Structural analysis of the lipopolysaccharide from Chlamydophila psittaci strain 6BC.

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.     

The characterization and distribution of hexahydropolyprenyl esters in cultures of Aspergillus fumigatus Fresenius

The total mycelial lipid of Aspergillus fumigatus was analysed and over half of its hexahydropolyprenol was shown to be esterified with fatty acids. Comparison of the fatty acid content of the prenyl esters with the sterol ester and the total lipid indicated a marked predominance of saturated fatty acids in the polyprenyl esters. The predominant acids esterified to the prenols were palmitic acid, linoleic acid, oleic acid, lignoceric acid, stearic acid and palmitoleic acid. Most of the unesterified polyprenol was found in the mitochondrial fraction, but the esterified prenol was equally distributed throughout the cell fractions. This distribution was unlike that found for ergosteryl ester in the same tissue.     

Changes in metabolism and hepatic ultrastructure induced by estradiol and testosterone in immature female Epinephelus akaara (Teleostei,Serranidae)

Summary Estradiol injections increase serum level of calcium, amino acid, glucose, protein, ammonia and creatinine in immature Epinephelus akaara, and also increase levels of total lipid, cholesterol, phospholipid and esterified fatty acids. Hepatic protein, glycogen and lipid concentrations also rise after estradiol treatment, and some hepatic enzymes participating in the metabolism of nitrogen, lipid and carbohydrate, show increased activity. Serum vitellogenin levels are increased. Testosterone treatment increases serum protein, total lipid, cholesterol, amino acid and ammonia levels, and also hepatic glycogen content, but in contrast to estradiol treatment, testosterone does not change serum vitellogenin, glucose, calcium, phospholipid, esterified fatty acid and creatinine levels, nor the hepatic lipid and protein content. A small number of hepatic enzymes shows an increased activity. Vitellogenic fish show biochemical changes similar to that of estradiol-treated fish, but are different from those of immature fish. Estradiol treatment induces ultrastructural changes in the hepatocytes of immature fish that are similar to those found in vitellogenic fish. These include a proliferation of rough endoplasmic reticulum and Golgi apparatus, and an increase in glycogen and lipid, all indicative of enhanced metabolic activity.     

The attractiveness of odorous esterified fatty acids to the potential biocontrol agent,Altica cyanea

The fatty acid composition of foliar buds, young, mature, and senescent leaves, and stem parts of the rice-field weed, Ludwigia adscendens L. (Onagraceae) was analyzed by thin layer chromatography and gas chromatography flame ionization detection. The analysis of fatty acid composition revealed that saturated fatty acids (i.e., C14:0, C16:0, and C18:0) were prevailing compounds among the all weed parts except senescent leaves where C18:1 was predominant. The esterified fatty acids isolated from different weed parts over the range of 10–100 μg/ml followed by individual synthetic esterified fatty acids that were identified from the esterified extracts of different weed parts, and a mixture of synthetic esterified fatty acids except esterified eicosenoic acid and docosahexaenoic acid were applied to identify their role as a chemical cue for a potential biocontrol agent, Altica cyanea (Weber) (Coleoptera: Chrysomelidae) in a Y-tube olfactometer under laboratory conditions. In this bioassay, the esterified fatty acids from mature leaves and stem parts of this weed attracted A. cyanea at 20–100 μg/ml and at 80 μg/ml concentrations, respectively. Clear attraction was recorded by female A. cyanea insects in the mixture of synthetic esterified fatty acids at 60, 80, and 100 μg/ml concentrations. It is thus concluded that A. cyanea rely on an effective proportion of esterified fatty acids as an olfactory cue for attraction.     

Serum and thyroid tissue lipids in patients with thyroid tumors in euthyroidism

Plasma lipids of 24 euthyroid subjects with thyroid adenoma and non-toxic primary differentiated thyroid carcinoma (papillary and follicular types) were assessed and compared with a similar investigation conducted on 20 normal subjects. A parallel study with thyroid tumor tissues examined the lipid changes which occurred in the same group of patients. These were compared with the picture seen in 5 normal thyroids. The investigations examined the changes in total lipids; total, free and esterified cholesterol; total phospholipids; lecithin; cephalin and sphingomyelin; triglycerides and free fatty acids in both serum and thyroid tissue. Compared with normal subjects, the serum lipids were almost identical except for the concentration of free fatty acids which showed significantly lower values in the group with neoplastic changes. Tissue analysis of the cancerous thyroid however revealed a marked rise in virtually all lipid fractions but the cholesterol seemed to dominate the picture. The differential studies of total phospholipids for thyroid tissue membrane lipids further revealed a significant increase in the lecithin and sphingomyelin components of total phospholipids as well as the esterified cholesterol fraction in thyroid carcinoma. The changes in thyrophospholipids were more marked in females normal thyroids than males. An attempt has been made through evaluation of the results derived from differential lipid studies to elucidate the role of some of the various fractions determined and the possible effect of the changes described on thyroid hormone metabolism.     

Chromatographic analysis of carotenol fatty acid esters in Physalis alkekengi and Hippophae rhamnoides

The carotenol fatty acid esters of two potentially valuable sources of plant carotenoids, sepals of Physalis alkekengi (Chinese lantern) and fruits of Hippophae rhamnoides (sea buckthorn), were separated by column chromatography and identified by HPLC-DAD and HPLC-MS. A chemical and an enzymatic hydrolysis were employed to identify the parent carotenoids and to remove the lipid components. Zeaxanthin and beta-cryptoxanthin esters represented the main fraction in P. alkekengi sepals and an important one in H. rhamnoides fruits. Beta-Cryptoxanthin palmitate and zeaxanthin dipalmitate were identified as major compounds in both plants. In P. alkekengi, the carotenoids were mainly (> 90%) esterified with palmitic acid, and a high proportion (> 80%) of saturated medium chain fatty acids was found (by GC-MS) in the total lipid extract. Although the total lipid extract of H. rhamnoides contained significant amounts of unsaturated fatty acids, especially oleic and palmitoleic acids, the xanthophylls were mainly esterified with saturated fatty acids. The oleoresins of both species represent potential sources of carotenoid esters and can be used as food additives, cosmetic ingredients or nutraceuticals.     

Studies on the chemical composition of lipopolysaccharide from Neisseria meningitidis group B.

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.     

Thehalose mycolates from Nocardia asteroides,Nocardia farcinica,Gordona lentifragmenta and Gordona bronchialis

Isolation of glycolipids from Nocardia asteroides, N. farcinica, Gordona lentifragmenta and G. bronchialis, by column chromatography of lipid extracts on a 50% (w/w) mixture of silicic acid and silica gel H, is described. The isolated materials were partially characterized by infrared spectroscopy, optical rotation and refractive index measurements, and by identifying the products of alkaline hydrolysis. Analytical studies showed that the glycolipids released only trehalose in the aqueous phase while mycolic acids were the constituent fatty acids identified.The isolated lipids are trehalose esters in which the trehalose molecule is esterified with mycolic acids.     

Changes in fatty acid composition of rat liver and serum induced by distal small bowel resection

The serum lipid composition and the fatty-acid profiles of the major lipid fraction (triglycerides, esterified cholesterol, and phospholipid) of liver and serum were examined 6 weeks after both 50% and 75% distal small bowel resection (DSBR). Total serum lipid content did not modify after DSBR. Esterified cholesterol and phospholipid levels of the serum did not significantly change after the operation. However, a significant increase in both free cholesterol and triglyceride levels was observed after DSBR. Different fatty acid changes in the liver and serum lipid fractions were found after DSBR, with the greatest differences in the hepatic esterified cholesterol fraction. These results suggest that DSBR affects both the lipid composition and the fatty acid composition of major lipid fraction of liver and serum.     

Possible existence of a novel amphipathic immunostimulator in the phenol-water extracts of Mycobacteriaceae

The extracts having diverse immunostimulating activities were obtained as a water-phase fraction from four bacterial species representing the 4 genera (Mycobacterium, Nocardia, Gordona, and Rhodococcus) of Mycobacteriaceae by the phenol-water method, which is commonly used for extraction of endotoxic lipopolysaccharides (LPS) from gram-negative bacteria and amphipathic substances from gram-positives. These fractions, especially those of G. aurantiaca and R. terrae, showed strong stimulatory effects on murine splenocytes, macrophages of mice and guinea pigs, the immunoadjuvant activities in guinea pigs and mice, and the distinct activities inducing a tumor necrosis factor and interferons alpha/beta and gamma in primed mice. The fractions from G. aurantiaca and R. terrae exhibited potent pyrogenicity and the ability to activate the clotting enzyme cascade of the horseshoe crab (Tachypleus tridentatus). Some of these biological activities were not very different from the potency of the reference endotoxic LPS derived from Escherichia coli or Fusobacterium nucleatum. But the test fractions neither showed the activity to prepare rabbit skin to the local Shwartzman reaction, nor reacted with anti-lipid A conventional and monoclonal antibodies. Furthermore, unlike LPS, these fractions stimulated the splenocytes of C3H/HeJ mice (LPS-Nonresponder). Although the fractions showing the above biological activities have not yet been adequately purified, they contained polysaccharides, whose main constituent sugar is mannose with a smaller amount of arabinose, fatty acids consisting primarily of palmitic, stearic, and tuberculostearic acids, and small amounts of peptides and amino sugars. Since components characteristic of known immunomodulators of bacterial origin, namely endotoxins (lipid A's), cell wall peptidoglycans, lipoteichoic acids, cord factors (trehalose dimycolates), or deoxyribonucleic acids, were practically not detected in these fractions, the agent responsible for the above bioactivities is considered to be a novel substance different from the known, bacterial immunomodulators.     

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.     

Diacylglycerol Levels Unchanged during Auxin-Stimulated Growth of Excised Hypocotyl Segments of Soybean

Diacylglycerol contents of excised soybean (Glycine max L.) hypocotyl segments, incubated for 4 hours in the presence or absence of a growth promoting concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) were monitored by three different methods as a sensitive measure of the action in vivo of C-type phospholipases. By all three methods, steady state levels of diacylglycerols representing about 3% of the total lipids or about 7% of the neutral lipids, depending on method of assay, declined 18% over 4 hours of incubation as determined by extraction of total lipids and analysis by thin layer chromatography and densitometry. The average decline with 2,4-D-treated segments was less but the difference from controls was not significant. In those experiments where a small effect of 2,4-D was noted, the fraction showing an elevated diacylglycerol level in response to 2,4-D, after separation into membrane and supernatant fractions, was the supernatant and not the membranes. Results were confirmed from analyses of total fatty acids in each of the major lipid fractions and from diacylglycerol assays by conversion into phosphatidic acid upon incubation with [γ-³²P]ATP and purified diacylglycerol phosphokinase from Escherichia coli. In the presence of 2,4-D, the diacylglycerol content of the membranes was unchanged compared to membranes from control segments. As with the densitometric method, the small 2,4-D induced increase in diacylglycerols, when observed, was insignificant and in the supernatant. The only membrane-associated lipid fraction consistently showing a response to 2,4-D was the fraction containing sterols esterified with fatty acids. Either total microsomes or purified plasma membranes when incubated for 10 to 20 minutes with 1 micromolar 2,4-D showed no accelerated formation of diacylglycerols compared to membranes not incubated. The results do not support operation during auxin growth of the animal paradigm where diacylglycerol activation of C-type protein kinases occurs in response to activated phospholipase C breakdown of phosphoinositides.     

Biochemical studies on the cell wall lipopolysaccharides (O-antigens) of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba).

Lipopolysaccharides were isolated from the cell walls of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba). Chemical analysis revealed the presence of glucose, fructose, mannose, heptose, rhamnose, ethanolamine, fatty acids and glucosamine. The lipopolysaccharides do not contain 2-keto-3-deoxyoctonate, the typical linking sugar of polysaccharide and lipid moieties of enterobacterial lipopolysaccharides. Galactose, a typical core polysaccharide component of many gram-negative bacteria was also absent from lipopolysaccharides of these organisms. By hydrolysis in 1% acetic acid, the lipopolysaccharides have been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Components of degraded polysaccharide and lipid A moiety were identified and determined. The lipid A fractions contained fatty acids, phosphorus and glucosamine. All the neutral sugars detected in lipopolysaccharides were shown to be the constituents of its polysaccharide moiety. The fatty acid analysis of lipopolysaccharide and lipid A showed the presence of both hydroxy and non hydroxy acids. They were different from those of lipids extracted from cell walls before the extraction of lipopolysaccharides. 3-Hydroxylauric and 3-hydroxymyristic acids predominated in lipopolysaccharide and lipid A of Vibrio cholerae and El-tor (Inaba).     

Rapid separation of serum lipids for fatty acid analysis by a single aminopropyl column.

A rapid and simple method for separation of serum lipid classes for fatty acid analysis with a single aminopropyl solid phase glass column is described. The recoveries of cholesteryl esters, triglycerides, free fatty acids, and phospholipids were all at least 98%. Coefficients of variation less than 10% were obtained for absolute and relative amounts of most individual fatty acids analyzed after separation of serum lipid classes. This method provides an efficient and convenient tool to follow fatty acid patterns in serum lipid fractions.     

Factors affecting the elicitation of sesquiterpenoid phytoalexin accumulation by eicosapentaenoic and arachidonic acids in potato

Eicosapentaenoic and arachidonic acids in extracts of Phytophthora infestans mycelium were identified as the most active elicitors of sesquiterpenoid phytoalexin accumulation in potato tuber slices. These fatty acids were found free or esterified in all fractions with elicitor activity including cell wall preparations. Yeast lipase released a major portion of eicosapentaenoic and arachidonic acids from lyophilized mycelium. Concentration response curves comparing the elicitor activity of the polyunsaturated fatty acids to a cell-free sonicate of P. infestans mycelium indicated that the elicitor activity of the sonicated mycelium exceeded that which would be obtained by the amount of eicosapentaenoic and arachidonic acids (free and esterified) present in the mycelium. Upon acid hydrolysis of lyophilized mycelium, elicitor activity was obtained only from the fatty acid fraction. However, the fatty acids accounted for only 21% of the activity of the unhydrolyzed mycelium and the residue did not enhance their activity. Centrifugation of the hydrolysate, obtained from lyophilized mycelium treated with 2n NaOH, 1 molarity NaBH₄ at 100°C, yielded a supernatant fraction with little or no elicitor activity. Addition of this material to the fatty acids restored the activity to that which was present in the unhydrolyzed mycelium. The results indicate that the elicitor activity of the unsaturated fatty acids is enhanced by heat and base-stable factors in the mycelium.     

Triglyceride metabolism in 3T3-L1 cells. An in vivo 13C NMR study.

13C nuclear magnetic resonance spectroscopy has been used to study triglyceride metabolism in 3T3-L1 cells incubated with [1-13/14C] acetate, myristate, palmitate, stearate, or oleate. Labeled cells embedded in agarose filaments were perfused in a specially fitted NMR tube within the spectrometer magnet. Incubation of 3T3-L1 cells with a specific fatty acid enriched the cellular triglycerides with that fatty acid; the NMR signal observed in the carbonyl region of the cell spectrum was due in large part to that fatty acid. NMR data demonstrated that cellular enzymes preferentially esterified saturated fatty acids at the glyceride sn-1,3 position and unsaturated fatty acids at the sn-2 position. cellular triglyceride hydrolysis by hormone-sensitive lipase was monitored by measuring the decrease in the integrated intensities of resonances arising from fatty acyl carbonyls esterified at glycerol carbons sn-1,3 and sn-2. Under basal conditions, the time courses were first-order, and the average rates were 0.14% of signal/min at both carbonyl positions. Under isoproterenol stimulated conditions, these rates were still first-order and increased 6.4-fold at the sn-1,3 position and 2.4-fold at the sn-2 position. The observation that the hydrolysis time courses were first-order suggested that only a small amount of cellular triglyceride was available to hormone-sensitive lipase, supporting the view that lipolytic enzymes operate at lipid surfaces where only small amounts of neutral lipid may be soluble. Attempts to correlate the measured rates with the rates of hydrolysis at the sn-1,3 and sn-2 positions were hindered by the fact that the chemical shifts of the carbonyl carbons of the diglyceride hydrolysis product did not overlie those of the triglyceride. Analysis of hydrolysis kinetics revealed that hormone-sensitive lipase exhibited little preference for a particular esterified fatty acid under basal conditions; however, under stimulated conditions, the enzyme exhibited a preference for certain triglyceride species.     

27-Hydroxyoctacosanoic acid is a major structural fatty acyl component of the lipopolysaccharide of Rhizobium trifolii ANU 843

A new hydroxylated, very long-chain fatty acid has been isolated and characterized from the lipopolysaccharide (LPS) of Rhizobium trifolii ANU 843. The lipid A of the organism was degraded by mild alkali and borohydride and the products methylated, peracetylated, and fractionated on a C18 reverse-phase column. The major lipid fraction was reduced with lithium triethylborohydride, methylated, peracetylated, and subjected to thin layer chromatography. The methylated peracetylated acid and the reduced diacetylated diol (1,27-dihydroxyoctacosane diacetate) were isolated and characterized by mass spectrometry and 1H NMR spectroscopy using homonuclear decoupling. The identity and linkage of the new fatty acid in the lipopolysaccharide was confirmed by 1H NMR spectroscopy of purified lipid A fractions and similar NMR studies of lipid A after acylation by phenylisocyanate. In the native LPS, the 27-hydroxy C-28 fatty acid is acylated at the 27-hydroxy position by other 3-hydroxy fatty acids. About 50% of the total fatty acid content of the LPS of R. trifolii ANU 843 is 27-hydroxyoctacosanoic acid. This oxyacyloxy structure involving 27-hydroxyoctacosanoic appears to be the major structural feature of the lipid A of this organism.     

Lipids of Pinus halepensis pollen

The total lipids of Pinus halepensis pollen were separated into individual classes of neutral and polar lipids and the components of each class were identified and determined quantitatively. Free fatty acids, waxes and triacylglycerols were found as the main constituents of neutral lipids and phosphatidylcholine and phosphatidylethanolamine of polar lipids. Glycerylether derivatives were detected in neutral and polar lipid fractions. Free and esterified volatile fatty acids were also found in pollen and its neutral lipid fraction.     

Fatty acid and lipid analysis of the house cricket,Acheta domesticus

The fatty acid content and composition of the house cricket Acheta domesticus have been investigated in entire insects at different developmental stages and in selected organs of male and female adults. We have also determined the fatty acid composition of the various lipid classes within extracts of the organs of adult female insects. Fatty acids were analysed by capillary gas chromatography or mass spectrometry as their methyl esters (FAMEs) after direct transesterification of insect material or separated lipid classes.The major esterified fatty acids in all extracts were palmitate (C_16:0), stearate (C_18:0), oleate (C_18:1) and linoleate (C_18:2). Levels of esterified fatty acid varied considerably between organs but the fatty acid compositions showed only small variations. The levels of polyunsaturated fatty acids of the C₁₈ series were considerably higher in phospholipid fractions than in other lipid classes. Triacylglycerols formed the major lipid class in ovaries, fat-body and newly-laid eggs, whereas diacylglycerols and phospholipid predominate in the haemolymph. Triacylglycerols, phospholipids, diacylglycerols and free fatty acids were all found in significant amounts in the gut tissue.     

Identification of albumin-bound fatty acids as the major factor in serum-induced lipid accumulation by cultured cells

Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty acids, the rabbit liver cells grown on the former media accumulated more lipid than cells grown on the latter media. This difference was shown to be due to the higher concentration of albumin per micro mole of fatty acid in horse serum as compared with rabbit serum. Consequently, the albumin to fatty acid ratio also controls the lipogenic activity of a serum. A linear relationship is presented which relates the cell lipid content to the molar ratio of albumin to free fatty acids and to the absolute concentration of free fatty acids in the medium.