Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very-long-chain fatty acids in mammalian testes and spermatozoa.

Polyenoic very-long-chain fatty acids (VLCFA) have been shown to be localized in unusual molecular species of sphingomyelin in the testes and spermatozoa of the ram, bull, rat, and boar and in the spermatozoa of man. The composition of polyenoic VLCFA-sphingomyelin was comparable in the testes and spermatozoa of each mammalian species; however, the sphingolipid was more concentrated in spermatozoa. The composition of testicular and spermatozoan polyenoic VLCFA-sphingomyelin differed considerably between animal types. Human spermatozoa mainly contained n-6 polyenoic VLCFA with two to four double bonds and even-carbon chain lengths up to 32. In ram and bull testes and spermatozoa, n-3 and n-6, tetra-, penta-, and hexaenoic VLCFA with even-carbon chain lengths up to 34 predominated. In rat and boar testes and spermatozoa, the polyenoic VLCFA were mainly n-6 derivatives with three to five double bonds and even- and odd-carbon chain lengths up to 34. The testes and spermatozoa of the latter two animal species contained 2-hydroxylated, in addition to non-hydroxylated, polyenoic VLCFA in sphingomyelin. This is the first time that 2-hydroxylated polyenoic VLCFA have been recognized in biological systems. Non-hydroxylated polyenoic VLCFA were initially observed in the sphingomyelin of rat testes 25 days after birth, followed by 2-hydroxylated derivatives at 30 days. The total amount of polyenoic VLCFA associated with rat testicular sphingomyelin increased dramatically from 25 to 40 days of postnatal life and then remained constant to 60 days (sexual maturity). The ratio of 2-hydroxylated to non-hydroxylated polyenoic VLCFA increased during this period. Polyenoic VLCFA-sphingomyelin seems to occur exclusively in the testes and spermatozoa of mammals, and it is postulated that this lipid plays a role in reproduction.     

Detection of a homologous series of C26-C38 polyenoic fatty acids in the brain of patients without peroxisomes (Zellweger''s syndrome).

The brains of patients with inherited abnormalities in peroxisomal structure and function contain greatly increased proportions of a homologous series of unique polyenoic fatty acids with carbon chain lengths ranging from 26 to 38. Based on evidence by chemical ionization and electron impact mass spectrometry before and after catalytic hydrogenation, and argentation t.l.c., these lipids have been tentatively identified as 26:5, 28:5, 30:5, 30:6, 30:7, 32:5, 32:6, 32:7, 34:5 and 34:6 fatty acids. A further two fatty acids eluting at very high temperatures from gas chromatography columns have been tentatively identified on the basis of their chemical ionization mass spectra as 36:6 and 38:6 fatty acids.     

Structure and lipid distribution of polyenoic very-long-chain fatty acids in the brain of peroxisome-deficient patients (Zellweger syndrome).

The polyenoic fatty acids with carbon chain lengths from 26 to 38 (very-long-chain fatty acids, VLCFA) previously detected in abnormal amounts in Zellweger syndrome brain have been shown to be n-6 derivatives and therefore probably derived by chain elongation of shorter-chain n-6 fatty acids such as linoleic acid and arachidonic acid. Polyenoic VLCFA are also present in Zellweger syndrome liver, but this tissue differs significantly from brain in that the saturated and mono-unsaturated derivatives are the major VLCFA. Zellweger syndrome brain polyenoic VLCFA are present in the neutral lipids predominantly in cholesterol esters, with smaller amounts in the non-esterified fatty acid and triacylglycerol fractions. These fatty acids are barely detectable in any of the major phospholipids, but are present in significant amounts in an unidentified minor phospholipid. The polyenoic VLCFA composition of this lipid differs markedly from that observed for all other lipids, as it contains high proportions of pentaenoic and hexaenoic fatty acids with 34, 36 and 38 carbon atoms. A polar lipid with the chromatographic properties in normal brain contains similar fatty acids. It is postulated that the polyenoic VLCFA may play an important role in normal brain and accumulate in Zellweger syndrome brain because of a deficiency in the peroxisomal beta-oxidation pathway, although a possible peroxisomal role in the control of carbon-chain elongation cannot be discounted.     

The occurrence of polyenoic very long chain fatty acids with greater than 32 carbon atoms in molecular species of phosphatidylcholine in normal and peroxisome-deficient (Zellweger''s syndrome) brain.

The n-6 tetra- and pentaenoic fatty acids with carbon chain lengths greater than 32 found in normal brain are located predominantly in a separable species of phosphatidylcholine. A similar phospholipid is found in increased amounts in the brain of peroxisome-deficient (Zellweger's syndrome) patients, but the fatty acid composition differs in that penta- and hexaenoic derivatives predominate. Our data strongly suggest that the polyenoic very long chain fatty acids are confined to the sn-1 position of the glycerol moiety, while the sn-2 position is enriched in saturated, monounsaturated and polyunsaturated fatty acids with less than 24 carbon atoms. It is postulated that these unusual molecular species of phosphatidylcholine may play some, as yet undefined, role in brain physiology.     

Occurrence of unusual molecular species of sphingomyelin containing 28-34-carbon polyenoic fatty acids in ram spermatozoa.

The high levels of very long chain fatty acids found in ram spermatozoa are located almost exclusively in one of two separable species of sphingomyelin. Mass spectral analysis, including fast atom bombardment of the purified sphingomyelin, has shown the fatty acids to have a carbon chain length of between 28 and 34, with between four and six double bonds, and to belong predominantly to the n-3 series.     

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ∼ 21–22°C), the rest remaining in the liquid state in the 5°C–45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.     

Oxidation of very-long-chain fatty acids in rat brain: cerotic acid is beta-oxidized exclusively in rat brain peroxisomes

We studied the effect of sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent inhibitor of mitochondrial carnitine palmitoyltransferase I, on fatty acid oxidation by rat brain cells. In cultured glial cells as well as in dissociated brain cells from adult rats palmitic acid (16:0) oxidation was inhibited by about 85% of control values when 25 microM POCA was added to the medium, whereas no inhibition of cerotic acid (26:0) oxidation was observed. Furthermore, omission of carnitine from the culture medium resulted in a 57.7% decrease in palmitic acid oxidation in cultured glial cells, whereas cerotic acid oxidation was not influenced. These results indicate that rat brain peroxisomes contribute only little (about 15%) to palmitic acid oxidation and provide conclusive evidence that cerotic acid is oxidized exclusively in rat brain peroxisomes.     

Occurrence of novel nonmethylene-interrupted C24 polyenoic fatty acids in female gonad lipids of the limpet Cellana grata

The occurrence of positional isomers of minor C(24) unsaturated fatty acids in female gonad lipids of the limpet Cellana grata was clarified by gas chromatography-mass spectrometry of the combination of their 4,4-dimethyloxazoline and picolinyl ester derivatives. In this study, in addition to 5,9-24:2, 9,15-24:2, 5,9,15-24:3, and 5,9,17-24:3, previously identified, 24:4n-6, 24:5n-3, and four novel nonmethylene-interrupted fatty acids, 9,17-24:2, 9,15,18-24:3, 5,9,15,18-24:4, and 5,9,15,18,21-24:5, were newly recognized. All C(24) unsaturated fatty acids detected were present only in triacylglycerols.     

Very long chain (C24 to C36) polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated phosphatidylcholines from bovine retina

A complete series of even-carbon chain polyenoic fatty acids having 20-36 carbons occur in dipolyunsaturated molecular species of phosphatidylcholine from bovine retina. Using oxidative ozonolysis, it is shown that very long chain tetraenes belong to the n-6 series, hexaenes to the n-3 series, and major pentaenes to the n-3 series of fatty acids (very long chain n-6 pentaenes also occur). Molecular ions are obtained by electron impact mass spectrometry of methyl ester derivatives which conclusively identify the major components of this novel group of fatty acids. Mass spectral patterns are similar for the major very long chain tetraenes, for the pentaenes, and for the hexaenes, but different for each group of unsaturation. Very long chain (C24 to C36) polyenes account for about half the weight (40 mol %) of the acyl chains of major dodecaenoic, undecaenoic, and decaenoic molecular species of bovine retina phosphatidylcholine, the other half being made up by docosahexaenoate (22:6 n-3).     

Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes

About one-fourth the phosphatidylcholines (PCs) from bovine disk photoreceptor membranes contain very long chain (24-36 carbons) polyunsaturated (4, 5, and 6 double bonds) fatty acids of the n-3 and n-6 series (VLCPUFA). Such fatty acids, exclusively occurring in dipolyunsaturated species, are esterified to the sn-1 position of their glycerol backbone, docosahexaenoate being the major fatty acid at sn-2. Chromatographically, such PCs display a weakly polar character relative to other species, ascribable to their exceedingly large number of carbons. After hexane extraction of lyophilized disks, PC is the major component of the fraction of lipids that remains associated with rhodopsin, followed by phosphatidylserine, while a large proportion of the phosphatidylethanolamine is removed. The fatty acid composition of the hexane-removable and protein-bound lipid fractions markedly differs, the latter being enriched in lipid species containing long-chain and very long chain polyenes. This is observed for all lipid classes except free fatty acids. VLCPUFA-containing PCs are the most highly concentrated species in the rhodopsin-associated lipid fraction. The very long chain polyenes these PCs have at sn-1 may account for their resistance to being separated from the protein. It is hypothesized that their unusually long polyenoic fatty acids could be well suited to partially surround alpha-helical segments of rhodopsin.     

Polyunsaturated fatty acids (PUFA) regulate neurotransmitter contents in rat brain

The effects of feeding of 6-propylthiouracil (6-PTU) and polyunsaturated fatty acids (PUFA) independently and in combination and administration (ip) of a single dose of triiodothyronine (T3) (2.5 microg/100 g body wt) along with feeding of 6-PTU and PUFA were studied in rat brain. Dopamine (DA), 5-hydroxytryptophan (5-HTP), serotonin (5-HT), 5-hydroxy indole acetic acid (5-HIAA), norepinephrine (NE) and epinephrine (EPI) contents were assayed in the hypothalamus and cerebral cortex regions. It was found that 6-PTU feeding resulted in decrease in dopamine, 5-HT, 5-HTP and 5-HIAA in both regions. In animals fed with PUFA followed by administration of T3, the DA level was found normal.     

Shape changes in human erythrocytes induced by replacement of the native phosphatidylcholine with species containing various fatty acids

Phosphatidylcholine-specific transfer protein from beef liver has been used to replace native phosphatidylcholine (PC) molecules from intact human erythrocytes by a variety of PC species differing in fatty acid composition. These replacements changed neither the total phospholipid content of the membrane, nor the composition of this fraction in terms of the various phospholipid classes. The morphology of the erythrocyte was not modified when native PC was replaced by 1-palmitoyl,2-oleoyl PC, 1-palmitoyl,2-linoleoyl PC, egg PC, or PC isolated from rat liver microsomes. Replacement with the disaturated species 1,2-dimyristoyl PC, 1,2-dipalmitoyl PC, and 1,2-distearoyl PC resulted in the formation of echinocytes and, at higher levels of replacement, in spheroechinocytes. Echinocyte-like erythrocytes were also observed after replacement with 1-palmitoyl,2-arachidonoyl PC, whereas stomatocytes were formed upon replacement with PC species containing two unsaturated fatty acids, e.g., 1,2-dioleoyl PC and 1,2-dilinoleoyl PC. The observations show that the erythrocyte membrane structure and the overall discoid cell shape of the human erythrocyte are optimally stabilized by PC species that contain one saturated and one mono- or diunsaturated fatty acid, and that the cell tolerates only limited variations in the species composition of its PC.     

Dehydration of 3-hydroxyacyl-CoA in brain very-long-chain fatty acid synthesis.

Rat brain microsomes actively dehydrate 3-hydroxyacyl-CoAs. Using chemically synthesized [1-(14)C] (R,S) 3-hydroxyeicosanoyl-CoA, we investigated the biochemical characteristics of the dehydration and reduction steps of stearoyl-CoA elongation. The reaction products, separated and identified as trans2,3-enoyl-CoAs and, in the presence of NADPH, as saturated acyl-CoAs, were released from the enzyme as thioesters which were partly hydrolysed. A kinetic analysis of the two coupled reactions showed that the 3-hydroxyacyl-CoA dehydrase catalysed a reversible reaction with kinetic constants of about 0.045 min(-1) for forward reaction (dehydration) and 0.025 min(-1) for reverse reaction (hydration); Vmax of the dehydration reached 20 nmoles/min/mg and the apparent Km was 44 microM. In the presence of NADPH, the kinetic constants for the dehydrase were unchanged and that for the trans2,3-enoyl-CoA reductase was 0.025 min(-1). The relative proportion of trans2,3-enoyl-CoA and saturated acyl-CoA depended on the protein amount. An inhibition of the reduction step was observed for substrate concentrations above 15 microM. The 3-hydroxyacyl-CoA dehydrase used (R) rather than (S) 3-hydroxyacyl-CoA. Furthermore, the elongation of (R) 3-hydroxyeicosanoyl-CoA yielded saturated very-long-chain acyl-CoA. These results demonstrated that 3-hydroxyacyl-CoAs entered the elongating complex exclusively at the level of the dehydrase and not of the condensing enzyme.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.