains. Their patterns were not identical to those of galactosylceramide, but were similar in all brain fractions.
With the exception of sphingomyelin and dihexosylceramide, which contained small amounts of C₂₀-sphingosine, all sphingolipids analyzed contained mostly sphingosine and dihydrosphingosine.
We conclude that the distribution of sphingolipids in the oligodendroglia is characteristic, but the lipophilic residues of these lipids are not cell-specific.     

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.     

CHANGES IN FREE FATTY ACIDS OF BRAIN BY DRUG-INDUCED CONVULSIONS, ELECTROSHOCK AND ANAESTHESIA

Abstract— The size of the free fatty acid pool in rat brain was significantly increased following convulsions induced by pentylenetetrazol as well as by electroconvulsive shock. Other convulsants such as d -methionine- dl -sulphoximine and the dibutyryl analog of adenosine 3',5'-monophosphate did not alter the levels of free fatty acids.
Diethyl ether anaesthesia suppressed the stimulatory effect of electroshock on the generation of free fatty acids in brain, but the effect was not seen with the anaesthetic pentobarbitone sodium. The lack of an inhibitory effect of either anaesthetic on the free fatty acid production which was induced in brain by ischaemia supported the view that the action of electroshock was not merely the result of anoxia.
The prominent increase in size of the free fatty acid pool in brain thus appeared to be specific for electroshock- and pentylenetetrazol-convulsed rats.
We have proposed that the changes in the free fatty acids might be involved in the regulation of membrane functioning.     

THE EFFECT OF HYPERCAPNIC ACIDOSIS UPON SOME GLYCOLYTIC AND KREBS CYCLE-ASSOCIATED INTERMEDIATES IN THE RAT BRAIN

Abstract— In order to study the influence of intracellular pH on the carbohydrate metabolism of brain tissue, the concentrations of glucose, glucose-6-phosphate, pyruvate, lactate, citrate, α-oxoglutarate, malate, glutamate, aspartate and ammonia were measured in rats exposed to 6–40% CO₂, for 45 min. Hypercapnia of increasing severity gave rise to progressive increases in the concentrations of glucose, glucose-6-phosphate and ammonium ion and to progressive decreases in the concentrations of all metabolic acids measured. The results fit with aH⁺ inhibition of a rate-limiting step between glucose-6-phosphate and pyruvate, and by inference from the results published by others it may be assumed that this step is the phosphofructokinase reaction. Since the proportionally largest decrease occurred in a α-oxoglutarate, the results might be compatible either with an inhibition of a second rate-limiting step such as isocitrate dehydrogenase, or with a loss of α-oxoglutarate through carboxylation to citrate.     

THE DISTRIBUTION OF LIPIDS IN THE HUMAN NERVOUS SYSTEM-V. GANGLIOSIDES AND ALLIED NEUTRAL GLYCOLIPIDS OF INFANT BRAIN

—Gangliosides and allied neutral glycosylceramides were isolated from human infant (2-24 months of age) cerebral cortex and white matter. The individual glycolipids were separated quantitatively by a combination of column and thin-layer chromatographic methods on silica gel, DEAE-cellulose and Sephadex G-25. In cerebral cortex G_D1a and G_M1 were the major fractions and constituted more than 70 per cent of the total gangliosides. The concentrations of neutral glycolipids, except for galactosylceramides, were very low: lactosylceramide and glucosylceramide comprised 30 and 5 nmol/g wet weight, respectively. In white matter their concentrations were 10 times higher. The ganglioside concentration was only 50 per cent of that in cerebral cortex: the difference was accounted for mainly by the much lower content of the major di- and trisialogangliosides. Stearic acid was the predominant fatty acid of all brain gangliosides. G_M3, and G_D3 had a considerable content of the very long-chain fatty acids, C₂₂-C₂₄, particularly in the white matter. Glucosylceramide and lactosylceramide had almost identical fatty acid patterns between each other in cerebral cortex and white matter. In the cerebral cortex stearic acid and in the white matter the very long-chain acids predominated. d20:1 Sphingosine comprised more than 20 per cent of total sphingosine in all the gangliosides of the G_l- and G₂-series. G_M3, and G_D3 like lactosylceramide contained significantly less of d20:1 sphingosine. The findings suggest the existence of separate compartments for the biosynthesis of the gangliosides. Glucosylceramides and lactosylceramides of white matter have the same ceramide composition as the galactosylceramides with normal fatty acids and are thus unlikely to be intermediates in the metabolism of the major brain gangliosides which have a completely different fatty acid composition.     

THE MEASUREMENT OF TRIGLYCERIDE IN BRAIN AND THE METABOLISM OF BRAIN TRIGLYCERIDE IN VITRO

Abstract—

• 1 Triglyceride has been isolated from brain by thin-layer chromatography and determined by absorption of the carbonyl group at 1740 cm^?1. The means of yields from whole mouse brain, whole rat brain, rat brain grey matter, rat brain stem, and incubated slices of rat brain cortex were 0.15–0.17 μmole/g tissue.
• 2 The distribution of fatty esters varied from preparation to preparation. Palmitate, stearate and oleate usually occurred in greatest amounts. Hydrolysis of a preparation of triglyceride from whole rat brain with pancreatic lipase indicated that palmitate was equally distributed between the α and β esters.
• 3 [1-¹⁴C]Acetate was rapidly incorporated into triglyceride of slices of incubated rat brain cortex. When the resulting triglyceride was hydrolysed with pancreatic lipase the distribution of radioactivity amongst the hydrolysis products was consistent with both the α and β esters of the triglyceride having been radioactively labelled.

     

LIPID COMPOSITION IN GRAY AND WHITE MATTER OF THE BRAIN IN MENKES' DISEASE

Abstract— Lipid composition has been determined in brain frontal lobe gray and white matter from a 5-month-old patient who died from Menkes' disease, and from a normal control patient of the same age.
Total cholesterol and the amount of cholesterol esters were significantly increased in the case of Menkes' disease, whereas the values for free cholesterol were nearly unchanged.
In white matter a decrease in total galactolipids was observed in the pathological brain.
The values for total phospholipids were unchanged for the tissues, but the ratio between phosphatidylcholines and phosphatidylethanolamines (including ethanolamineplasmalogens) in white matter from the patient seemed increased. The fatty acid pattern of phosphatidylethanolamines (including ethanolamineplasmalogens), phosphatidylcholines and sphingomyelin were similar to those of the normal control. Phosphatidylethanolamines from pathological tissues contained 25–30 per cent polyunsaturated fatty acids with four, five or six double bonds.     

PELIZAEUS-MERZBACHER DISEASE: BRAIN LIPID AND FATTY ACID COMPOSITION

Abstract— Biochemical analysis of the leukodystrophy brain from a case of Pelizaeus-Merzbacher disease, classical type, was performed. A decrease in the amount of solid material present was found. The lyophilized brain weight was reduced to 76% of normal with a slightly greater decrease in the amount of extractable lipid. Total myelin was diminished to 7% of normal. Among specific lipids plasmalogens were present in slightly lowered amounts. Cerebrosides and sulphatides were drastically reduced to 8% of normal, whereas sphingomyelin was less severely affected. Fatty acids from phospholipids were close to normal, only enols being slightly diminished. Analysis of pure cerebrosides and sulphatides revealed that the a-hydroxylated compounds as well as very long chain fatty acids (over C₁₈, especially C₂₃ to C₂₆) were greatly reduced. For chain lengths over C₁₈, the ratio of leukodystrophy fatty acid to normal fatty acid was close to 10%. The defect in very long chain fatty acids is estimated at 99.2% in total brain.
Thus, we have found a marked decrease in the amount of very long chain fatty acids and a less marked decrease in sphingolipids. The reduced amount of these acids appears to be partially offset by an increase in the amount of medium-chain fatty acids in sphingolipids. We conclude that one aspect of Pelizaeus-Merzbacher disease may be a defect in the synthesis of myelin very long chain fatty acids (as these acids are far much reduced than any other myelin molecule).