Brain cardiolipin: isolation and fatty acid positions

Abstract— Cardiolipin (diphosphatidyl glycerol) was isolated from bovine brain grey matter in pure form by column chromatography. A combination of a DEAE cellulose column, an acid-silicic acid column and a bicarbonate-treated silicic acid column was used for the isolation. Analytical data on cardiolipin (and lyso-cardiolipin), including phosphorus and fatty ester values, infrared and ultraviolet spectra, and chromatographic behaviour of intact cardiolipin, lyso-cardiolipin and their water-soluble hydrolysis products gave results which were consistent with the diphosphatidylglycerol structure proposed by MacFarlane. Cardiolipin constituted 1·2 per cent of the total lipids from grey matter, or 0·43 per cent of the dry weight. No aldehydes were detected in purified cardiolipin. The major fatty acids were 16:0, 16:1, 18:1, 18:2 and 20:4. Cardiolipin contained much higher concentrations of 18:2 (17·8 per cent) than any of the other major grey matter glycerophosphatides. Cardiolipin was hydrolysed by incubation with snake venom (Trimeresurus flavoviridis) phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) to determine the distribution of fatty acids on the carbons of its diglyceride moieties. The fatty acids were not distributed randomly; 16:0, 18:0 and 18:2 were predominantly localized in the α and α positions, while 18:1 and 20:4 were predominantly localized in the β and β positions.     

Effects of postnatal age and diet on the fatty acid composition of plasma lipid fractions in preterm infants

Diet and postnatal age effect the fatty acid composition of plasma and tissue lipids. This work was designed as a transversal study to evaluate the changes in the fatty acid composition of plasma phospholipids, cholesteryl esters, triglycerides and free fatty acids in preterm infants (28-35 weeks gestational age), fed human milk (HM) and milk formula (MF) from birth to 1 month of life. Sixteen blood samples were obtained from cord, and 19 at 6-8 h after birth, 14 at 1 week and 9 at 4 weeks from HM-fed infants and 18 at 1 week and 14 at 4 weeks from MF-fed ones. Groups had similar mean birth weight, gestational age and sex ratio. The MF provided 69 kcal/dl and contained 16% of linoleic acid and 1.3% of alpha-linolenic acid on the total fat. Plasma lipid fractions were extracted and separated by thin-layer chromatography and fatty acid methyl esters were quantitated by gas liquid chromatography. In plasma phospholipids, linoleic acid (18:2 omega 6) continuously increased from birth to 1 month of age, but no changes were seen as related to type of diet; polyunsaturated fatty acids greater than 18 carbon atoms of both the omega 6 and omega 3 series (PUFA omega 6 greater than 18 C and omega 3 greater than 18 C) dropped from birth to 1 week and continued to decrease in MF-fed infants until 1 month; eicosatrienoic (20:3 omega 6), arachidonic (20:4 omega 6) and docosahexaenoic (22:6 omega 3) were the fatty acids implicated. In cholesteryl esters palmitoleic (16:1 omega 7) and oleic (18:1 omega 9) acids decreased from birth to 1 month and linoleic acid increased and arachidonic acid dropped, especially in MF fed infants. In triglycerides, palmitic, palmitoleic and stearic acid (18:0) decreased during the first month of life; oleic acid remained constant and linoleic acid increased in all infants, but arachidonic acid decreased only in those fed formula. Free fatty acids showed a similar behavior in fatty acids and in plasma triglycerides. Preterm neonates seem to have special requirements of long-chain PUFA and adapted MF should contain these fatty acids in similar amounts to those of HM to allow the maintenance of an adequate tissue structure and physiology.     

The effect of cold stress on ganglioside fatty acid composition and ganglioside-bound sialic acid content of rat brain subcellular fractions

The effect of cold stress on the ganglioside fatty acid composition and sialic acid content of brain subcellular fractions and homogenate of rats was studied, the animals were kept in a cold room with 12h light-dark cycles at 3 and 10 degrees C for 2 weeks. (1) The rat brain homogenate, synaptosomes and myelin of rats exposed to 3 degrees C contained significantly higher amounts of ganglioside-bound sialic acid per mg of protein than these fractions of control rats kept at 23 degrees C; the differences were less pronounced in rats exposed to 10 degrees C. (2) A small, but significant, diminution of relative palmitic acid content and an increase of stearic acid content was found to take place in gangliosides from rat brain synaptosomes, synaptosomal plasma membranes and homogenate as a result of the exposure of animals to 3 degrees C and to a lesser extent to 10 degrees C. (3) The content of unsaturated fatty acids in gangliosides from brain subcellular fractions was approximately the same in cold exposed and control rats.     

Leishmania species: fatty acid composition of promastigotes

The fatty acid composition of the total lipid fractions of five different Leishmania organisms grown on Eagle's medium was determined by gas chromatography. The major fatty acids identified in the total lipid fractions of L. donovani, L. tropica major, L. tropica minor, L. tropica (England strain), and L. enriettii were C_12:0, C_13:0, C_14:0, C_15:0, C_16:0, C_17:0, C_18:0, C_18:1, C_18:2, and C_18:3. The statistical differences among the fatty acid methyl esters of different Leishmania organisms are discussed.Gas chromatographic analysis of the fatty acid methyl esters of the total lipid fractions of the original Eagle's medium and the media after harvesting of various Leishmania species revealed the presence of C_18:3 fatty acid in the total lipid fraction of the medium of L. donovani and the complete absence of 18-carbon unsaturated fatty acids in the total lipid fraction of the medium of L. enriettii. The use of such differences in the differentiation of various Leishmania species is discussed.     

Methods of isolation and polypeptide composition of membrane fractions of Rickettsia prowazekii

The methods of cell lysis by lysozyme in tris-EDTA-sucrose with the consequent disruption of spheroplasts by the osmotic shock were used to obtain the total membranes from the intact or temperature-inactivated Rickettsia prowazekii. Detergents solubilization methods were used for analysis of outer membrane proteins. Sarcosyl insoluble material is shown to contain the main 134, 31, 29.5 and 25 Kd proteins, the minor 78, 60, 42, 17 Kd proteins, while the mixture of both membranes possess a more complex composition. Treatment of total membranes by the 2% octylglycoside results in elimination of the 31 Kd polypeptide. Inactivated Rickettsia can be used for isolation of the outer layer proteins diminishing the risk of working with this pathogenic microorganism.     

Interaction between isolation source, cellular fatty acid composition and stress tolerance in Saccharomyces cerevisiae and its subspecies

The physiological properties and fatty acid content of 59 strains of Saccharomyces cerevisiae isolated from soft-drink factories, a fruit puree factory, a fuel-alcohol distillery and a winery were compared. Discriminant analysis of the results allocated the strains to four groups according to their source. Resistance to preservatives and temperature stress were correlated with differences in fatty acid composition. The fatty acid C18: 1Δ11, growth at pH 2 and in the presence of 200–600 mg 1^-1 benzoate or sorbate, and maximal growth rate at 42°C were characteristics associated with yeasts from particular environments. However, tolerance of thermal stress and content of the C18: 2 fatty acid were associated with subspecies: the former species S. capensis, S. chevalieri , etc. The relative content of C10 : 0, C12 : 0 and C18 : 0 acids varied according to both isolation source and subspecies.     

Higher fatty acid composition of enterococci

The content of unsaturated fatty acids in enterococcal cells has been found to have no essential relation to the composition of the culture medium. When cultivated in the same media, S. faecium had the degree of lipid unsaturation 1.5-2 times higher than S. faecalis. Mobile enterococci are sharply differentiated from immobile species by the content of cyclopropanic acid with 19 carbon atoms, constitute a heterogenous group and consist of at least 2 taxons, differing in the content of acids with 18 carbon atoms and the degree of lipid unsaturation.     

Changes in fatty acid composition of peripheral nerve myelin in essential fatty acid deficiency

Severe essential fatty acid deficiency (EFAD) was induced by feeding weanling rats a diet free of essential fatty acids 8 months after weaning. The fatty acid compositions of phospholipids and glycosphingolipids in peripheral nerve myelin were compared in rats with and without EFAD. With the deficient diet, 20:3ω9 was found in the major myelin phospholipids. The level of 18:1 was increased and the levels of 18:2ω6, 20:4ω6, and 22:4ω6 were decreased. Both sphingomyelin and cerebroside showed higher proportion of 24:1 and lower proportions of 24:0 in EFA-deficient rats than in control rats. The fatty acid chain elongating system in myelin cerebroside was also depressed by EFAD. A two- to sevenfold increase of the ratio 20:4ω6 to 20:3ω6 was found in myelin phospholipids of regenerated nerve from rats fed control diet. However, this ratio was suppressed by EFAD diet. The biochemical index (20:3ω9/20:4ω6) for EFAD was not affected by crush injury. These results suggest that dietary EFAD in postweaning rats can induce fatty acid alterations in peripheral nerve myelin without resulting in detectable changes in function or structure and that myelin lipids may be sequestered and reused during nerve degeneration and regeneration.     

Variation in the lipid and fatty acid composition in purified membrane fractions from Sarcina aurantiaca in relation to growth phase

S. aurantiaca membrane lipid contains both branched and straight-chain fatty acids from C9 to C22 with the saturated branched C15 predominating in almost all of the lipid fractions studied. Unsaturated fatty acids are only present in low concentrations. Significant amounts of straight-chain, even-numbered acids, more common in gram-negative and gram-variable bacteria, are also present. All lipid fractions show a marked change in their fatty acid profiles from the exponential to stationary phase of growth. At least 88% of the total lipid is free lipid, and of this material, at least 62% is neutral lipid. The amount of the latter decreases, with a corresponding increase in phospholipid as cells go into stationary phase. During this time, there is a slight fall in the amount of glycolipid which contains predominately mannose, but also glucose and galactose.     

Sperm fatty acid composition in subfertile men

INTRODUCTION: The lipid composition of spermatozoa plays an important role for successful fertilization. PATIENTS AND METHODS: In the present study, we analyzed the fatty acid (FA) composition of spermatozoa of normozoospermic, asthenozoospermic, oligozoospermic and oligoasthenozoospermic men. RESULTS: Spermatozoa from asthenozoospermic (P<0.01), oligozoospermic (P<0.05) and oligoasthenozoospermic men (P<0.05) had lower levels of docosahexaenoic acid (22:6w3, DHA) than those from normozoospermic men. In oligozoospermic and asthenozoospermic men, spermatozoa 18:0 content was higher than that of normozoospermics (P<0.01 and P<0.001, respectively). 18:1w9 was higher in oligoasthenozoospermic and oligozoospermic samples when compared with normozoospermic samples (P<0.05 for both). While from the point of view of total w6 FAs there was no significant difference among the groups, the w6/w3 ratio was significantly higher in asthenozoospermic samples than in normozoospermic samples (P<0.05). Monounsaturated fatty acids (MFA) were higher in oligozoospermic samples (P<0.05) than in normozoospermic samples, polyunsaturated fatty acids (PUFA) were lower in asthenozoospermic (P<0.01), oligoasthenozoospermic (P<0.05) and oligozoospermic samples (P<0.05) than in normozoospermic samples. Saturated fatty acids (SFA) were significantly higher in asthenozoospermic (P<0.01) and oligozoospermic samples (P<0.05) compared with normozoospermic samples. In correlation analysis, there were significant positive correlations between DHA with sperm motility (r=0.53), sperm concentration (r=0.36) and normal sperm morphology (r=0.30). In addition, there were significant correlations between PUFA with sperm motility (r=0.50), sperm concentration (r=0.35), and normal sperm morphology (r=0.28), and between w6/w3 with sperm motility (r=-0.47), sperm concentration (r=-0.27), and normal sperm morphology(r=-0.24). DISCUSSION: These suggest that decreased DHA and PUFA, and increased w6/w3 in spermatozoa may be related to infertility in oligo- and/or asthenozoospermic men.     

Effect of acute streptozotocin diabetes on fatty acid content and composition in different lipid fractions of rat skeletal muscle.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on long chain fatty acid content and composition in different lipid classes of particular muscle types in the rat. Two days after streptozotocin administration, rats were anesthetised, and the white and red sections of the gastrocnemius, the soleus and the blood were taken. Lipids were extracted with chloroform/methanol and separated into different fractions (phospholipids, free fatty acids, di- and triacylglycerols) by means of thin layer chromatography. Fatty acids of each fraction were identified and quantified by means of gas-liquid chromatography. The diabetes resulted in elevation of the concentration of blood glucose (over four-fold) and the plasma free fatty acid (over two-fold). Total free fatty acid content in the muscles of diabetic rats increased by 26% in the white, 24% in the red gastrocnemius and 21% in the soleus. There were also changes in the composition of that fraction in each muscle. Diacylglycerol fatty acid content was elevated in both parts of the gastrocnemius (the white part by 15%, the red part by 44%) and remained stable in the soleus of the diabetic rats. The content of triacylglycerol fatty acids was elevated only in the red gastrocnemius in the diabetic group (by 112%), but changes in fatty acid composition in this fraction occurred in each muscle. The content of phospholipid fatty acids was elevated in the white gastrocnemius (by 13%) and remained stable in other muscles. There were only minor changes in phospholipid fatty acid composition in the diabetic rats. We concluded that acute insulin deficiency changes fatty acid content and composition in skeletal muscle lipids. The changes depend both on lipid fraction and muscle type.     

Sarcolemmal phospholipid fatty acid composition and permeability

In this study, the mechanism of ischaemia-induced increased sarcolemmal permeability, as manifested by release of intracellular enzymes, was investigated. The role of changes in the sarcolemmal phospholipid bilayer in this process was evaluated by experimental modulation of the phospholipid fatty acid composition. The isolated perfused rat heart subjected to low-flow hypoxia, was used as a model of global ischaemia. Glucose as well as saturated (palmitate) and unsaturated (linoleate) long-chain fatty acids were used as substrates. Hearts perfused with palmitate or linoleate (1.5 mM, fatty acid/albumin ratio, 3.4) showed a significantly higher rate of lactate dehydrogenase release in both control and ischaemic conditions than hearts perfused with glucose (10 mM). Lactate dehydrogenase release in the fatty acid-perfused hearts was associated with a significant increase in the percentage unsaturation of the sarcolemmal phospholipid fatty acids. Glucose-perfused hearts, on the other hand, showed only minor changes in the sarcolemmal phospholipid fatty acid composition. Attempts to correlate enzyme release directly with an increase in the percentage unsaturation of phospholipid fatty acids failed, since enzyme release was also stimulated in control fatty-acid-perfused hearts which (when compared with glucose) contained a higher percentage saturated phospholipid fatty acids. The results suggest that myocardial ischaemia, apart from changes in the sarcolemmal phospholipid fatty acid composition, also induces several other changes in sarcolemmal composition (e.g., cholesterol loss) which may affect is permeability for macromolecules.