In vivo metabolism of 3-ketoceramide in rat brain

Eight hours after intracerebral injection of a double-labeled 3-ketoceramide⁴, [1-¹⁴C]lignoceroyl 3-keto [1-³H]sphingosine, various brain sphingolipids were isolated. Free ceramide and the ceramide portions of nonhydroxy cerebroside and sphingomyelin were further fractionated into subgroups containing longer-chain or shorter-chain fatty acids. Nonhydroxy ceramide, nonhydroxy cerebroside and sphingomyelin containing longer-chain fatty acids had significant quantities of radioactivity with ³H/¹⁴C ratios similar to each other but lower than that of the injected material. The sphingolipids containing shorter-chain fatty acids were also significantly labeled; however, the ³H/¹⁴C ratios were much higher than that of the injected material. Hydroxy-ceramide and sulfatides contained very little radioactivity. However, hydroxy-cerebroside contained an amount of radioactivity comparable to that of the longer-chain nonhydroxy cerebroside with a similar ³H/¹⁴C ratio. It is proposed that the injected 3-ketoceramide was converted into ceramide, cerebroside, and sphingomyelin and that the fatty acids of these lipids were partly replaced by other fatty acids during the metabolic conversions.     

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.     

Triethyllead treatment of cultured brain cells

Cultured cells from chick embryo brains were studied for their sensitivity to triethyllead. Triethyllead chloride (3.16 M) was added to the nutrient medium and incubated for 48 hr with the cells. Morphological changes in light microscope and radioactive labeling of galactolipids were assayed. Triethyllead treatment reduced the number of neuronal cells with processes. Morphological changes were not observed in glial cells. The [³⁵S]sulfate labeling of sulfatides was reduced to 50%. The [³H]serine labeling of cerebrosides with alpha-hydroxy fatty acids was not influenced, while the [³H]serine labeling of cerebrosides with nonhydroxy fatty acids was inhibited 40% in one- and two- but not in three-week-old cultures. The results indicate that the nerve cell response to triethyllead in cultures is selective, since the neurons are more sensitive than the glia cells and the labeling of sulfatides is more sensitive than that of cerebrosides.     

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.     

THE METABOLISM OF PALMITIC ACID IN THE PHOSPHOLIPIDS, NEUTRAL GLYCERIDES AND GALACTOLIPIDS OF MOUSE BRAIN

Abstract— Following intracerebral injection, [¹⁴C]palmitic acid was rapidly incorporated into a variety of brain lipids. After 12 hr, 78 per cent of the lipid radioactivity was in phospholipids, 15 per cent was in triacylglycerols, 1 per cent each was in free fatty acids and galactolipids, and the remainder was in other neutral glycerides. Over 65 per cent of the phospholipid radioactivity was found in the choline phosphoglycerides but this proportion decreased substantially with time. At later times, increasing portions of the radioactivity were present in the monounsaturated acyl groups and the alkenyl groups but no radioactivity was detected in cholesterol or polyunsaturated acyl groups. These results indicate that most of the extensive recycling of radioactivity took place without oxidative degradation of the palmitoyl groups. The relative rates of incorporation of radioactivity were compared at 12 hr after injection. The specific radioactivities of the serine, ethanolamine, and choline phosphoglycerides had ratios of 6:3:2 based on the palmitoyl group content and 1:2:4 based on their phosphorus content. The specific radioactivities of galactolipids with O -acyl groups were higher than the specific radioactivitiesof cerebrosides or cerebroside sulphates. A new solvent mixture for thin-layer chromatography of brain galactolipids was described (chloroform-acetone-methanol-water, 60:20:20:1, by vol.).     

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.     

Synthesis and Transport of Cerebrosides and Sulfatides in Rat Brain During Development

Synthesis and transport of nonhydroxy fatty acid (NFA)- and hydroxy fatty acid (HFA)-containing ceramides, cerebrosides, and sulfatides were studied in vivo in rat brain during development. After an intracerebral injection of [3H]serine, incorporation into these lipids of microsomal and myelin membranes was analyzed after HPLC. Distribution of amounts and incorporation of radioactivity were also determined in individual molecular species of these lipids. The results showed that HFA-ceramides and long-chain NFA-ceramides have small pool sizes and rapid turnover rates in the microsomal membranes and are preferentially utilized for the synthesis of long-chain (greater than or equal to 20:0) HFA- and NFA-galactocerebrosides of both microsomal and myelin membranes. Glucocerebrosides are not expressed in myelin and their synthesis in microsomal membranes is predominant before the onset of myelination. With development, synthesis and accumulation of HFA-cerebrosides increase over NFA-cerebrosides in both microsomal and myelin membranes. In myelin, incorporation of radioactivity into HFA-cerebrosides is even higher than that expected by transport alone from microsomal membranes and it is possible that part of the HFA-cerebrosides in myelin could be due to de novo synthesis by myelin itself. The amount of NFA- and HFA-sulfatides is about equal, both in myelin and microsomal membranes, and this relative proportion does not change with development. Similar relative rates of incorporation of radioactivity into sulfatides of microsomal and myelin membranes are consistent with the notion that both NFA and HFA sulfatides are synthesized in the microsomal (Golgi) membranes and are transported to myelin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Brain cerebrosides: preparative purification using a new chromatographic support--magnammsil

A preparative method for isolation and purification of cerebrosides from bovine brain is described. A crude cerebroside fraction is precipitated with ether from a chloroform-methanol extract and purification is achieved by column chromatography on a new column support, "magnammsil." A partial separation of hydroxy and nonhydroxy cerebrosides is achieved. The method is more economical for a large-scale preparation than previously published methods.     

Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes

Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, coupled to lissamine-rhodamine, fluoresceine, eosine and NBD. The spectroscopic properties of these compounds, in different solvent systems and when incorporated into micelles of a non-ionic detergent or liposomes of a phospholipid, are reported. Incubation of these respective sulfatides with a human leukocyte preparation, resulted in the formation of the corresponding fluorescent cerebrosides.     

IN VIVO METABOLISM OF [G-3H]LIGNOCERIC ACID IN DEVELOPING RAT BRAIN1

Abstract— [G-³H]Lignoceric acid (tetracosanoic acid) was injected into the brains of 20-day-old rats, and the animals were killed after 8, 24, or 72 h. Various lipids were isolated from these brains, and the distribution of radioactivity was determined. The injected free acid rapidly disappeared, and the radioactivity was incorporated into varying chain-length nonhydroxy- and hydroxy saturated fatty acids of sphingolipids and phospholipids. Little radioactivity was found in unsaturated acids, sphingo-sine, and cholesterol. A time-dependent shift of the label among various fatty acids was relatively small 8 h after injection, probably because of the metabolic stability of the brain sphingolipids. In cerebrosides, the radioactivity was equally distributed between nonhydroxy and x-hydroxy fatty acids of all chain lengths. C₂₃ and C₂₂ fatty acids contained equal total radioactivities; C₂₃ and C₂₄ fatty acids contained similar specific activities. These results confirm the significant role of a-hydroxylation and 2-oxidation in the synthesis of very long-chain fatty acids in brain. In total lipid fatty acids, docosanoic acid (22:0) contained more radioactivity than its α-oxidation precursor, α-hydroxytricosanoic acid (23h:0) at all times. In sphingolipid fatty acids, the specific activity of 21:0 was always higher than that of its ct-oxidation precursor 22:0. These observations indicate that part of the 22:0 and 21:0 was derived by β-oxidation from the injected lignoceric acid or its α-oxidation product, respectively.     

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.     

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.     

Preparation and analysis of benzoylated cerebrosides

The benzoylation of cerebrosides with benzoyl chloride and with benzoic anhydride is described, and the derivatives that contained hydroxy and nonhydroxy fatty acids were isolated by high pressure liquid chromatography (HPLC). Studies on the structures of these derivatives and the products formed by mild alkaline hydrolysis are reported. Reaction of cerebrosides containing nonhydroxy fatty acids with benzoyl chloride results in amide-acylation in addition to normal O-acylation. Mild alkali treatment of the N-diacyl derivative results in the formation of N-benzoyl psychosine. Derivatization with benzoic anhydride avoids amide-acylation. These derivatives are useful for the HPLC analysis of cerebrosides.     

6-acyl galactosyl ceramides of pig brain: structure and fatty acid composition

Two glycolipids were isolated from pig brain and were shown to be the fatty acid esters of kerasin and cerebron in which the second fatty acid moiety is attached to the 6-position of the galactose. The point of attachment was shown in two ways: by permethylation and by cleavage with periodate. Methanolysis of the permethylated cerebroside esters yielded O-methyl sphingosines, methyl esters of nonhydroxy or 2-methoxy acids, and methyl 2,3,4-trimethyl galactoside. Cleavage of the cerebron ester with periodate, followed by treatment with sodium borohydride and dilute HCl, yielded ceramide plus 1-monoglyceride. The ester-linked fatty acids were primarily 16:0, 18:0, and 18:1, while the amide-linked fatty acids showed the wide assortment of chain lengths typical of brain cerebrosides. The methylation step, with silver oxide and methyl iodide, yielded two derivatives with the cerebroside esters, but the structural explanation for the difference was not elucidated. The galactose in the cerebron ester was shown to exist in the beta-pyranoside form.     

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.     

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.     

Reptile brain cerebrosides and cerebroside sulfates

Studies have been made on the content of cerebrosides and cerebroside sulfates, as well as on their fatty acid composition in the brain of reptiles, subclass Anapsida (tortoises Emys orbicularis and Testudo horsfieldi) and subclass Lepidosauria (lizards Agama caucasica, A. sanguinolenta, Phrynocephalus mystaceus and snake Natrix tesselata). Total content of cerebrosides and cerebroside sulfates is higher in the brain of Lepidosaurians than in that of Anapsids. In the brain of tortoises, the content of cerebroside fraction with hydroxy fatty acids is significantly higher than of the fraction with normal fatty acids, which is also typical of the brain of homoiothermic mammals and birds. In the brain of Lepidosaurians, concentration of hydroxycerebrosides is considerably lower than of cerebrosides with normal fatty acids, which is similar to lower vertebrates -- amphibians and fishes. Low content of hydroxycerebrosides was found in all the Lepidosaurians investigated, irrespectively of their ecological conditions, being therefore dependent on their phylogenetic position. The composition of fatty acids, both normal and hydroxyderivates, as well as that of glycolipids from the brain of Anapsids and Lepidosaurians is essentially similar. However, some interspecific differences were noted in the pattern of fatty acids of cerebrosides and cerebroside sulfates of the brain, which concern the content of saturated and long chain fatty acids.     

An improved procedure for the quantitative determination and characterization of sulfatides in rat kidney and brain by high-performance liquid chromatography

A significant improvement has been made in the desulfation step of our previously published HPLC determination of cerebrosides, sulfatides, and monogalactosyl diacylglycerols (Nonaka, G. and Kishimoto, Y., Biochim. Biophys. Acta 572 (1978) 423-431). Instead of the original two-phase reaction, a solution of trifluoroacetic acid in ethyl acetate is used for the solvolysis in the new method. The revised method was used to determine the levels of cerebrosides and sulfatides in rat kidney. Among four individual glycosphingolipids studied, hydroxysulfatide was present at the highest level (0.7-1.3 nmol/mg of dry tissue), followed by nonhydroxysulfatide (0.3-0.8 nmol/mg of dry tissue). Hydroxycerebroside (0.09-0.16 nmol/mg of dry tissue) and nonhydroxycerebroside (0.03-0.09 nmol/mg of dry tissue) were present in smaller quantities. There appear to be no significant differences between male and female animals of different ages (30-120 days), although the amounts decreased slightly in older animals and there was a higher concentration in female than in male kidney. Tissue size was significantly smaller in females. The homolog composition of rat kidney sulfatide was studied by reverse-phase HPLC, and was found to be significantly different from that reported in human kidney. Rat sulfatides contained fatty acids with a higher degree of saturation and longer chain length. Preliminary studies indicated that rat kidney contained unusually large quantities of C25:1 and C27:1 fatty acids and also that there was more C26:1 than C24:1 acid. In brain of the same animals the ratio of nonhydroxy to hydroxysulfatide decreased with age (1.5:1 in 30-day-old brain; 1:1 at 90 days).     

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.