Fatty Acid Composition of Endoneurium and Perineurium from Adult Rat Sciatic Nerve

Reports on lipid composition of peripheral nervous system have generally been restricted to the saturated fatty acids of the endoneurium. In this work we attempt to determine the fatty acid composition of the different lipid classes in both endo- and perineurium from sciatic nerve microdissection on adult rats. Unsaturated fatty acids were found to make up around 60% of total fatty acids in samples of endoneurium and perineurium, with monounsaturated fatty acids forming 40-50% of total unsaturated fatty acid content. Although the same fatty acids were present in both tissues there was a striking difference in C 18:1 (n-9) and C 18:2 (n-6) ratio between endoneurium and perineurium, which is particularly rich in linoleic acid. The nonpolar perineurial lipids were found to be richest in linoleic acid. Phospholipids were present in the perineurium, and they contained high proportions of saturated and medium-chain monounsaturated fatty acids.     

Purification and characterization of C28-55 fatty acids from Mycobacterium smegmatis

The nonmycolic C16 to C55 fatty acids obtained from Mycobacterium smegmatis ATCC 356 by saponification were enriched with respect to the C28 to C55 acids by successive chromatography on silicic acid and Sephadex LH-20 columns. These partially purified fatty acids were then derivatized to the p-bromophenacyl ester and further fractionated by argentation thin-layer chromatography and reverse-phase high-performance liquid chromatography into their individual components. The esters were characterized by electron impact mass spectrometry. Two structural series of C28:1 to C42:1 and C45:2 to C55:2 fatty acids were identified as possible precursors of the monoenyl and dienyl mycolic acids, respectively. These acids were structurally related to the alpha-alkylhydroxyl group of the corresponding mycolic acid. The results suggest that these C28 to C55 fatty acids (meromycolic acids) of M. smegmatis might be precursors of mycolic acids.     

Cell-free synthesis of mycolic acids in Mycobacterium aurum: radioactivity distribution in newly synthesized acids and presence of cell wall in the system

Distribution of radiolabelling in different parts of the newly synthesized mycolic acids, by a cell-free system from Mycobacterium aurum previously described, is examined, [1-14C]acetate being the precursor. By oxidation cleavage of mycolic acids and examination of the fragments, it was shown that acetate was not uniformly incorporated into the molecule: the methyl terminal part was not labelled, while the central fragments--between unsaturations or between oxygenated functions (oxo or ester) and unsaturations--presented the major part of radioactivity, suggesting the elongation of a preformed compound that the cell-free extract was unable to synthesize. Moreover, the side-chain R2-CH2-COOH was only weakly labelled compared to the central fragments. Since non-hydroxylated fatty acids were not synthesized by the system, it is suggested that de novo C18 fatty acids may be elongated with C2 units by the cell-free extract into C22 fatty derivative, only a low level of labelling being recorded (two C2 units for all the molecule). A scheme is proposed to summarize the main results. Identification of meso-DAP which is a characteristic amino-acid of the peptidoglycan in Actinomycetes and analysis of the profiles of total fatty esters, demonstrated that the cell-free extract is partly constituted by fragments of the cell wall as has already been noticed by examination of micrographs of the extract.     

Identification of mouse palmitoyl-coenzyme A Delta9-desaturase

Stearoyl-coenzyme A desaturase (SCD) catalyzes the desaturation of saturated fatty acids to monounsaturated fatty acids in mammalian cells. Currently, there are four known enzymatic isoforms (SCD1-SCD4) in the mouse genome. The physiological roles for multiple SCD isoforms and their substrate specificities are unknown at present. We report here distinct substrate specificities for the mouse SCD isoforms. Each SCD isoform was able to complement the ole1 mutation in Saccharomyces cerevisiae through heterologous expression of transgenic SCD. Fatty acid analysis showed that mouse SCD1, SCD2, and SCD4 desaturate both C18:0 and C16:0, whereas mouse SCD3 uses C16:0 but not C18:0. We identify SCD3 as a mammalian palmitotyl-CoA Delta9-desaturase, and its existence in mouse helps explain distinct physiological roles for each SCD isoform.     

Mycolic acid analysis in Nocardia species. The mycolic acid compositions of Nocardia asteroides, N. farcinica, and N. nova.

Mycolic acids from twelve Nocardia species were analyzed for structure using capillary gas chromatography and mass spectrometry. This high-resolution procedure permitted good separation of the trimethylsilyl (TMS) ether derivatives of mycolic acid methyl ester according to the total number of carbon and double bonds. The profiles of the mycolic acid molecular species were used as models to illustrate the difference in the structures of each species, even in the case of N. asteroides complex; N. asteroides, N. farcinica and N. nova. Although N. asteroides and N. farcinica had similar lengths of carbon skeleton, i.e., 51.9-53.7 was the average carbon number (Av.Nc.), they had different compositions of unsaturated acids. Mycolic acids from N. asteroides were composed of abundant saturated acids and less than 1% tetraenoic acids; mycolic acids from N. farcinica were composed of unsaturated acids, which were composed of abundant dienoic acids, 2-12% of tetraenoic acids and a trace of pentaenoic acids. In contrast, Av.Nc. of mycolic acids from N. nova were 55.7-56.3, which were relatively longer than those from N. asteroides or N. farcinica. Regarding the characteristics of the structure of alpha-branch, major components were C16:0 and C18:0 for N. asteroides 23206T, and C16:0 and C14:0 for N. farcinica 23157T, respectively. The presence of monounsaturated alpha-branch (C18:1 and C16:1) was characteristic of N. nova.     

Time course dependent changes in contents and physical properties of glycolipid species in Rhodococcus rhodochrous.

The yield of trehalose dimycolate (TDM), the major glycolipid species elaborated by Rhodococcus rhodochrous, a producer of approx. C40-mycolic acid, was not constant in cells cultured for different periods of time. From cells collected at 24, 36, 72, 144 and 172 h of cultivation the following percentages of TDM in diethyl ether soluble lipids (DESL) were found: 10.8%, 23.4%, 10.0%, 9.0% and 5.0%, respectively. In turn, the cellular content accounted for approx. 0.6%, 1.2%, 0.9%, 0.6% and 0.2%, respectively. On the other hand, the yield of galactose monomycolate (GalMM), a minor glycolipid species maintained at approx. 3.4% in DESL during the different periods of time examined; this value represented about 0.3% of the cellular content. The melting temperatures of TDMs fell between 37 degrees C to approximately 97 degrees C with the lowest value from cells grown for 36 h, whereas the melting temperatures of the GalMMs were in a narrow range between 56 degrees C and 64 degrees C. The methyl ester derivatives of the constituent fatty acid moieties of DTMs and GalMMs migrated on thin layer chromatography like methyl esters of C40-C46 mycolic acids, therefore faster than methyl esters of C28-C34 mycolic acids but slower than methyl esters of C50-C56 mycolic acids. Further analysis of the products of pyrolysis of the methyl ester derivatives of the fatty acid moiety released from TDM after alkaline hydrolysis was carried out using gas chromatography combined mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

De novo synthesis and elongation of fatty acids by subcellar fractions of monkey aorta

Subcellular fractions of aorta of squirrel monkey (Saimiri sciureus) were examined for their ability to synthesize and elongate fatty acids. High-speed supernate (HSS) incorporated substantial quantities of malonyl CoA into fatty acids while acetyl CoA was much less effectively utilized. Acetyl-CoA carboxylase activity exceeded the amount of acetyl CoA incorporated into fatty acids and thus does not account for the low incorporation of this substrate. Microsomes used malonyl CoA and acetyl CoA equally well; mitochondria incorporated either acetyl CoA or acetate. The amounts of substrate incorporated into fatty acids (m micro moles/mg of protein per hr) were 2.3 for HSS, 1.2 for microsomes, and 0.9 for mitochondria. The synthesized fatty acids were separated by gas-liquid chromatography, radioassayed, extracted from the scintillation fluid, and decarboxylated. HSS completely synthesized palmitic and stearic acids from malonyl CoA. Microsomes and mitochondria utilized acetyl CoA to elongate endogenous fatty acids and gave mainly palmitic, stearic, and C(18) and C(20) monoenoic acids, with lesser amounts of other saturated and unsaturated fatty acids. A significant quantity of malonyl CoA was utilized by microsomes to yield a fatty acid tentatively identified as docosapentaenoic. Radioactive fatty acids are incorporated into various lipid classes by the particulate preparations. These studies demonstrate that aortic tissue in a nonhuman primate is able to carry out several processes of fatty acid metabolism and that the aortic synthesis and elongation of fatty acids may play an important role in providing fatty acids for incorporation into aortic lipids.     

Mycolic acid synthesis by Mycobacterium aurum cell-free extracts

The first cell-free system capable of synthesizing whole mycolic acids: (R1CH(OH)CH(R2)COOH, with 60 to 90 carbon atoms) from [1-14C]acetate is described and preliminary investigations into some of its requirements and properties are reported. Biosynthetic activity for mycolic acids occurred in an insoluble fraction (40 000 X g pellet) from disrupted cells of Mycobacterium aurum (ATCC 23366-type strain); it produced mycolic acids, but a very small amount of non-hydroxylated fatty acids. The predominant product was unsaturated mycolic acid (type I), while oxo- (type IV) and dicarboxy- (type VI) mycolic acids were synthesized to a lesser extent. When [1-14C]palmitic acid was used as a marker, no labelled mycolic acid was detected. The reaction required a divalent cation (Mg2+ or Mn2+), KHCO3 and O2. Neither CoA, NADH, NADPH nor ATP were necessary, but CoA rather increased the synthesis of non-hydroxylated fatty acids. Glucose or trehalose were not required. Avidin inhibited the biosynthesis of the three types of mycolic acid indicating the presence of a biotin-requiring enzyme in the reaction sequence and therefore a carboxylation step, but citrate had no allosteric effect. Iodoacetamide inhibited the system. These first data are in favor of a complex multienzyme system.     

Molecular weight determination of methyl esters of mycolic acids using thermospray mass spectrometry.

Methyl esters of normal fatty acids, corynomycolate and corynomycolenate were used as model compounds for thermospray mass spectrometric procedures for molecular weight determination of the related nocardial mycolic acids. By using ammonium acetate at the positive ion generator, in both cases, a family of ions was produced. The following members were found and corresponded to the adducts: (1) M + H; M + NH4 and M + H + NH4 for methyl esters of normal fatty acids, whereas M + H, M + 2H and M + H + NH4 were the adducts most frequently observed with methyl corynomycolates. The methyl esters of C40-C48 mycolic acids from Rhodococcus rhodochrous exhibited prominent peaks corresponding to adducts M + H + NH4 whereas those corresponding to M + 2H showed slightly lower intensities. The structure M + H had no significant representatives with this subclass of mycolic acids. A similar pattern was observed with methyl esters of C50-C54 mycolic acids from Nocardia asteroides GUH-2. Ion peaks C50-C54 representing adducts M + 2H and M + H + NH4 prevailed in the mass spectrum. In this case, the intensities of peaks corresponding to M + 2H were slightly higher than those of the M + H + NH4. Essentially three main species of nocardomycolic acids were detected: (1) monounsaturated C50:1, C52:1 and C54:1; (2) diunsaturated C50:2, C52:2 and C54:2 and (3) triunsaturated C52:3 and C54:3 mycolic acids. The most abundant mycolic acid was C52:2 followed in decreasing abundance by C52:1, C54:2, C50:2, C52:3 and C54:3 mycolic acids.     

Fatty acid composition of platelet phospholipids and plasma lipids in obese Zucker rats

The purpose of this work was to see whether hyperlipaemia observed in genetically obese Zucker rats (fa/fa) was associated with differences in fatty-acid composition of plasma triacylglycerols, plasma phospholipids and of platelet phospholipids, in comparison with the control lean rats (Fa/-). Results showed that plasma triacylglycerols and phospholipids were increased in obese rats. In triacylglycerols, the amount of saturated and monounsaturated fatty acids was highly increased whereas the amount of the n-6 and n-3 polyunsaturated fatty acids was little modified. In plasma phospholipids, saturated and monounsaturated fatty acids were also increased, as were the n-3 fatty acids (except C 18:3 n-3); the n-6 fatty acids were little increased except C 20:3 n-6 which was markedly increased. These results concerning the amounts of fatty acids have their counterpart in their relative proportions of fatty acids. Data thus obtained suggest that conversion of linoleic acid (C 18:2 n-6) into arachidonic acid (C 20:4 n-6) was decreased in obese rats, particularly the delta 5 desaturation step. On the contrary, conversion of linolenic acid (C 18:3 n-3) into higher polyenes seemed increased. Thrombocytosis was not modified in the obese rat, but the volume of the platelets was increased. Platelet phospholipids exhibited the same modifications as plasma phospholipids but with different magnitude. Saturated and monounsaturated fatty acids were little augmented, n-3 fatty acids were more augmented (except C 18:3 n-3 acid which was unchanged); n-6 fatty acids were not modified except C 20:3 n-6 acid which was highly increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gaschromatography of Constitutive Fatty Acids in Mycobacterium leprae

A constitutive saturated and monounsaturated fatty acid pattern of Mycobacterium leprae, isolated from the liver of a nine-banded armadillo with experimental leprosy, was analyzed gaschromatographically and compared with that of cultured M. lepraemurium, M. avium, M. bovis, strain BCG and M. smegmatis. In comparing the fatty acid pattern thus obtained and the known structure of mycolic acids in these mycobacteria, an experiential rule that each species of mycobacteria has a relatively high content of normal (straight-chained) saturated fatty acid having two more carbons than those of the α-branch in this species' mycolic acids, coincided well for all mycobacteria tested. In particular, M. leprae was found to contain a relatively high content of behenic acid (n-C_22:0) and the carbon-number of the α-branch in this species' mycolic acids is 20 as we previously reported. These data suggested the possibility of simple detection of M. leprae by gaschromatography, and results sustaining this possibility were obtained.     

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-¹⁴C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2-¹⁴C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-¹⁴C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2-¹⁴C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols.     

Changes in molecular species composition of nocardomycolic acids in nocardia rubra by the growth temperature

Nocardomycolic acids from Nocardia rubra were fully separated and characterized by a combination of argentation thin-layer chromatography and gas chromatography — mass spectrometry (GCMS). The occurrence of 20 or more different molecular species of mycolic acids was demonstrated. GCMS analysis of each subclass of mycolic acids after separation on AgNO₃ thin-layer chromatography revealed that in general the major species consisted of the even-carbon mycolic acids ranging from C₃₈ to C₅₂. However, the most abundant species differed by the subclasses; C₄₄ being in saturated, C₄₆ in monoenoic and C₄₆ in dienoic mycolic acids, respectively. All these acids were shown to possess C₁₂ or C₁₄ alkyl branch at 2 position, while double bonds were located in longer straight chain alkyl unit.By using this method, distinctive changes in mycolic acid composition by growth temperature were observed. The ratios of saturated, monoenoic to dienoic mycolic acids in a mixture of certain carbon numbered mycolic acids varied greatly, according to the shift of growth temperature. The mass fragmentographic analysis, monitoring M-15 ions derived from the loss of methyl group from the molecular ions showed the lower temperature (15°C) grown cells contained more unsaturated (especially dienoic) mycolic acids, while the higher temperature (40°C) grown cells contained more saturated mycolic acids in both extractable and cell-wall bound lipids. These changes in mycolic acid composition occurred shortly after shifting up the growth temperature from 20°C to 43°C at a logarithmic stage of the bacterial growth.     

Chemotaxonomic differentiation of coryneform bacteria isolated from biofilters.

Coryneform bacteria that were isolated from biofilters which are used for waste gas treatment of animal-rendering plant emissions were differentiated and partially identified by using chemotaxonomic methods. On the basis of the results of a numerical analysis of whole-cell fatty acid profiles, 79 isolates were divided into two major groups; the members of the first group contained saturated and monounsaturated fatty acids, whereas the members of the second group were characterized by iso- and anteiso-branched fatty acids. Division into subclusters was based mainly on quantitative differences in fatty acid composition and was confirmed by the results obtained for additional chemical markers (e.g., respiratory quinones, mycolic acids, polar lipids, cell wall amino acids, and whole-cell sugar patterns). By combining the results obtained for chemotaxonomic analyses that were performed for strains containing saturated and monounsaturated fatty acids, we were able to identify the genus Corynebacterium (two Corynebacterium species were differentiated on the basis of the occurrence of tuberculostearic acid), the genus Gordona, and the genus Mycobacterium. Among the strains that produced iso-anteiso fatty acid patterns, one subgroup was affiliated with the "nicotianae" group of the genus Arthrobacter; however, some strains contained a new combination of chemical markers. Peptidoglycan type A4 alpha, L-Lys-Gly-L-Glu was combined with menaquinones MK-7 and MK-8, whereas peptidoglycan type A4 alpha, L-Lys-L-Glu occurred together with MK-8 and MK-9. The second subgroup was characterized by a new type B peptidoglycan and MK-11, as well as small amounts of MK-12. Differentiation that was based first on chemotaxonomy and second on physiology gave reliable results. Thus, coryneform strains with new characteristics were isolated from biofilters.     

Effect of frozen storage on fatty acid composition and changes in lipid content of Scomberomorus commersoni and Carcharhinus dussumieri

Investigated were the changes in fatty acid composition, oxidation and enzymatic deterioration of lipids in frozen (−30°C) fish fillets from the Persian Gulf. The narrow barred Spanish mackerel ( Scomberomorus commersoni ) and white cheek shark ( Carcharhinus dussumieri ) were tested with storage times of 0, 1, 2, 3, 4, 5 and 6 months at −18°C. Statistical results showed that the major fatty acids among the saturated and monounsaturated fatty acids of each fish species were palmitic (C16:0) and oleic (C18:1n-9) acids, respectively. Both linoleic acid (C18:2n-6) and arachidonic acid (AA) (C20:4n-6) were predominant in total n-6 polyunsaturated fatty acids in both mackerel and shark. The EPA (eicosapentaenoic acid; C20:5 n-3) and DHA (docosahexaenoic acid; C22:6 n-3) acids were the major fatty acids among total n-3 acids in both fishes. During frozen storage, the PUFA (40.1 and 23.94%), n-3 (48 and 42.83%), ω 3/ ω 6 (41.36 and 50%), PUFA/SFA (56 and 42.23%) and EPA + DHA/C16 (55.55 and 46.66%) contents decreased in S. commersoni and C. dussumieri , respectively. Also peroxide, thiobarbituric acid (TBA) and free fatty acid (FFA) values significantly increased (P < 0.01) with the time of storage.     

Preparative fractionation of triglyceride mixtures according to acyl carbon number, using hydroxyalkoxypropyl Sephadex

The present paper describes the use of hydroxyalkoxypropyl Sephadex in a liquid chromatography system. When the column is held at 40 degrees C, and when elution is made with a linear gradient of two solvents, an excellent separation of saturated triglycerides in the region C(9)-C(56) is obtained in 24 hr, even with sample loads as high as 0.5 g/cm(2) of column. Triglycerides containing unsaturated fatty acids are eluted more rapidly than their saturated homologs, one C-C double bond being equivalent to -1.42 fatty acid carbon atoms.     

Crystal structures of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids.

The primary ligands of human serum albumin (HSA), an abundant plasma protein, are non-esterified fatty acids. In vivo, the majority of fatty acids associated with the protein are unsaturated. We present here the first high-resolution crystal structures of HSA complexed with two important unsaturated fatty acids, the monounsaturated oleic acid (C18:1) and the polyunsaturated arachidonic acid (C20:4). Both compounds are observed to occupy the seven binding sites distributed across the protein that are also bound by medium and long-chain saturated fatty acids. Although C18:1 fatty acid binds each site on HSA in a conformation almost identical with that of the corresponding saturated compound (C18:0), the presence of multiple cis double bonds in C20:4 induces distinct binding configurations at some sites. The observed restriction on binding configurations plausibly accounts for differences in the pattern of binding affinities for the primary sites between polyunsaturated fatty acids and their saturated or monounsaturated counterparts.     

Comparison of long term lipogenic effects of two different medium-chain triglycerides (tri C8: O and tri C12 : O) in the growing rat.

During the growth (35 g-340 g), and as compared to results obtained with a lipid-free diet or a diet containing long-chain fatty acids, high levels of Tri C8 : O or Tri C12 : O did not change the quantitative aspects of proteinogenesis and lipogenesis balances. The incorporation of Tri C8 : O into the diet did not change the fatty acid composition of body lipid stores while the incorporation of Tri C12 : O induced a lipogenesis characterized by the disappearance of about 50% of the n-9 and n-7 unsaturated fatty acids, the emergence of an equivalent amount of saturated fatty acids in C12 and C14, and the decrease of hexadecanoic or palmitic acid concentration. Titers of saturated fatty acids with a melting point higher than 40 degrees C increased from 34% to 64%. Results suggested an efficient inhibition of fatty acid biosynthesis de novo by C12 : O, associated with an impossibility for microsomal enzymes to assume the elongation of a sufficient amount of C12 : O to maintain C16 : O concentration and to furnish an important amount of substrate (C18 : O) to delta-9-stearoyl coenzyme A desaturase for oleic acid synthesis. Introducing dodecanoic acid into the diet of growing animals appears to be the most efficient method for increasing the degree of saturation of body lipids without changing the concentrations of long-chain saturated fatty acids.     

Mass spectrometry as a tool for identifying group D2 corynebacteria by their fatty acid profiles

Corynebacterium group D2 (CGD2) are lipophilic antibiotic-multiresistant bacteria involved in some infections of immunocompromised patients. The fatty acid composition and structure of different strains was established by several mass spectrometric methods, particularly negative ion tandem mass spectrometry coupled with capillary gas chromatography. Non-hydroxylated fatty acid profiles of three strains of CGD2 (ATCC 43042, ATCC 43043, ATCC 43044) were almost identical and revealed the presence of several straight chain unsaturated fatty acids from the omega-9 series, with even carbon numbers ranging from 14 to 24. Branched saturated fatty acids were mainly anteiso-heptadecanoic acid and tuberculostearic acid. Surprisingly, a relatively large quantity of 10-methylene octadecanoic acid was found. The non-hydroxylated fatty acid profile of one rare beta-lactam susceptible strain (SC1) was different; 10-methylene octadecanoic acid was lacking whereas tuberculostearic acid was much more abundant. In contrast, the four CGD2 strains displayed highly similar mycolic acid patterns. The major mycolic acid species corresponded to C32, C30 and C28 bis-unsaturated with a double bond on each branch at the omega-9 position. The comparison of the mycolic acid composition and structure with those of other medically important corynebacteria strains, revealed a characteristic pattern for CGD2 strains, and CGD2 strains were easily distinguished from Corynebacterium jeikeium (CIP 82.51).