Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo beta-oxidation but are first subjected to fatty acid alpha-oxidation in which the terminal carboxyl-group is released as CO(2). The mechanism of alpha-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid alpha-oxidation, and the complete alpha-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid alpha-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo β-oxidation but are first subjected to fatty acid α-oxidation in which the terminal carboxyl-group is released as CO₂. The mechanism of α-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid α-oxidation, and the complete α-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid α-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

The Kinetics of Fatty Acid Uptake By Paramecium Tetraurelia

ABSTRACT. The kinetics of radiolabeled fatty acid uptake by the ciliate Paramecium tetraurelia was examined on a homologous series of saturated, straight chain fatty acids of even carbon numbers. Uptake rates increased with chain length from acetate to palmitate. Saturation kinetics was demonstrated for most fatty acids examined, thus ruling out simple diffusion as the major mechanism for fatty acid transport and implicating carrier-mediated, facilitated transport as the major mechanism. Data from most competitive inhibition experiments were too scattered to determine the number of transporter systems present. Cholesterol uptake also exhibited saturation kinetics and hence other sterols, which can satisfy this nutritional requirement, may also be transported by a carrier-mediated mechanism. the uptake of the essential fatty acid oleate was faster than those observed for the saturated acids and could not be explained by only one transport mechanism. Therefore, fatty acid transport also occurs via other kinetically significant routes.     

Nutritional Alteration of the Fatty Acid Composition of a Thermophilic Bacillus Species

The fatty acid composition of a thermophilic Bacillus sp. was altered by the addition of isobutyrate, isovalerate, alpha-methylbutyrate, leucine, and isoleucine to the growth medium. With isobutyrate, 81% of the fatty acids had 16 carbon atoms and 79% were iso-fatty acids with an even number of carbon atoms. With leucine, 58% of the fatty acids had 15 carbon atoms and 86% were iso-fatty acids with an odd number of carbon atoms. With isoleucine, 72% of the fatty acids had 17 carbon atoms and 88% were anteiso-fatty acids with an odd number of carbon atoms. Thus, by altering the composition of the growth medium, cells were produced in which the majority of the fatty acids had either 15, 16, or 17 carbons and belonged to each of the three groups of branched-chain fatty acids. The wide variation observed in the fatty acid composition makes it unlikely that any specific branched-chain fatty acid is required for vital functions.     

Anaerobic transformation of alkanes to fatty acids by a sulfate-reducing bacterium,strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and (13)C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and (13)C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-(13)C(2)]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [(13)C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a (13)C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-(13)C(2)]hexadecane-derived fatty acids contained either two (13)C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Anaerobic Transformation of Alkanes to Fatty Acids by a Sulfate-Reducing Bacterium, Strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and ¹³C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and ¹³C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-¹³C₂]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [¹³C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a ¹³C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-¹³C₂]hexadecane-derived fatty acids contained either two ¹³C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Noninvasive analyses of polyunsaturated fatty acids in human oral mucosa in vivo by Fourier-transform infrared spectroscopy

The aim of this study was to develop a noninvasive method to observe polyunsaturated fatty acids (PUFAs) behavior in the human body using Fourier transform infrared spectroscopy. For the noninvasive measurement of human oral mucosa, we have used infrared spectroscopy with a suitable attachment for an in vivo attenuated total reflectance system. The fatty acid contents in the tissues were determined by gas-chromatography mass-spectrometry after methylation. The alkene C-H stretching vibrations of unsaturated fatty acids in dietary oils showed infrared absorption bands with various peak positions and intensities at around 3010 cm(-1) depending on the extent of unsaturation and their species. The diurnal fluctuation of the alkene peak position of oral mucosa suggested that the contents of PUFAs were increased gradually in the early afternoon, and these data were supported by the direct determination of fatty acid species in oral mucosa where the relative increase of arachidonic and docosahexaenoic acids was observed in the early afternoon. This diurnal change of alkene peak position resembled the pattern of a "lipid factor" change calculated with the factor analysis applied to the overall infrared spectrum. We could monitor the diurnal fluctuations of PUFA contents of human oral mucosa noninvasively using a reagent-free infrared analysis system. The measurement of alkene and methylene infrared bands may provide a useful tool for detecting changes in PUFA balance in the human body.     

Quadrupole ion-trap mass spectrometry to locate fatty acids on lipid A from Gram-negative bacteria

The structure of lipid A released by mild acid hydrolysis from lipopolysaccharide from two strains of Shigella flexneri with different degrees of acylation was characterized using electrospray ionization (ESI) and ion-trap mass spectrometry. The lipid A was analyzed underivatized with ESI in negative-ion mode. With multiple stages of fragmentation (MS(n)), both the degree of acylation and the positions of the fatty acids on the disaccharide backbone could be determined. It was possible to determine the degree of acylation by the MS(n) technique, where in each MS stage the parent ion was an ion where one fatty acid had been eliminated. One way to determine the location of the fatty acids was by identifying cross-ring fragments of the reducing sugar from parent ions containing different numbers of fatty acids. Another was by identifying a possible charge-driven release of fatty acids situated close to a phosphate group. The fatty acids were otherwise eliminated by a charge-remote fragmentation mechanism. The combined data show the usefulness of ion-trap mass spectrometers for this type of analysis.     

Solubility of saturated fatty acids in water at elevated temperatures

The solubility in water of saturated fatty acids with even carbon numbers from 8 to 18 was measured in the temperature range of 60 to 230 degrees C and at a pressure of 5 or 15 MPa. The pressure had no significant effect on the solubility. The solubility of the fatty acids increased with increasing temperature. At temperatures higher than about 160 degrees C, the logarithm of the solubility in mole fraction was linearly related to the reciprocal of the absolute temperature for each fatty acid, indicating that the water containing solubilized fatty acid molecules formed a regular solution at the higher temperatures. The enthalpy of a solution of the fatty acids in water, which was evaluated from the linear relationship at the given temperatures, increased linearly with the carbon number of the fatty acid.     

Solubility of Saturated Fatty Acids in Water at Elevated Temperatures

The solubility in water of saturated fatty acids with even carbon numbers from 8 to 18 was measured in the temperature range of 60 to 230°C and at a pressure of 5 or 15 MPa. The pressure had no significant effect on the solubility. The solubility of the fatty acids increased with increasing temperature. At temperatures higher than about 160°C, the logarithm of the solubility in mole fraction was linearly related to the reciprocal of the absolute temperature for each fatty acid, indicating that the water containing solubilized fatty acid molecules formed a regular solution at the higher temperatures. The enthalpy of a solution of the fatty acids in water, which was evaluated from the linear relationship at the given temperatures, increased linearly with the carbon number of the fatty acid.     

Synthesis in vitro of very long chain fatty acids in Vibrio sp. strain ABE-1

The activity of fatty acid synthetase (FAS) from Vibrio sp. strain ABE-1 required the presence of acyl carrier protein and was completely inhibited by thiolactomycin, an inhibitor specific for a type II FAS. These observations indicate that this enzyme is a type II FAS. Analysis by gas-liquid chromotography of the reaction products synthesized in vitro from [2-¹⁴C]malonyl-CoA by the partially purified FAS revealed, in addition to 16-and 18-carbon fatty acids which are normal constituents of this bacterium, the presence of fatty acids with very long chains. These fatty acids were identified as saturated and mono-unsaturated fatty acids with 20 up to as many as 30 carbon atoms. The longest fatty acids normally found in this bacterium contain 18-carbon atoms. These results suggest that the FAS from Vibrio sp. strain ABE-1 has potentially the ability to synthesize fatty acids with very long chains.Abbreviations ACP acyl carrier protein - FAME fatty acid methyl ester - FAS fatty acid synthetase - FID flame ionization detection - GLC gas-liquid chromatography - TLC thin-layer chromatography - In designations of fatty acids, such as 16:0, 16:1, etc the colon separates the number that denotes the number of carbon atoms and the number that denotes the number of double bonds, respectively, in the molecule - 16:0-CoA CoA ester of 16:0     

Microbial Assimilation of Hydrocarbons I. Fatty Acids Derived from Normal Alkanes

Fatty acids derived from Micrococcus cerificans growing at the expense of odd- and even-carbon normal alkanes were studied. Results demonstrated that cultures grown with a variety of nonhydrocarbon substrates serving as sole carbon and energy source yielded only even-carbon fatty acids. Even-chain alkanes, dodecane through octadecane serving as sole carbon source, resulted in even-carbon fatty acids with direct correlation between carbon number of the major fatty acid species and carbon number of the alkane substrate. Odd-carbon alkanes, undecane through heptadecane serving as sole carbon source, yielded both odd- and even-carbon fatty acids. A transitional shift from even-carbon fatty acids to odd-carbon fatty acids was observed as the carbon number of the alkane substrate increased. Unsaturated fatty acids were found to comprise a significant percentage of all profiles. Analysis of unsaturated fatty acids showed all odd- and even-carbon acids analyzed were Delta(9) monounsaturated fatty acids.     

Utilization of C20-polyunsaturated fatty acids by a yeast fatty acid desaturase mutant

A study was made of the utilization of C₂₀-polyunsaturated fatty acids by the $\text{S. cerevisiae}$ fatty acid desaturase mutant $\text{olel-1}$, Arachidonic acid, 8,11,14-eicosatrienoic acid, and 5,8,11,14,17-eicosapentaenoic acid were about equally effective in supporting growth with lactate as the carbon source. The relative proportion of these fatty acids in total cell fatty acids was $\text{ca}$. 50%. 5,8,11-eicosatrienoic acid synthesized from oleate was less effective. Very little growth occurred with 11,14,17-eicosatrienoic acid or with 11,14-eicosadienoic acid. These results indicate the usefulness of the yeast mutant as a eucaryotic model for study of membrane systems enriched in specific C₂₀-polyunsaturated fatty acids.     

Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue

The principal supply of carbon precursors for fatty acid synthesis in leaf tissue has been a much debated topic, with some experiments suggesting a direct supply from the C3 products of photosynthetic carbon fixation and colleagues suggesting the utilization of free acetate (for which concentrations in leaves in the range of 0.05-1.4 mM have been reported). To address this issue we first reassessed the in vivo rate of fatty acid synthesis using a new method, that of [13C]carbon dioxide labeling of intact Arabidopsis plants with the subsequent analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS). This method gave an average value of 2.3 mmoles carbon atoms h-1 mg chlorophyll-1 for photosynthetic tissues. The method was extended by isotopic dilution analysis to measure the rate of fatty acid synthesis in the dark. There was negligible fatty acid synthesis (< 5% of the rate in the light) in the dark. In addition, the method allowed an estimate of the absolute rate of fatty acid degradation of about 4% of the total fatty acid content per day. With the in vivo rate of fatty acid synthesis in the light defined, if the bulk tissue acetate concentration available for fatty acid synthesis is 1 mM, this acetate pool can sustain fatty acid synthesis for approximately 60 min. When the leaves of Arabidopsis, barley and pea were given a 5 min pulse of [14C]carbon dioxide, the label rapidly appeared in fatty acids with a lag phase of less than 2-3 min. Continuous labeling with [14C]carbon dioxide, for up to 1 h, showed a similar result. Furthermore, 14C-label in free acetate was less than 5% of that in fatty acids. In conclusion, these data suggest that either the bulk pool of acetate is not involved in fatty acid synthesis or the concentration of acetate must be less than 0.05 mM under strong illumination.     

Anaerobic 1-Alkene Metabolism by the Alkane- and Alkene-Degrading Sulfate Reducer Desulfatibacillum aliphaticivorans Strain CV2803T

The alkane- and alkene-degrading, marine sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803^T, known to oxidize n-alkanes anaerobically by fumarate addition at C-2, was investigated for its 1-alkene metabolism. The total cellular fatty acids of this strain were predominantly C-(even number) (C-even) when it was grown on C-even 1-alkenes and predominantly C-(odd number) (C-odd) when it was grown on C-odd 1-alkenes. Detailed analyses of those fatty acids by gas chromatography-mass spectrometry after 6- to 10-week incubations allowed the identification of saturated 2- and 4-ethyl-, 2- and 4-methyl-, and monounsaturated 4-methyl-branched fatty acids with chain lengths that correlated with those of the 1-alkene. The growth of D. aliphaticivorans on (per)deuterated 1-alkenes provided direct evidence of the anaerobic transformation of these alkenes into the corresponding 1-alcohols and into linear as well as 10- and 4-methyl-branched fatty acids. Experiments performed with [¹³C]bicarbonate indicated that the initial activation of 1-alkene by the addition of inorganic carbon does not occur. These results demonstrate that D. aliphaticivorans metabolizes 1-alkene by the oxidation of the double bond at C-1 and by the subterminal addition of organic carbon at both ends of the molecule [C-2 and C-(ω-1)]. The detection of ethyl-branched fatty acids from unlabeled 1-alkenes further suggests that carbon addition also occurs at C-3. Alkylsuccinates were not observed as potential initial intermediates in alkene metabolism. Based on our observations, the first pathways for anaerobic 1-alkene metabolism in an anaerobic bacterium are proposed. Those pathways indicate that diverse initial reactions of 1-alkene activation can occur simultaneously in the same strain of sulfate-reducing bacterium.     

A role for hormone-sensitive lipase in the selective mobilization of adipose tissue fatty acids.

The mobilization of fatty acids from rat and human fat cells is selective according to molecular structure, and notably carbon atom chain length. This study aimed at examining whether the release of individual fatty acids from triacylglycerols (TAG) by hormone-sensitive lipase (HSL) plays a role in the selectivity of fatty acid mobilization. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 18 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation degree from 0 to 3 double bond(s), was measured by comparing the composition of non-esterified fatty acids (NEFA) to that of the original TAG. The relative hydrolysis (% in NEFA/% in TAG) differed between fatty acids, being about 5-fold and 3-fold higher for the most (18:1n-7) than for the least (24:0) readily released fatty acid by recombinant rat and human HSL, respectively. Relationships were found between the chain length of fatty acids and their relative hydrolysis. Among 12-24 carbon atom saturated fatty acids, the relative hydrolysis markedly decreased (by about 5- and 3-times for recombinant rat and human HSL, respectively) with increasing chain length. We conclude that fatty acids are selectively released from TAG by HSL according to carbon atom chain length. These data provide insight on the mechanism by which fatty acids are selectively mobilized from fat cells.     

The sources of carbon and reducing power for fatty acid synthesis in the heterotrophic plastids of developing sunflower (Helianthus annuus L.) embryos

The provision of carbon substrates and reducing power for fatty acid synthesis in the heterotrophic plastids of developing embryos of sunflower (Helianthus annuus L.) has been investigated. Profiles of oil and storage protein accumulation were determined and embryos at 17 and 24 days after anthesis (DAA) were selected to represent early and late periods of oil accumulation. Plastids isolated from either 17 or 24 DAA embryos did not incorporate label from [1-(14)C]glucose 6-phosphate (Glc6P) into fatty acids. Malate, when supplied alone, supported the highest rates of fatty acid synthesis by the isolated plastids at both stages. Pyruvate supported rates of fatty acid synthesis at 17 DAA that were comparable to those supported by malate, but only when incubations also included Glc6P. The stimulatory effect of Glc6P on pyruvate utilization at 17 DAA was related to the rapid utilization of Glc6P through the oxidative pentose phosphate pathway (OPPP) at this stage. Addition of pyruvate to incubations containing [1-(14)C]Glc6P increased OPPP activity (measured as (14)CO(2) release), while the addition of malate suppressed it. Observations of the interactions between the rate of metabolite utilization for fatty acid synthesis and the rate of the OPPP are consistent with regulation of the OPPP by redox control of the plastidial glucose 6-phosphate dehydrogenase activity through the demand for NADPH. During pyruvate utilization for fatty acid synthesis, flux through the OPPP increases as NADPH is consumed, whereas during malate utilization, in which NADPH is produced by NADP-malic enzyme, flux through the OPPP is decreased.     

Fatty acid induced hemolysis. Protective action of ceruloplasmin, albumins, thiols and vitamin C

The hemolytic effect of saturated fatty acids increased rapidly, when the number of carbon atoms in the chain exceeded 12. At low fatty acid concentrations (less than 60 microM) the hemolytic effect decreased with increasing number of double bonds in the carbon chain (cis-form fatty acids). A more complex pattern was observed at higher fatty acid concentrations. Trans-unsaturated fatty acids were more hemolytic than cis-analogs. Ceruloplasmin, a serum protein with no fatty acid binding capacity, reduced the hemolytic effect of fatty acids; possibly by interacting with the cell membrane. Reducing compounds (thiols, vitamin C) also protected against fatty acid induced hemolysis.     

Stability of the fatty acid composition of actinomycetes

The stability of fatty acid composition of total extractable lipids was studied in Streptomyces cultures. The type of fatty acid composition typical of the Streptomyces genus remains stable when the actinomycetes were grown as submerged cultures in various synthetic media: saturated fatty acids with methyl branching in the chain predominated in all of the cases, and fatty acids with an uneven number of carbon atoms in the chain prevailed in most of the cases. Fatty acids with the anteiso structure predominated among the acids with a branched chain, amounting to more than a half of the latter and reaching sometimes 50% of the total fatty acid content. Methyl branchings were located in the anteiso position in fatty acids with an uneven number of carbon atoms, and in the iso position in fatty acids with an even number of carbons. Unsaturated fatty acids were found as a minor component.