Brain lipid biochemistry of the sturgeons Acipenseridae]

Brain lipids of 4 species of sturgeons-Acipenser güldenst?dti, A. stellatus, A. ruthenus and Huso huso-have been investigated. In whole brain homogenates the content of the basic phospholipid families, their fatty acid composition as well as the content of glycosphingolipids-gangliosides, cerebrosides and sulfatides were studied. The quantitative relations between different molecular ganglioside fractions and their fatty acid composition, as well as the relative content of hydroxycerebrosides and cerebrosides with normal fatty acids were determined. The brain lipids of Acipenseridae were compared to previously obtained data on the brain of Elasmobranch and Teleost fishes. The brain lipid pattern of the four sturgeon species is very similar, but it differs greatly from that of Elasmobranch and of Teleostean brain, being in some respect more like the type of bony fish brain, in others the kind of brain of sharks and rays, and frequently considerably differing from both these types. The conspicous peculiarity in the content and molecular characteristics of the brain lipids of Acipenseridae compels us to agree with the opinion expressed by the late A.N. Belozerski and his coworkers [26, 27], based on a detailed study of fish DNA nucleotides, that the Ganoid fish are to be regarded as an independent class.     

Comparative research on the cerebroside and sulfocerebroside content of the mammalian brain

Data are presented on cerebroside and sulfocerebroside content of the brain for 31 mammalian species from 8 orders. The increase in concentration of both glycolipids in the brain of mammals in phylogenesis was demonstrated. Low levels of cerebrosides and sulfocerebrosides were found in the brain of lower mammals (Insectivora, Chiroptera) and high ones--in the brain of higher mammals (Carnivora, Primates). Irrespectively from taxonomic position and ecological factors, in the brain of all mammals investigated higher content of cerebrosides with hydroxy acids was found as compared to the content of cerebrosides with normal fatty acids. The ratio of these cerebrosides in the brain of terrestrial mammals is 2-3 times higher than in aquatic and semi-aquatic ones. The data obtained are discussed in relation to the development of the brain of mammals during their phylogenesis.     

Cerebrosides of<Emphasis Type="Italic"> Candida lipolytica</Emphasis> yeast

Candida lipolytica yeast was grown batchwise on glucose medium. Cerebrosides were isolated from the sphingolipid fraction of total lipids using column chromatography and separated into two compounds by high-performance thin-layer chromatography. Glucose was detected as the sole sugar constituent in cerebrosides. The fatty acid composition of cerebrosides was characterised by a predominance of saturated fatty acids and by a high proportion of fatty acids with 16 carbon atoms. The dominant fatty acid was h16:0. The principal long-chain base components of both cerebroside species were trihydroxy bases, 18- and 20-phytosphinosine. The unique characteristic of cerebrosides was the presence of a high proportion of sphingosine (one-fourth of the total long-chain bases), which is a common characteristic of mammalian sphingolipids and rarely occurs in yeast cerebrosides. The ceramide moiety profile of cerebrosides is similar to that of epidermal ceramides, which implies a possibility for their application in care cosmetics.     

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.     

Long-chain and very long-chain fatty acyl-coa synthetase activity in microsomes of jimpy mouse brain

The objective of this study was to determine whether the conversion of free, very long chain fatty acids (C₂₂–C₂₆) to their CoA-esters are involved in cerebroside synthesis, since cerebrosides are uniquely rich in very long chain fatty acids including lignoceric acid (C_24:0). We have studied lignoceroyl-CoA synthetase activity in the microsomes isolated from normal and jimpy mouse brain. The jimpy mouse lacks the ability to make myelin and is deficient in enzyme activities involved in the synthesis of myelin components, including cerebrosides. Unexpectedly, the lignoceroyl-CoA synthetase activity in jimpy brain microsomes was slightly higher than that in control microsomes. The palmitoyl (C_16:0)-CoA synthetase activity in jimpy brain was not different from the control. The level of cerebrosides in microsomes was grossly lower in jimpy brain. The implication of these findings and the involvement of lignoceric acid activation in cerebroside synthesis is discussed.     

LIPID COMPOSITION OF THE CENTRAL NERVOUS SYSTEM OF MARINE VERTEBRATES

—The lipid composition of the central nervous system of some marine vertebrates and two mammalian species (rat and man) was analysed by one- and two-dimensional quantitative thin-layer chromatography, and the cerebroside fatty acids were analysed by gas chromatography. The concentrations of sphingomyelin and cerebrosides are higher in mammals than in fishes, while no significant differences are observed for other lipid classes. Furthermore, in mammals the ratio between hydroxy and normal fatty acids in the cerebrosides is much higher than in fishes. The cerebrosides of mammals contain more very long chain fatty acids than those of marine vertebrates.     

Cerebrosides of human aorta: isolation, identification of the hexose, and fatty acid distribution

Cerebrosides have been isolated from adult human aortic tissue. Each aorta was divided into portions classified as normal, fatty streaks, fibrous plaques, or complicated lesions. The cerebrosides were isolated by Florisil column chromatography, mild alkaline methanolysis, a second Florisil column, and preparative thin-layer chromatography. The concentration of cerebrosides was higher in fatty streaks than in the more advanced plaques; apparently normal tissue gave the same cerebroside content as plaques found in the same aorta. The quantities of cerebrosides ranged from 0.01 to 0.73% of the total lipid. Of the 16 cerebroside samples isolated, 10 contained glucosyl ceramide, 1 contained galactosyl ceramide, and 5 were not analyzed for specific hexose. The fatty acid distribution was determined for 11 of the samples; it was similar to that of spleen cerebrosides. We suggest that aortic cerebrosides originate in the plasma. "Normal tissue" cerebrosides contained less unsaturated fatty acid than cerebrosides from a diseased area of the same aorta. Preparative thin-layer chromatography, the last step of cerebroside isolation, always separated at least two unidentified substances. One of these substances yielded both glucose and galactose on acid hydrolysis. Their removal from the cerebrosides accounts for the lower values for cerebroside compared to other authors' determinations.     

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.     

Content of phospholipid, cerebroside and cerebroside sulfate in the central nervous system of mice with acute experimental viral demyelination

A study was made of the content of phospholipids, cerebrosides and cerebroside sulfates in the central nervous system of mice with experimental acute viral encephalomyelitis. No considerable changes in phospholipid content were revealed. A significant drop in the content of cerebrosides and cerebroside sulfates was defected in the CNS, being more pronounced in the spinal cord of sick animals. The reduction in the content of glycolipids can be explained by myelin disintegration and by the effect of viruses on the olygodendrocytes in which cerebrosides and cerebroside sulfates are synthesized.     

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.     

Changes in the fatty acid composition of cerebrosides and sulfatides of human nervous tissue with age

Sphingogalactolipids (galactocerebrosides and sulfatides) have been isolated in almost quantitative yields from normal human nervous tissue (mostly brain) at different ages and their fatty acid compositions have been determined by gas-liquid chromatography. The ratio of hydroxy acids to normal acids increased slightly during myelination and then remained rather constant; in adults the ratio for cerebrosides was about 2, and for sulfatides, 0.6-0.8. In adult nervous tissue the two predominant fatty acids of cerebrosides and sulfatides were the C(24) monounsaturated and 2-hydroxy saturated acids. The infant brain galactolipids had (compared with child and adult) a lower percentage of C(22)-C(26) fatty acids and a much lower percentage of monoenoic acids, both of normal and hydroxy acids. Low activities of fatty acid elongation and desaturation systems during myelination are inferred. Fatty acid changes with age were the same for cerebrosides and sulfatides but occurred later in the sulfatides, which supports the hypothesis that the cerebrosides are precursors of the sulfatides. The adult pattern of fatty acid composition with regard to degree of unsaturation and total percentage of C(22)-C(26) acids was reached as early as at 2 yr of age, but the percentage of odd-numbered (C(23) and C(25)) fatty acids continued to increase up to the age of 10-15 yr. The fatty acid composition of the galactolipids of peripheral nerves differed mainly in its lower percentages of C(25) and C(26) acids and higher percentages of C(22) and C(16) acids. This composition is thus intermediate between those of brain and of extraneural organs.     

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     

FATTY ACID COMPOSITION OF CEREBROSIDES, SULPHATIDES AND CERAMIDES IN MURINE SUDANOPHILIC LEUCODYSTROPHY: THE JIMPY MUTANT

The fatty acid composition of cerebrosides, sulphatides and ceramides was determined at 15-16 days post partum in the brain of the Jimpy mutant and in littermate controls. There was a marked deficit in the long chain fatty acids (C₂₂-C₂₄) of cerebrosides and sulphatides of Jimpy brain, with the unsubstituted fatty acids affected more than the alpha-hydroxy fatty acids. A decrease of long chain normal fatty acids was also found in the ceramides of Jimpy brain. The deficit of long chain fatty acids in these sphingolipids of the Jimpy brain was more severe than that found in the Quaking mutant which has a less extensive disorder of myelin formation.     

A method for fractionation of cerebrosides into classes with different fatty acid compositions

A method is described for the separation of beef brain cerebrosides into three fractions containing different classes of fatty acids: nonhydroxy (I), unsaturated nonhydroxy (II), and hydroxy fatty acid cerebrosides (III). The procedure consists of benzoylation of either crude or purified cerebrosides, followed by column chromatographic separation of benzoylated derivatives containing nonhydroxy acids from those containing hydroxy fatty acids. The benzoyl groups are removed by sodium methoxide-catalyzed transesterification; from the reaction mixtures, fractions I and III precipitate. The fraction II present in mother liquor of I was shown to contain mainly short-chain and unsaturated nonhydroxy fatty acid cerebrosides. The fatty acid composition of each fraction was obtained by gas-liquid chromatography.     

Concentration and fatty acid composition of cerebrosides and sulfatides in mature and immature human brain

The fatty acid composition of cerebrosides and sulfatides from frontal lobe gray and white matter was determined for five fresh and four formalinized adult brains and for eight infants. Fatty acid patterns were unaffected by formalinization, but varied considerably from one another in the proportion of saturated to unsaturated fatty acids. The percentages of 24:0 and 24:1 increased with age. Cerebrosides obtained from areas such as the brainstem and cerebellum, where myelination was more advanced, tended to have a larger proportion of long-chain fatty acids than samples extracted from frontal or parietal lobe white matter. Hydroxy fatty acids showed an adult pattern in all instances in which amounts sufficient for accurate quantification could be isolated. Lipid hexose, cerebroside + sulfatide hexose, and methanoleluted hexose were measured in the brains of 12 infants ranging in age from a 4 month fetus to 2 yr. In the most immature, the majority of lipid hexose was in the form of glycolipids more polar than cerebrosides and sulfatides. These have tentatively been identified as hematosides and globosides. With maturation, cerebrosides and sulfatides increased progressively, but the amounts of the more polar glycolipids remained constant in relation to the total lipid content of tissue.     

The Lipid Composition of Urodele Myelin Which Lacks Hydroxycerebroside and Hydroxysulfatide

The concentrations of cerebrosides and sulfatides were measured in the nervous systems of urodeles and related orders with a high performance liquid chromatographic technique. The peripheral and central nervous systems of all three urodele species, Necturus maculosis (mud puppy, a salamander), Notophthalmus viridescens (eastern red spot newt), and Desmognathus ochropheus (mountain salamander), were found to be completely devoid of alpha-hydroxy fatty acid-containing cerebrosides and sulfatides. All species of reptiles and fish classes close to urodeles contain these galactolipids. The levels of nonhydroxy fatty acid-containing cerebrosides and sulfatides are essentially similar in both urodeles and reptiles. Myelin isolated from Necturus spinal cord had a specific density of 1.07, lighter than mammalian myelin. Except for the absence of hydroxycerebrosides and hydroxysulfatides, the lipid composition of Necturus spinal cord myelin is essentially similar to that of frog and rat myelin. The fatty acids of nonhydroxycerebrosides are rich in monounsaturated homologs of C22-C25, and the sphingoid base consists of both sphinganine and sphingosine. Electron microscopic examination of the sciatic nerve showed that the general structure and interlamellar distances of salamander and newt myelin are identical to those of frog, chameleon, and rat. Necturus myelin, therefore, can be used as a model for the study of the functional and structural role of hydroxygalactolipids.     

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.     

Phase behavior of galactocerebrosides from bovine brain

Bovine brain cerebrosides (BOV-CER) were separated by high-performance liquid chromatography into cerebroside fractions with a single acyl chain type or with a relatively homogeneous acyl chain distribution. The thermal behavior of these isolated cerebroside fractions was studied by differential scanning calorimetry. Nonhydroxy (n-acyl) fatty acid cerebrosides (NFA-CER) possessing a saturated acyl chain (C16:0, C18:0, C24:0) exhibit their major order-disorder transition temperature TM at 83 degrees C, independent of chain length. NFA-CER possessing primarily unsaturated acyl chains (C24:1) exhibits TM at 70 degrees C. 2-Hydroxy fatty acid cerebrosides (HFA-CER), which possess a saturated hydroxyacyl chain (C18:0h, C24:0h), exhibit TM at 70-72 degrees C. Thus, naturally occurring cerebrosides exhibit high TM's that do not depend significantly on acyl chain length and that depend only to a small degree on unsaturation and the presence of a 2-hydroxy branch in the amide-linked chain. Isolated NFA-CER's each exhibit metastable polymorphism of the type previously described for unfractionated NFA-CER [Curatolo, W. (1982) Biochemistry 21, 1761]. Polymorphism in HFA-CER is complex, with a different type of thermal behavior observed for each isolated acyl chain fraction studied. On prolonged storage at low temperature, unfractionated HFA-CER and unfractionated BOV-CER reach a highly ordered gel state similar to that which is readily reached by NFA-CER's. These results indicate that all cerebrosides exhibit metastable polymorphism. However, the kinetic barriers to reaching the stable gel state are greater for HFA-CER and BOV-CER than for NFA-CER.     

Fatty acids of glycosphingolipids from pig blood fractions

Four glycolipids have been isolated from three fractions of pig blood. The glycolipids were presumably cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The blood fractions were erythrocytes and plasma high and low density lipoproteins. Fatty acid distributions were determined for each glycolipid as a means to assist in identifying relationships among the several glycolipids. Normal fatty acids predominated in all glycolipids except the globosides from erythrocytes in which the amount of hydroxy acids was slightly greater than the amount of normal acids. Hydroxy acids appeared to be present in all the glycolipids, but the concentration was very low in cerebrosides isolated from high density lipoproteins and erythrocytes, and in diglycosyl ceramide and globoside of the low density lipoproteins. In general, the average fatty acid chain length increased from cerebroside to globoside. This was most apparent in erythrocytes and also greater for normal acids than for hydroxy acids. Fatty acid distributions of erythrocyte glycolipids had sufficient variation to make a metabolic relationship by simple addition of a hexose appear doubtful. While the fatty acid distributions found in plasma lipoproteins were more similar, some means of acyl group selection is probably present for either the synthesis or degradation of these glycolipids.     

Globoid cell leukodystrophy (Krabbe's disease): isolation of myelin with normal glycolipid composition

Myelin was isolated from the brain of a patient with Krabbe's globoid cell leukodystrophy at 0.4% of the normal yield. Despite the exceedingly low yield, the fraction appeared morphologically clean, and consisted mostly of well-preserved myelin lamellae and few contaminating structures. Total lipid and cholesterol were slightly lower than in normal myelin. Total phospholipid was normal, but the ratio of ethanolamine phospholipid to lecithin was reversed. Total galactolipid was normal, and consisted only of cerebroside and sulfatide in normal proportions. The only sugar in cerebroside and sulfatide was galactose. The fatty acid composition of cerebroside and sulfatide was essentially normal with no deficiency of long-chain fatty acids and only with a reversed ratio of C(24:0) to C(24:1) in cerebroside. These data appear to exclude the previous postulate that abnormally rapid breakdown of myelin occurs in this disorder as the result of the formation of chemically abnormal myelin, deficient in sulfatide.