Rapid β-oxidation of eicosapentaenoic acid in mouse brain: An in situ study

Analyses of brain phospholipid fatty acid profiles reveal a selective deficiency and enrichment in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively. In order to account for this difference in brain fatty acid levels, we hypothesized that EPA is more rapidly β-oxidized upon its entry into the brain. Wild-type C57BL/6 mice were perfused with either ¹⁴C-EPA or ¹⁴C-DHA via in situ cerebral perfusion for 40 s, followed by a bicarbonate buffer to wash out the residual radiolabeled polyunsaturated fatty acid (PUFA) in the capillaries. ¹⁴C-PUFA-perfused brains were extracted for chemical analyses of neutral lipid and phospholipid fatty acids. Based on the radioactivity in aqueous, total lipid, neutral lipid and phospholipid fractions, volume of distribution (V_D, μl/g) was calculated. The V_D between ¹⁴C-EPA- and ¹⁴C-DHA-perfused samples was not statistically different for total lipid, neutral lipids or total phospholipids. However, the V_D of ¹⁴C-EPA in the aqueous fraction was 2.5 times higher than that of ¹⁴C-DHA (p=0.025), suggesting a more extensive β-oxidation than DHA. Furthermore, radiolabeled palmitoleic acid, a fatty acid that can be synthesized de novo, was detected in brain phospholipids from ¹⁴C-EPA but not from ¹⁴C-DHA-perfused mice suggesting that β-oxidation products of EPA were recycled into endogenous fatty acid biosynthetic pathways. These findings suggest that low levels of EPA in brain phospholipids compared to DHA may be the result of its rapid β-oxidation upon uptake by the brain.     

Dependence of Arbuscular-Mycorrhizal Fungi on Their Plant Host for Palmitic Acid Synthesis

Lipids are the major form of carbon storage in arbuscular-mycorrhizal fungi. We studied fatty acid synthesis by Glomus intraradices and Gigaspora rosea. [¹⁴C]Acetate and [¹⁴C]sucrose were incorporated into a synthetic culture medium to test fatty acid synthetic ability in germinating spores (G. intraradices and G. rosea), mycorrhized carrot roots, and extraradical fungal mycelium (G. intraradices). Germinating spores and extraradical hyphae could not synthesize 16-carbon fatty acids but could elongate and desaturate fatty acids already present. The growth stimulation of germinating spores by root exudates did not stimulate fatty acid synthesis. 16-Carbon fatty acids (16:0 and 16:1) were synthesized only by the fungi in the mycorrhized roots. Our data strongly suggest that the fatty acid synthase activity of arbuscular-mycorrhizal fungi is expressed exclusively in the intraradical mycelium and indicate that fatty acid metabolism may play a major role in the obligate biotrophism of arbuscular-mycorrhizal fungi.     

Effect of Ethrel and Ceratocystis fimbriata on the Synthesis of Fatty Acids and 6-Methoxy Mellein in Carrot Root

The rate of incorporation of ¹⁴C from acetate-1-¹⁴C into fatty acids by carrot root discs, 18 hours after inoculation with Ceratocystis fimbriata, was 9-fold greater than that in freshly cut discs. The rate in discs treated with water or Ethrel was 3-fold greater. The rate of incorporation of ¹⁴C from glucose-U-¹³C into fatty acids was 3-fold greater 18 hours after any of the above treatments. The rate of ¹⁴C incorporation from malonate-2-¹⁴C into fatty acids 24 hours after inoculation with C. fimbriata or treatment with water was 25 and 60%, respectively, of that in freshly cut discs. Linoleic acid was the principal fatty acid in carrot root, but incorporation of ¹⁴C from acetate-1-¹⁴C into the acid was low until 18 hours after inoculation with C. fimbriata or treatment with Ethrel. Turnover rates of the fatty acids appeared low and were similar for all treatments.     

Effects of light and temperature on fatty acid desaturation during the maturation of rapeseed

In a winter variety of rapeseed, low temperatures enhance fatty acid desaturation as evidenced by ¹⁴C-acetate incorporation into fatty acids or ¹⁴C-oleate desaturation in vivo; similarly, low temperatures favour polyunsaturated fatty acid accumulation during the maturation of the seeds. Oleate desaturation was slightly higher under 16 hr daylight exposure than under 9 hr treatment.     

Metabolism of [4-14C]levulinic acid by etiolated and greening leaves of Hordeum vulgare

When etiolated barley (Hordeum vulgare L. var. Larker) shoots are incubated with [4-¹⁴C]levulinic acid, ¹⁴CO₂ is evolved, and amino and organic acids are labelled. Respiratory inhibitors and short-chain fatty acids, similar in size to levulinic acid, reduce the production of ¹⁴CO₂ from [4-¹⁴C]levulinic acid, while δ-aminolevulinic acid treatment or illuminating the tissue increase ¹⁴CO₂ evolution. The contribution of levulinic acid metabolism to α-aminolevulinic acid biosynthesis is no greater than that of a general cellular metabolite. The data suggest that fatty acid oxidation and the citric acid cycle are involved in levulinic acid metabolism.     

Further evidence for an elongation-decarboxylation mechanism in the biosynthesis of paraffins in leaves

In isolated tobacco leaves l-valine-U-¹⁴C gave rise to labeled even-numbered isobranched fatty acids containing 16 to 26 carbon atoms and iso C₂₉, iso C₃₁, and iso C₃₃ paraffins. l-Isoleucine-U-¹⁴C on the other hand produced labeled odd-numbered anteiso C₁₇ to C₂₇ fatty acids and anteiso C₃₀ and C₃₂ paraffins. Trichloroacetic acid inhibited the incorporation of isobutyrate into C₂₀ and higher fatty acids and paraffins without affecting the synthesis of the C₁₆ and C₁₈ fatty acids. Thus the very long branched fatty acids are biosynthetically related to the paraffins. In Senecio odoris leaves acetate-1-¹⁴C was incorporated into the paraffins (mainly n-C₃₁) only in the epidermis although acetate was readily incorporated into fatty acids in the mesophyll tissue. Similarly only the epidermal tissue incorporated acetate into fatty acids longer than C₁₈ suggesting that the epidermis is the site of synthesis of both paraffins and the very long fatty acids. In broccoli leaves n-C₁₂ acid labeled with ¹⁴C in the carboxyl carbon and ³H in the methylene carbons was incorporated into C₂₉ paraffin without the loss of ¹⁴C relative to ³H. Since n-C₁₈ acid is known to be incorporated into the paraffin without loss of carboxyl carbon these results suggest that the condensation of C₁₂ acid with C₁₈ acid is not responsible for n-C₂₉ paraffin synthesis in this tissue. Thus all the experimental evidence thus far obtained strongly suggests that elongation of fatty acids followed by decarboxylation is the most likely pathway for paraffin biosynthesis in leaves.     

Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

Effects of Cyanide on Rates and Products of Fatty Acid Synthesis by Chloroplasts Isolated from Spinacia oleracea

Cyanide inhibited unesterified fatty acid synthesis but stimulated glyceride synthesis from [1-¹⁴C]acetate when Spinacia oleracea chloroplasts were incubated in basal media. Both unesterified fatty acid and glyceride accumulation were inhibited when chloroplasts were incubated in a diacylglycerol mode. Stimulation of chloroplast fatty acid synthesis by either exogenous coenzyme A or Triton X-100 was almost completely abolished in the presence of cyanide. Stearoyl-ACP desaturation is considered to be inhibited to a greater extent than is fatty acid synthesis de novo.     

The incorporation of radioactive fatty acids into the phospholipids of nerve-cell-body membranes in vivo. Evidence for highly labelled neuronal nuclei

1. Nerve cell bodies were isolated in bulk from cerebral cortices of 15 day-old rabbits after intrathecal injections of [³H]plamitate, [³H]oleate or [³H]arachidonate and [¹⁴C]glycerol. 2. Nuclear, microsomal and two mitochondrial fractions were isolated from homogenates of the radioactively labelled nerve cell bodies by using differential and discontinuous-gradient centrifugation. 3. After 7.5min in vivo, a high percentage (>80%) of the total ³H-labelled fatty acid radioactivity was found in the membrane fractions of the nerve cell bodies, whereas after 60min in vivo 50% of the total [¹⁴C]glycerol radioactivity was found in the high-speed supernatant. 4. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and the radioactivity in neutral lipid and non-esterified fatty acid fractions were determined in the four subfractions, as were the distributions of several marker enzymes and nucleates. 5. With respect of ³H-labelled fatty acid, the phospholipids of the nuclear fraction had the highest specific radioactivities of the four subfractions. However, for [¹⁴C]glycerol labelling, generally the ¹⁴C specific radioactivities for individual phospholipids were comparable in the four subfractions. This latter observation suggests transport of phospholipids synthesized de novo between membranes of the nerve cell body. 6. Double-labelling experiments demonstrated that individual phospholipids and the combined neutral lipids of the nuclear fraction had higher labelling ratios of ³H-labelled fatty acid/[¹⁴C]glycerol than did the corresponding lipids of the microsomal or mitochondrial fractions. 7. On the basis of the labelling results and the marker studies, it is proposed that it is indeed the nuclei of the nuclear fraction that have these lipids highly labelled with ³H-labelled fatty acid, and the existence of nuclear acyl transferases that are responsible for this fatty acid incorporation is suggested.     

Incorporation of intravenously injected (2-(14)C) acetate into tissue lipids of hamsters during the last hour of 3-, 6-, and 24-hour periods of hypothermia.

The in vivo incorporation of total lipid ¹⁴C from [2-¹⁴C]acetate is decreased in kidney, liver, and small intestine tissue from 3-, 6-, and 24-hr hypothermic hamsters compared to tissues from normothermic animals. The length of time in hypothermia affects hamster tissues differently; thus, ¹⁴C activity: decreases with time in kidney; increases with time in liver; and increases at 3 and 6 hr but decreases from 6 to 24 hr of hypothermia in small intestine.Tissues from hypothermic hamsters incorporated a greater percentage of [2-¹⁴C] acetate into free sterols and diglycerides and a smaller percentage into phospholipid than did corresponding tissues from normothermic hamsters.The percentage of total fatty acid ¹⁴C activity found as polyunsaturated fatty acid ¹⁴C activity increases in hypothermic kidney, liver, and small intestine with a decrease in the percentage of ¹⁴C activity measured in the saturated fatty acids. Esterification of fatty acid was inhibited in all tissues taken from hypothermic hamsters.     

Suppression of long chain acyl-CoA synthetase blocks intracellular fatty acid flux and glucose uptake in skeletal myotubes

Alterations in fatty acid metabolism are associated with impaired glucose uptake in skeletal muscle. Long-chain acyl-CoA synthetase (Acsl) 6 is the one of the Acsl isoforms expressed in skeletal muscle although its role in muscle energy metabolism has not been studied. Thus, the aims of this study were to investigate the role of Acsl6 in fatty acid partitioning and glucose uptake in differentiated skeletal myotubes using a siRNA-mediated knockdown approach. Compared with cells transfected with control siRNA, cells transfected with Acsl6 siRNA exhibited reduced intracellular triacylglycerol (TAG) accumulation. The initial rate of [1‑¹⁴C]‑oleic acid uptake was not altered while the incorporation of [1‑¹⁴C]‑acetic acids into total cellular lipids decreased under Acsl6 knockdown (p < 0.05). In a metabolic labeling study, Acsl6 suppression decreased the incorporation of [1‑¹⁴C]‑oleic acids and [1‑¹⁴C]‑acetic acids into TAG and diacylglycerol (DAG) (p < 0.05). During the chase period of a pulse-chase experiment, Acsl6 suppression increased the intracellular free fatty acids and decreased the fatty acid channeling toward the reacylation of TAG (p < 0.05). The incorporation of the labeled fatty acids into acid-soluble metabolites, β-oxidation product, was not changed under Acsl6 knockdown. Acsl6 siRNA decreased the insulin-induced uptake of [1‑¹⁴C]‑2‑deoxyglucose (p < 0.05) but did not change the glucose uptake in the presence of acipimox, inhibitor of lipolysis. Suppression of Acsl6 deteriorated Akt phosphorylation and Glut4 mRNA expression in response to insulin. These results suggest that Acsl6 activates and channels fatty acids toward anabolic pathways and has a role in glucose and fatty acid cycling through the re-esterification of fatty acids in skeletal muscle.     

Metabolism of alkyl glyceryl ethers and their noninvolvement in alkane biosynthesis in plants

[1-¹⁴C]Octadecyl glyceryl ether did not label alkanes in the leaves of Brassica oleracea and Pisum sativum while [1-¹⁴C]octadecanol and [1-¹⁴C]octadecanoic acid readily labeled the alkanes. About 40% of the exogenous-labeled glyceryl ether was incorporated intact into choline phosphatide while 10–20% was converted into fatty acids and alcohols. [1-¹⁴C]octadecanol was not converted into alkyl glyceryl ether, but it was oxidized to the corresponding acid and then incorporated into alkanes. These results show that alkyl ether is not an intermediate in alkane biosynthesis. When [1-¹⁴C-1-³H]-octadecanol was fed to the leaves of B. oleracea and P. sativum, only the ¹⁴C and no ³H was incorporated into alkanes, ketones, and secondary alcohols. These results show that fatty alcohols are first oxidized to the acid before being incorporated into alkanes, ruling out fatty alcohol, alkyl ether, and alk-1-enyl ether as intermediates in alkane biosynthesis. The exogenous alcohols were also readily esterified into wax esters in both tissues.     

Galactolipid Synthesis in Vicia faba Leaves: II. Formation and Desaturation of Long Chain Fatty Acids in Phosphatidylcholine, Phosphatidylglycerol, and the Galactolipids

The labeling kinetics of the fatty acids of phosphatidylcholine (PC), phosphatidylglycerol (PG), monogalactosyldiglyceride (MGDG), and digalactosyldiglyceride (DGDG) were examined after ¹⁴CO₂ feeding and incubation of leaf discs of Vicia faba over 72 hours in continuous light. The results indicate a rapid accumulation and turnover of radioactivity into PC and PG fatty acids (oleic acid in PC and oleic and palmitic acids in PG). Radioactivity accumulates in MGDG and DGDG fatty acids much more slowly and continuously over 72 hours. Most of this activity is found in linoleic and linolenic acids; very little activity is found in the more saturated fatty acids. Little or no desaturation occurs in situ in conjunction with the galactolipids. The results suggest that PC and PG may act as “carriers” for MGDG and DGDG fatty acid synthesis. Analyses of the labeling patterns of the molecular species of MGDG after ¹⁴CO₂ and ¹⁴C-acetate feeding confirm that MGDG is formed by galactosylation of a preformed diglyceride containing predominantly unsaturated fatty acids.     

Stability of Lactic Acid Bacteria to Freezing as Related to Their Fatty Acid Composition

The viability of Streptococcus lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h was better preserved when the cells were grown in medium supplemented with oleic acid or Tween 80 (polyoxyethylene sorbitan monooleate). A pronounced change in the cellular fatty acid composition was noted when the bacteria were grown in the presence of Tween 80. In S. lactis the ratio of unsaturated to saturated fatty acids increased from 1.18 to 2.55 and in Lactobacillus sp. A-12 it increased from 0.85 to 1.67 when Tween 80 was added to the growth medium. The antibiotic cerulenin markedly inhibited the growth of lactic acid bacteria in tomato juice (TJ) medium but had almost no effect on the growth of the bacteria in TJ medium containing Tween 80 (or oleic acid). The antibiotic inhibited markedly the incorporation of [1-¹⁴C]acetate but had no inhibitory effect on the incorporation of exogenous [1-¹⁴C]oleate (or [1-¹⁴C]palmitate) into the lipid fractions of lactic acid bacteria. Thus, the fatty acid composition of lactic acid bacteria, inhibited by the antibiotic cerulenin, can be modulated by exogenously added oleic acid (or Tween 80) without the concurrent endogenous fatty acid synthesis from acetate. The data obtained suggest that cerulenin inhibits neither cyclopropane fatty acid synthesis nor elongation of fatty acid acyl intermediates. The radioactivity of cells grown in the presence of [1-¹⁴C]oleate and cerulenin was associated mainly with cyclopropane Δ19:0, 20:0 + 20:1, and 21:0 acids. As a consequence, cerulenin caused a decrease in the ratio of unsaturated to saturated fatty acids in lactic acid bacteria as compared with cells grown in TJ medium plus Tween 80 but without cerulenin. Cerulenin caused a decrease in the viability of S. lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h only when Tween 80 was present in the growth medium. We conclude that the sensitivity of lactic acid bacteria to damage from freezing can be correlated with specific alterations in the cellular fatty acids.     

The biosynthesis of the hydrocarbons in males and females of the millipede Graphidostreptus tumuliporus

The in vivo hydrocarbon biosynthesis in the millipede Graphidostreptus tumuliporus was studied after the injection of 1-¹⁴C-acetate, 16-¹⁴C-, and 1-¹⁴C-palmitic acid.From all precursors used an active incorporation into the unsaturated hyrocarbons (alk-1-enes, alkadienes, and alkatrienes) was observed, whereas no radioactivity was incorporated into the saturated alkanes at all, in accordance with their supposed exogenous origin (food). From the distribution of the radiolabel over both the various hydrocarbon classes and the individual hydrocarbon components it was concluded that in this millipede hydrocarbons are synthesized from fatty acids (irrespective of their chain structure) by an elongation-decarboxylation mechanism in which an α-oxidation step is involved, whilst during the decarboxylation process a terminal double bond is introduced. Thus, saturated fatty acids give rise to alk-1-enes (as is evidenced by an overwhelming incorporation of palmitic acid into the alk-1-enes), monoenoic fatty acids to alkadienes, and dienoic fatty acids to alkatrienes.The proposed mechanism for hydrocarbon biosynthesis in G. tumuliporus has not yet been described in other organisms.     

On the chemical structure of the apical droplet from eggs of Culex pipiens

The chemical nature of the apical droplet from eggs of Culex pipiens was investigated by chromatographic techniques. Results indicated that the hydrolysate of the apical drop contains C-12, C-14, C-16, and C-18 straightchain aliphatic fatty acids. A C-12β-hydroxy fatty acid was also found, but the largest component of the fatty acid mixture of the apical drop was shown to be a C-14β-OH fatty acid. Two other fractions appear to be unsaturated fatty acids, probably C-12 and C-14. Quantitative estimation of the percentage of each fatty acid in the mixture showed that about 85 per cent of the fatty acid content of the apical drop consisted of hydroxy fatty acids. By thin-layer chromatography, the largest component coincided with β-OH myristic acid.Glycerol was confirmed to be present in the hydrolysate. Feeding studies with radioactive ³²PO₄^?3 and ³⁵SO₄^?2 showed no significant incorporation of phosphorus, but a sulphur-containing anionic compound could be detected in the apical drop. Infrared analysis showed the presence of an ester group, double bond, primary and secondary alcohol groups, suggesting the presence of hydroxy-, unsaturated-, saturated straight-chain fatty acids, as well as mono-and diglycerides. The structural evidence explains in part the surfactant properties of the apical drop.     

A microsomal fatty acid synthetase coupled to acyl-CoA reductase in Euglena gracilis

Microsomal particles from dark-grown Euglena gracilis incorporated malonyl-CoA into fatty acids and fatty alcohols in the presence of acetyl-CoA, NADH, NADPH, and ATP with an optimum pH of 8.0. Schmidt degradation of the individual fatty acids derived from [l,3-¹⁴C]malonyl-CoA showed that the microsomal fatty acid synthesis was a de novo type. Detailed analysis of the products formed in the absence of various cofactors showed that the role of ATP was specifically in the formation of fatty alcohols and that fatty acid reduction specifically required NADH.The major aliphatic chains synthesized by the microsomes were C₁₆, C₁₈, and C₁₄ in both the acyl portions and alcohols. Although relative concentrations of acetyl-CoA and malonyl-CoA influenced the chain length distribution of products, C₁₆remained the major product in both the alcohol and the acid fractions. Effects of NADPH and NADH concentrations on malonyl-CoA incorporation suggested that the two reductive steps involved in the microsomal fatty acid synthesis have different pyridine nucleotide specificity. The apparent K_m for malonyl-CoA was 4.2 × 10^?4m. Based on the experimental results a mechanism is suggested by which carbon is channeled into wax esters under conditions of nutritional abundance in dark-grown E. gracilis.     

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [¹⁴C]trioleoylglycerol, releasing [¹⁴C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [¹⁴C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [¹⁴C]oleic acid produced from the lipase reaction to [¹⁴C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.     

Studies in lipogenesis in vivo: Fatty acid and cholesterol synthesis during starvation and re-feeding

1. Lipogenesis in vivo has been studied in mice given a 250mg. meal of [U-¹⁴C]glucose (2·5μc) or given an intraperitoneal injection of 25μg. of [U-¹⁴C]glucose (2·0μc). 2. The ability to convert a [U-¹⁴C]glucose meal into fatty acid was not significantly depressed by 6–7hr. of starvation. In contrast, incorporation of ¹⁴C into fatty acid in the liver after the intraperitoneal dose of [¹⁴C]glucose was depressed by 80% and by more than 90% by 1 and 2hr. of starvation respectively. Carcass fatty acid synthesis from the [U-¹⁴C]glucose meal was not depressed by 12hr. of starvation, whereas from the tracer dose of [U-¹⁴C]glucose the depression in incorporation was 80% after 6hr. of starvation. 3. Re-feeding for 3 days, after 3 days' starvation, raised fatty acid synthesis and cholesterol synthesis in the liver fivefold and tenfold respectively above the levels in non-starved control mice. These increases were associated with an increased amount of both fatty acid and cholesterol in the liver. 4. After 18hr. of starvation incorporation of a [U-¹⁴C]glucose meal into carcass and liver glycogen were both increased threefold.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.