Preferential oxidation of linolenic acid compared to linoleic acid in the liver of catfish (Heteropneustes fossilis andClarias batrachus)

The fate of [1-¹⁴C] linoleic acid and [1-¹⁴C] linolenic acid in the liver slices and also in the liver tissues of live carnivorous catfish,Heteropneustes fossilis andClarias batrachus, was studied. Incorporation of the fatty acids into different lipid classes in the live fish differed greatly from the tissue slices, indicating certain physiological control operative in vivo. The extent of desaturation and chain elongation of linoleic and linolenic acids into long-chain polyunsaturated fatty acids was low. Linolenic acid was oxidized (thus labeling the saturated fatty acid with liberated¹⁴C-acetyl-CoA) in preference to linoleic acid, and this oxidation also seemed to be under physiological control since both of the fatty acids were poorly oxidized in the tissue slices and in the killed fish. These fish can therefore recognize the difference in the acyl chain structures of linoleate and linolenate. The higher oxidation of liolenic acid and poor capacity for its conversion to longer chain, highly unsaturated derivatives indicates a higher demand for the dietary supply of these essential fatty acids in these two species.     

The influence oftrans-acids on desaturation and elongation of fatty acids in developing brain

trans-Monounsaturated acids account for up to 3% of the total octadecenoic acyl chains of human brain lipids. To investigate the influence oftrans-acids on desaturation and chain elongation of fatty acids, in vitro and in vivo experiments with rat brain were performed. In the in vitro assays of Δ9 desaturation, Δ6 desaturation and chain elongation,trans,trans-dienoic acid was inhibitory, particularly to chain elongation. Slight differences between the inhibitory effects oftrans-monoenoic acids and theircis-isomers were observed. In an in vivo model, unlabeled fatty acid (stearate, oleate, elaidate, linoleate, linoelaidate, arachidonate, ortrans-monoene from margarine) was injected simultaneously with [1-¹⁴C] linoleic acid into the brains of suckling rats. Linoelaidate and oleate inhibited desaturation and elongation of linoleate, whereas elaidate, stearate andtrans-monoene from margarine were stimulatory. While the demonstration of differences betweencis andtrans monoenic isomers required relatively high levels of the test acids, it appears thattrans-acids can influence desaturation and elongation enzymes that lead to acyl chain modification in the central nervous system. Portions of this study were presented at the meeting of the American Oil Chemists' Society and the International Society for Fat Research in New York, NY, April 1980.     

Fatty acid biosynthesis in the developing endosperm ofCocos nucifera

Endosperm tissue of developing coconut endosperm incorporated [¹⁴C] acetate and [¹⁴C]-malonate into [¹⁴C]C₈-C₁₈ fatty acids. The distribution of [¹⁴C] label into the various fatty acids mimicked the distribution of endogenous fatty acids at early and middle stages of endosperm development. Although [¹⁴C] C₈-C₁₈ fatty acids were taken up rapidly by endosperm tissue slices, no elongation occurred; [¹⁴C] stearic acid was not desaturated to oleic. Cell free preparations have also been obtained from this tissue that readily incorporated [¹⁴C] malonyl-CoA into a range of [¹⁴C] fatty acids in the presence of ACP and NADH at pH 7.0. Employing this system, a number of experiments were designed to determine the mechanism of chain length termination. In contrast to intact tissue slice experiments, cell-free extracts yielded as principal products palmitic and stearic acid, although up to 20% were shorter chain acids. A number of possible mechanisms for chain length termination were proposed and tested. Supported in part by NSF Grant No. PCM76 01495.     

The monounsaturated acyl- and alkyl-moieties of wax esters and their distribution in commercial orange roughy (Hoplostethus atlanticus) oil

Wax esters were isolated from commercial orange roughy (Hoplostethus atlanticus) oil by column chromatography and fractionated by argentation thin layer chromatography. Following transesterification, the resultant fatty acid methyl esters and fatty alcohols were analyzed by gas chromatography. both acyl- and alkyl-moieties were mainly of the monoene structure within the 16∶1–22∶1 range. After derivatization, the positions of the double bonds of even numbered fatty acid and fatty alcohol isomers were located by chromatography-mass spectrometry and compared. Results of these positional analyses indicate that the primary desaturation reactions takes place in the Δ9 position of pre-existing (C₁₄ to C₂₄) acyl chains. It is proposed that acyl components from 18∶1 are subjected to chain elongation to form a mixture of 24∶1 isomers as the final product. Apart from the 24∶1 acyl moiety of the wax esters, in which the double bond was almost exclusively in the Δ15 position, de novo biosynthetic reactions on acids and alcohols appear to yield related acyl- and alkyl-moieties of resynthesized wax esters.     

p-Chlorophenoxyisobutyrate enhanced retention of homologous lipoproteins by human aortic smooth muscle cells

Human aortic smooth muscle cells (SMC) specifically bind and take up indiscriminately both the lipid and protein moietics of homologous²⁵I-very low density lipoproteins (VLDL) and¹²⁵I-low density lipoproteins LDL). Sixty-five to 80% of absorbed lipids are incorporated into the cell lipids, preferentially into the phospholipid fraction. Twenty to 35% of the lipid bound and the protein moiety are eliminated from the cells. Half of the eliminated protein label is recovered as TCA soluble products. Five mM of p-chlorophenoxyisobutyrate (CPIB) raise the level of intracellular radioactivity derived from the lipid moieties of VLDL and LDL by about 40% via a reduced elimination. The processing of the protein moiety and lipoprotein binding to the cell surface are not affected by 5.0 mM of CPIB. CPIB lowers the incorporation of¹⁴C-acetate,¹⁴C-pyruvate, and³²phosphate radioactivity into fatty acids and phospholipids of aortic SMC. Five mM of CPIB reduce the overall palmitic acid synthesis by shifting from de novo synthesis to the mechanism of chain elongation, although the further elongation to saturated C₁₈–C₂₄ fatty acids is also depressed. The CPIB-enhanced retention of the lipid-derived lipoprotein radio-activity is interpreted as a compensatory mechanism providing cellular fatty acids which are deficient as a result of the CPIB inhibited synthetic processes.     

Wax ester biosynthesis in the liver of myctophid fishes

The biosynthetic properties of wax esters in the liver were compared between two types of myctophid fishes having different body lipid composition, i.e., three triglyceriderich species (Lampanyctus jordani, Diaphus theta, and Symbolophorus californiensis) and three was ester-rich species (L. regalis, Stenobracius nannochir, and Stenobracius leucopsarus). n-Heptadecanol (17∶0-ALC) and/or 10-cis-heptadecenoic acid (17∶1-ACID) was incubated with liver homogenate of the six myctophid fishes and with co-factors such as NADPH and ATP for 2 to 5 h. Considerable amounts of wax esters with odd-numbered fatty acids and/or alcohols were produced in the liver homogenate of the was ester-rich species. Stenobracius nannochir and L. regalis, which exclusively contained wax esters as neutral lipids, showed the highest activity of wax ester synthesis, followed by S. leucopsarus, which contained triglyceride as the minor constituent. Only trace amounts at most of odd-numbered fatty acids and alcohols were incorporated into the wax esters after incubation with the liver homogenates of the triglyceride-rich fishes. Active interchange between the fatty acids and the alcohols occurred during wax ester biosynthesis in the wax ester-rich fishes. The chain elongation and shortening of acyl moieties were also observed during incubation. These results suggested that the deposition of lipids in myctophid fishes is mainly due to their biosynthetic activities.     

Different metabolism of saturated and unsaturated long chain plasma free fatty acids by intestinal mucosa of rats

During fat absorption, unsaturated long chain fatty acids are esterified at a higher rate than saturated fatty acids of similar chain length. This phenomenon has been attributed to differences in the binding affinity of fatty acids to a cytosolic fatty acid-binding protein. As intestinal mucosa utilizes plasma free fatty acids as well, we investigated whether long chainplasma free fatty acids of different degree of saturation are metabolized also at different rates.³H-Palmitic and¹⁴C-linoleic acid complexed to rat serum were injected rapidly into a tail vein of fasting rats. One, 2 and 4 min later there was no difference between³H and¹⁴C-radioactivity in intestinal mucosa, suggesting equal initial uptake of the two labeled fatty acids from plasma. Despite their equal uptake, the incorporation of the isotopes into ester lipids was significantly different, however: at 2 min, 53.1±3.9% of³H and 73.8±4.6% of¹⁴C were recovered in ester lipids. Phospholipids and triglycerides accounted for most of the mucosal³H and¹⁴C. At 4 min, a similar distribution of isotopes in intestinal mucosal metabolites was found. These data show that despite equal initial uptake by intestinal mucosa unsaturated long chain fatty acids taken up from plasma are esterified to a higher and oxidized to a lower extent than saturated plasma free fatty acids. Unsaturated plasma free fatty acids, therefore, may provide a more important source of fatty acids for endogenous intestinal lipoprotein lipids than saturated plasma free fatty acids. It is speculated that the fatty acid binding protein might be operative not only in the intracellular transport and metabolism of luminal fatty acids but of plasma free fatty acids as well.     

Biosynthesis of lipids by bovine meibomian glands

Isolated bovine meibomian glands incorporated exogenous [1-¹⁴C] acetate into lipids. Thin layer chromatographic analysis of the lipids showed that wax esters and sterol esters contained 61% of the total label. Radio gas liquid chromatographic analysis of the acid and alcohol moieties of both ester fractions showed the label was distributed equally between the two portions of the ester in both cases. Cholesterol and 5-α-cholest-7-en-3β-ol were the major labeled sterols, and anteiso-C25, anteiso-C27 and anteiso-C23 were the most highly labeled alcohols. The major labeled fatty acids in the wax esters were anteiso-C15,n-C16, anteiso-C17 andn-C18∶1, whereas anteiso-C25 and anteiso-C27 were the major labeled acids in the sterol esters. The diester region with 6% of the total label contained labeled fatty acids and fatty alcohols each with anteiso-C25 as the major component and ω-hydroxy acids in whichn-C32∶1 was the major labeled component. The trigly ceride fraction which contained 8% of the total lipids was composed of labeled fatty acids similar to those found in both sterol and wax ester fractions. Chromatographic analyses of the labeled lipids derived from exogenous labeled isoleucine showed that anteiso-branched products were preferentially labeled. The labeled triglyceride fraction derived from [U-¹⁴C] isoleucine also contained esterified C15, C13, C11, C9, C7 and possibly shorter anteisobranched acids.     

Tissue culture of cocoa beans (Theobroma cacao L.): Incorporation of acetate and laurate into lipids of cultured cells

Suspension cultures of cocoa bean tissue readily incorporated exogenous acetate into lipids. The distribution of radioactivity from acetate in individual lipid classes after 48 hr was 20, 5, 1, 15, 25, and 35% in triglycerides, diglycerides, free fatty acids, sterol esters, sterols and polar lipids, respectively. The labeled acetate was rapidly incorporated into various fatty acids within 2 hr. The [1-¹⁴C] saturated fatty acids declined slightly after 4 hr, whereas [1-¹⁴C] oleate declined significantly after 2 hr. There was a concomitant increase in [1-¹⁴C] linoleate. The radioactivity associated with linolenate was relatively high up to 4 hr, declined by 24 hr, and then increased again. The kinetics of fatty acid labeling suggested that biosynthesis of linolenic acid in cocoa bean suspension culture may occur via the desaturation of linoleic acid and the chain elongation of dodecatrienoic acid. The patterns of fatty acid radiolabeling following incubation of cells with [1-¹⁴C] laurate was consistent with this mechanism.     

Uptake and metabolism of fatty acids by Soybean suspension cells

Soybean suspension cultures very rapidly take up C₁₆ and C₁₈ fatty acids by a nonspecific, nonenzymic binding of exogeneously added fatty acids to cell walls and by a subsequent transfer into the cell where they are rapidly incorporated into triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines.¹⁴C-Palmitic and¹⁴C-stearic acids follow this sequence but are not desaturated, wherease¹⁴C-oleic and¹⁴C-linoleic acids are transferred more rapidly than the saturated fatty acids and are then further modified. All the data fit a sequence of events by which free oleic acid is first activated to a CoA thioester, and then desaturated to linoleyl-CoA; both thioesters are then transferred to triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine.     

Metabolism of arachidonate and stearate injected simultaneously into the mouse brain

The metabolism of a polyunsaturated and a saturated fatty acid in brain membrane phosphoglycerides was examined by injecting simultaneously a mixture of¹⁴C-arachidonate and³H-stearate into the mouse brain and isolating the microsomal and synaptosomal fractions at 1–40 min after injections. Both types of labeled fatty acids were utilized more readily in the microsomal than the synaptosomal fractions in brain. However, labeled arachidonate was incorporated more rapidly into membrane phosphoglycerides than was stearate. In both subcellular fractions, the relative specific radioactivity (³H and¹⁴C) of diacyl-glycerophosphorylinositol (diacyl-GPI) was higher than other types of phosphoglycerides such as diacyl-glycerophosphorylcholine (diacyl-GPC) and diacyl-glycerophosphorylethanolamine (diacyl-GPE). Furthermore, the apparent rates of incorporation of radioactivity into diacyl-GPI was more rapid for the¹⁴C-arachidonate than for the³H-stearate. Results of the experiment have demonstrated obvious differences in metabolism between stearate and arachidonate in brain. The more rapid transfer of arachidonate to diacyl-GPI is probably due to the presence of an acyl transferase system specially active for the transfer of arachidonyl groups to diacyl-GPI.     

Metabolism of ether linked glycerolipids in dogfish (Squalus acanthias) serum: Evidence for resistance of ether bond to cleavage in 1-alkyl-2,3-diacylglycerols

Rac-[1-¹⁴C]-palmitylglycerol (chimyl alcohol), rac-[1-¹⁴C]-palmityl-2,3-dioleoylglycerol, and rac-palmityl-2,3-[9, 10-³H]-dioleoylglycerol were incubated with dogfish (Squalus acanthias) serum for periods of up to 15.0 hr. The ether bond of the carbon 14 labeled chimyl alcohol was cleaved readily, and radioactivity was incorporated into free fatty acids and the acyl chains of the major glycerolipids of the serum. In sharp contrast, the ether bond of the corresponding dioleoyl derivative remained virtually intact. However, the tritium from the acyl chains was incorporated into glycerolipids via intermolecular rearrangements of fatty acids. The findings are consistent with previous findings with rat liver microsomes showing that the ether linkages of alkylglycerolipids are resistant to oxidative cleavage when acyl groups are present on the glycerol moiety. However, substantial differences may exist between the conditions required for oxidative cleavage of the ether linkage of alkylglycerols in mammals and primative fish. National Marine Fisheries Service, NOAA, U.S. Department of Commerce.     

Cuticular lipid constituents of cabbage seedpod weevils and host plant oviposition sites as potential pheromones

The cuticular lipids of cabbage seedpod weevils (Ceutorrhynchus assimilis Payk.) and those at their oviposition site, i.e., the seed pods of the host plant (Brassica napus L.), were analyzed by chromatographic techniques in conjunction with mass spectrometry (MS). Long chain hydrocarbons were most abundant in both lipids; however, the seed pods ofB. napus containedn-alkanes only, whereasC. assimilis showed a predominance of dimethylalkanes over internally branched methylalkanes as well as iso- andn-alkanes. The amounts of ketones, secondary alcohols and aldehydes, the usual plant components that are unique in insects, occurred in cuticular lipids of both organisms in approximately the same ratio. The composition ofn-alkanes,n-ketones, secondaryn-alcohols, iso- and anteisoaldehydes and esterified primary anteiso-alcohols of wax esters was similar betweenC. assimilis andB. napus. In both sources, qualitative but not quantitative similarities were observed in the composition ofn-aldehydes and esterified primaryn- and iso-alcohols, respectively. The esterified fatty acids of wax esters fromB. napus were composed of roughly equal proportions of saturated branched and unbranched components. The esterified fatty acids of wax esters and steryl esters fromC. assimilis consisted of major proportions of saturated as well as unsaturatedn-compounds, whereas iso- and anteiso structures were present in minor proportions only. Free fatty acids and traces of ethyl esters of fatty acids found inC. assimilis were mainly composed of unsaturatedn-compounds.     

Lipid metabolism of brain tissue in culture

Tissue explants from frontal lobes of rat brain were used for the study of cerebral fatty acid metabolism. After tissues had been maintained in serum-supplemented medium, a lipid free medium was substituted and metabolic studies were carried out. Under these conditions explants continued to take up lipid precursors for at least 48 hr. Stearic acid 1-C¹⁴, palmitic acid 1-C¹⁴ and lignoceric acid 1-C¹⁴ were bound to cells as the free fatty acids or incorporafed into neutral lipids (particularly triglycerides), glycolipids and phospholipids. In the galactolipid fraction, cerebrosides were the principal radioactive lipids. Choline phosphoglycerides, ethanolamine phosphoglycerides, inositol phosphoglycerides and serine phosphoglycerides were the principal radioactive phospholipids. Fatty acids were incorporated into cellular lipids either unchanged or after desaturation, chain elongation, or both. In a patient with a demyelinating disease, precursor uptake was reduced and chain elongation and desaturation of the fatty acid was diminished. In a patient with generalized G_M2 gangliosidosis, glycolipids other than cerebrosides were labeled to a greater extent than normal. These studies exemplify the usefulness of tissue explants for prolonged metabolic studies in normal and pathological specimens of brain. One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May, 1971.     

Biosynthesis of fatty acids in cell-free homogenates of lactating gerbil mammary gland

Cell-free homogenates of lactating mammary gland of gerbils maintained on a diet of sunflower seed, guinea pig chow and oats (Diet 1) or a diet of guinea pig chow and oats (Diet 2) and of rats maintained on laboratory chow (Diet 3) were incubated with¹⁴C-labeled acetate, acetyl CoA or malonyl CoA aerobically. A large proportion of the¹⁴C from¹⁴C-acetate and¹⁴C-acetyl CoA incorporated into fatty acids by homogenates from gerbils on Diet 1 was in unsaturated compounds, particularly in 18-carbon and 20-carbon dienoic acids, compared to preparations from animals on Diets 2 or 3. The two radioactive dienoic acids were proven to be Δ^9,12 18∶2 and Δ^11,14 20∶2, and the latter was shown to be a direct elongation product of Δ^9,12 18∶2 by the substrate¹⁴C-acetyl CoA. In all experiments¹⁴C from¹⁴C-malonyl CoA was incorporated predominantly into 14∶0 and 16∶0, and very little incorporation occurred into unsaturated fatty acids in homogenates made either from gerbil or rat. Total fatty acids isolated from homogenates and from milk fat (fat floating on the centrifuged homogenates) of gerbils on Diet 1 had a higher proportion of 18∶2 than animals on the other two diets, a reflection of the large dietary intake of linoleic acid by gerbils on Diet 1. Under these conditions the amount of 18∶2 in the mammary gland had a significant effect on the products of the incubation.     

Alteration of Wax Ester Content and Composition in Euglena gracilis with Gene Silencing of 3‐ketoacyl‐CoA Thiolase Isozymes

Euglena gracilis produces wax ester under hypoxic and anaerobic culture conditions with a net synthesis of ATP. In wax ester fermentation, fatty acids are synthesized by reversing beta‐oxidation in mitochondria. A major species of wax ester produced by E. gracilis is myristyl myristate (14:0‐14:0Alc). Because of its shorter carbon chain length with saturated compounds, biodiesel produced from E. gracilis wax ester may have good cold flow properties with high oxidative stability. We reasoned that a slight metabolic modification would enable E. gracilis to produce a biofuel of ideal composition. In order to produce wax ester with shorter acyl chain length, we focused on isozymes of the enzyme 3‐ketoacyl‐CoA thiolase (KAT), a condensing enzyme of the mitochondrial fatty acid synthesis pathway in E. gracilis. We performed a gene silencing study of KAT isozymes in E. gracilis. Six KAT isozymes were identified in the E. gracilis EST database, and silencing any three of them (EgKAT1‐3) altered the wax ester amount and composition. In particular, silencing EgKAT1 induced a significant compositional shift to shorter carbon chain lengths in wax ester. A model fuel mixture inferred from the composition of wax ester in EgKAT1‐silenced cells showed a significant decrease in melting point compared to that of the control cells.     

Studies on biosynthesis of waxes by developing jojoba seed. II. the demonstration of wax biosynthesis by cell-free homogenates

The following enzyme activities were demonstrated in cell-free homogenates from developing jojoba cotyledons: 1) elongation of long chain acyl-CoAs in the presence of malonyl-CoA and NADPH (or NADH), 2) NADPH-dependent reduction of long chain acyl-CoAs to the corresponding alcohols, 3) esterification of long chain acyl-CoAs and the alcohols produced from them into wax, 4) elongation of stearoyl-ACP to eicosanoate and docosanoate as well as reduction to stearyl alcohol, 5) desaturation of stearoyl-ACP to oleate in the presence of reduced ferredoxin, and 6) incorporation of malonyl-CoA into long chain fatty acids and alcohols in the presence of added acyl carrier protein. These activities were associated entirely with the floating wax pad after centrifugation of the cell-free homogenate at 12,000 g for 20 min. The relevance of the above reactions (1–6) to wax biosynthesis in vivo is discussed. Production of oleate from acetate by enzymes utilizing ACP-thioesters as substrates followed by conversion of oleyl-ACP to oleoyl-CoA (via free oleic acid) for subsequent elongation, reduction, and esterification, is presented as the most probable in vivo pathway, for wax biosynthesis. The substrate specificities of the elongation and reduction reactions utilizing acyl-CoAs as substrates are examined in terms of wax composition.     

Fatty acid synthesis from [2-14C]acetate in normal and peroxisome-deficient (Zellweger) fibroblasts

The incorporation of [2-¹⁴C]acetate into the lipids of normal and peroxisome-deficient (Zellweger's syndrome) skin fibroblasts was examined. Most of the label was incorporated into triacyglycerol fatty acids in normal as well as Zellweger's syndrome cells. Triacylglycerols and cholesteryl esters in Zellweger's syndrome cells contained increased levels of labelled saturated and monounsaturated very long-chain fatty acids (VLCFA, that is fatty acids with more than 22 carbon atoms), in particular hexacosanoic (26∶0) and hexacosaenoic (26∶1) acids. As traces of labelled VLCFA with up to 32 carbon atoms were detected in triacylglycerols even in control cells it is probable that these fatty acids are formed naturally during the elongation process. Our data suggest that peroxisomes are involved in the chain shortening of the saturated and monounsaturated VLCFA.     

Evidence for acyl transfer reactions between neutral glycerolipids in dogfish (Squalus acanthias) serum

Radioactively labeled triacylglycerols, 1,3-dioleoyl-2-[1-¹⁴C]-palmitoylglycerol and 1,3-[9,10-³H]-dioleoyl-2-palmitoylglycerol, were incubated with dogfish (Squalus acanthias) serum for periods of up to 10.0 hr. Changes in the positional distributions of carbon-14 and tritium within the triacylglycerols in 5.0 hr and 10.0 hr indicate that intermolecular and intramolecular shifts occur among the fatty acids. In addition, a maximum of 8.3% of the carbon-14 and 5.9% of the tritium was incorporated into 1-alkyl-2,3-diacylglycerols; essentially all of this incorporated radioactivity was associated with the acyl chains in the 1.5 and 5.0 hr incubations. In the 10.0 hr incubations, however, 25% of the tritium incorporated into the 1-alkyl-2,3-diacylglycerols was associated with the 0-alkyl chains. Radioactivity was not incorporated significantly into free fatty acids in the 1.5 and 5.0 hr incubations. These results indicate that acyl transfer reactions take place among molecular species of triacylglycerols, as well as between triacylglycerols and 1-alkyl-2,3-diacylglycerols. In the latter case, the conversions appear to be operative in the virtual absence of net biosynthesis.     

Biosynthesis of hydrocarbons in insects: Decarboxylation of long chain acids ton-alkanes inPeriplaneta

The biosynthetic pathway ofn-alkanes was investigated in the cockroachesPeriplaneta americana andPeriplaneta fuliginosa. Both sodium [1-¹⁴C] acetate and randomly tritiated long chain fatty acids were incorporated into the cuticular hydrocarbons of both species. The relative incorporation of acetate into each component of the hydrocarbon fraction was about the same as the relative amount of each component in the fraction. In contrast, [R-³H] hexacosanoic acid was preferentially incorporated inton-pentacosane inP. americana and [R-³H] tetracosanoic acid inton-tricosane inP. fuliginosa. Long chain ketones and secondary alcohols, likely intermediates in the proposed condensation-reduction pathway forn-alkane biosynthesis, were not incorporated into hydrocarbon. Results from experiments with dual labeled lauric aicd were also not consistent with the condensation-reduction pathway. The demonstration of the direct decarboxylation of long chain fatty acids ton-alkanes one carbon unit shorter and the lack of incorporation of proposed intermediates of a condensation-reduction pathway constitute the strongest evidence to date that insects utilize an elongation-decarboxylation pathway forn-alkane biosynthesis.