Tissue culture of cocoa bean (Theobroma cacao L.): Incorporation of fatty acids into lipids of cultured cells

Suspension cell cultures of cocoa bean rapidly incorporated palmitic, stearic, oleic and linoleic acids into cellular lipids. Thus, 75 and 20% of [1-¹⁴C] palmitic acid was incorporated into polar lipids and triglycerides, respectively, after 48 hr. When [1-¹⁴C] oleic and [1-¹⁴C] linoleic acid were added separately, polar lipids consistently contained most of the radioactive fatty acids. Ca. 60% of the stearic acid accumulated as unesterified fatty acid in the cells. Palmitic and stearic acid were not desaturated, but oleic acid and linoleic acid were further desaturated. The kinetics of conversion of oleic acid and linoleic acid suggested a sequential desaturation pathway of 18∶1→18∶2→18∶3 in cocoa bean cell suspensions.     

Polar and nonpolar lipids and their fatty acid composition of a fewFusarium species

A few species ofFusarium have been evaluated for their potential to produce lipids. The isolates under investigation exhibited wide variation with respect to the mycelial weight, total lipid content and percentage composition of polar and nonpolar lipids in which triglycerides were the major components (81–90%). Palmitic, stearic, oleic and linoleic acids were the major fatty acids in both the fractions. The polar lipids contained higher levels of linoleic acid, whereas nonpolar lipids contained oleic acid as the predominant acid. Nonpolar lipids were more saturated than polar lipids.     

Polyunsaturated fatty acids and neutral lipids in developing larvae of the oyster,Crassostrea virginica

The fatty acid composition of oyster larvae at various stages, as well as of the algal diet, were determined by gas liquid chromatography (GC). Saturated fatty acids are the major fatty acid components in all larval stages and account for 34–62%, 30–35% and 35–81% of the neutral, polar and total lipids of algal-fed larvae respectively. Weight percentage of saturated fatty acid in “starved” larvae was consistently higher (63–81%) during the whole period. The total polyunsaturated fatty acids were higher in the polar lipids than in the neutral lipids. The concentration of the ω3 fatty acids also was comparatively higher in the polar lipids than in the neutral lipids. In the total and neutral lipid fractions, the weight percentage of polyunsaturated and ω3 fatty acids was higher in the eyed than in the pre-eyed (pediveliger) larvae. Eicosapentaenoic acid (20∶5ω3) and 22∶6ω3 were not detected in lipids of “starved” and young larvae. There was an accumulation of 20∶5ω3, 22∶6ω3, and total ω3 fatty acids in the older larvae. Lipid classes were separated by thin layer chromatography (TLC). There was no qualitative change in lipid composition during larval development, but a marked increased of triacylglycerol in larvae up to the stage of maturation in algae-fed larvae. Contribution number 1195 of the Virginia Institute of Marine Science, Gloucester Point, VA 23062     

Variation in fatty acid composition of the different acyl lipids in seed oils from fourSesamum species

Seeds from different collections of cultivatedSesamum indicum Linn. and three related wild species [specifically,S. alatum Thonn.,S. radiatum Schum and Thonn. andS. angustifolium (Oliv.) Engl.] were studied for their oil content and fatty acid composition of the total lipids. The wild seeds contained less oil (ca. 30%) than the cultivated seeds (ca. 50%). Lipids from all four species were comparable in their total fatty acid composition, with palmitic (8.2–12.7%), stearic (5.6–9.1%), oleic (33.4–46.9%) and linoleic acid (33.2–48.4%) as the major acids. The total lipids from selected samples were fractionated by thin-layer chromatography into five fractions: triacylglycerols (TAG; 80.3–88.9%), diacylglycerols (DAG; 6.5–10.4%), free fatty acids (FFA; 1.2–5.1%), polar lipids (PL; 2.3–3.5%) and steryl esters (SE; 0.3–0.6%). Compared to the TAG, the four other fractions (viz, DAG, FFA, PL and SE) were generally characterized by higher percentages of saturated acids, notably palmitic and stearic acids, and lower percentages of linoleic and oleic acids in all species. Slightly higher percentages of long-chain fatty acids (20∶0, 20∶1, 22∶0 and 24∶0) were observed for lipid classes other than TAG in all four species. Based on the fatty acid composition of the total lipids and of the different acyl lipid classes, it seems thatS. radiatum andS. angustifolium are more related to each other than they are to the other two species.     

The fatty acid composition of three unicellular algal species used as food sources for larvae of the American oyster (Crassostrea virginica)

The total lipid and fatty acid content of 3 algal species,Pyramimonas virginica, Pseudoisochrysis paradoxa andChlorella sp., which have been successful as food sources for rearing larvae of the American oyster,Crassostrea virginica, was determined. Of the fatty acids of ω6 and ω3 families which have been shown to be essential fatty acids for normal growth in many animals, only the ω6 fatty acids were found to be higher in these 3 species of algae than in the traditional oyster larvae diet which consists of the algaeMonochrysis lutheri andIsochrysis galbana. The major fatty acid constituents of the total lipids of the 3 species were the C12, C14, C16 and C18 saturated fatty acids and the C16 and C18 mono- and polyunsaturated acids. These components constituted 70–93% of the total lipid in cultures of all ages. There were modest amounts of C20 and C22 polyunsaturated acids; some of these existed only in trace amounts. InP. virginica andChlorella sp., hexadecanoic acid was dominant (23–39%). The presence of large quantities of tetradecanoic acid (22–26%) and oleic acid (17–21%) was characteristic ofP. paradoxa. Chlorella sp. had the highest proportion of octadecatrienoic acid (18∶3ω3) which accounted for up to 17% of the total lipids. γ-Linolenic acid (18∶3ω6) was found only inChlorella sp., but in the 5th-day culture only. The lowest proportion of total polyethylenic acid was inP. paradoxa; however, lipid analyses showed this alga had the most lipid/individual cell. Some variations were observed in the fatty acid composition with age of the culture. Contribution No. 883 of the Virginia Institute of Marine Science, Gloucester Point, VA 23062.     

Trans-6-hexadecenoic acid and the corresponding alcohol in lipids in the sea anemoneMetridium dianthus

Trans-6-hexadecenoic acid was found in polar lipids, triglycerides, was esters and diacylglyceryl ethers of the sea anemoneMetridium dianthus from Passamaquoddy Bay. The corresponding alcomaquoddy Bay. The corresponding alcohol also apparently occurs in the wax esters of this species. The long-chain (C₂₀, C₂₂) monoethylenic alcohols reported for other species of sea anemones from neighboring waters were absent and the major alcohol and glyceryl ether chain both had 16∶0 structures. The isomers of C₁₈ and C₂₀ monoethylenic fatty acids in polar lipids and triglycerides were unusual in their high proportion of theω ₇ isomer. These two lipids also contained higher proportion of the polyunsaturated fatty acids than the others.     

New branched-chain fatty acids from the senegalese gorgonianLeptogorgia piccola (white and yellow morphs)

Fatty acids from total lipids of the gorgonianLeptogorgia piccola (white and yellow morphs), collected from the same area at two different periods with regard to the average water temperature, were studied. More than fifty fatty acids were identified as methyl esters andN-acyl pyrrolidides by gas chromatography and gas chromatography/mass spectrometry. Three new, branched-chain unsaturated fatty acids were identified in addition to the unusual 7-methyl-6-hexadecenoic acid, namely 10-methyl-6-hexadecenoic, 7,9-dimethyl-6-hexadecenoic, and 10-methyl-6,9-heptadecadienoic acids. Also 6,9-heptadecadienoic acid was identified. The fatty acid patterns of specimens harvested in colder waters were quite different from those harvested in warmer waters in that the former contained high amounts of methylene-interrupted polyunsaturated acids, including tetracosapolyenoic acids, especially 6,9,12,15,18–24∶5 (up to 15.8% of the total acid mixture) and 6,9,12,15,18,21–24∶6 (up to 5.3%). Arachidonic acid was, nevertheless, a major component in all the fatty acid mixtures studied (13.6–20.5%). Based on gas chromatography/Fourier transform infrared experiments, the double bonds were assigned the (Z) configuration. Several fatty aldehydes and their dimethyl acetals were also detected, of which the most abundant was octadecanal.     

The lipid composition of microsomal preparations from lactating bovine mammary tissue

The microsomes isolated from lactating bovine mammary tissue contained 4.3 mg lipid per milligram nitrogen. Phospholipids comprised 83% of the lipids. The neutral lipids were composed of triglycerides (20–30%), diglycerides (5–10%), free fatty acids (15–30%, cholesterol (35–40% and cholesterol esters (10–12%, respectively. Phosphatidylcholine was the predominant phospholipid component (>50%), and the remainder consisted of phosphatidylethanolamine (21–13%), phosphatidylserine (4–6%), phosphatidylinositol (8%), sphingomyelin (9%) and lysophosphatidylcholine (2%) respectively. The composition of the microsomal phospholipids was similar to that of isolated mammary cells and tissue homogenates but quite different from milk and fat globule membrane phospholipids. The triglycerides contained short chain fatty acids but their relative concentrations were lower than in milk triglycerides. The various lipid fractions had a variable proportion of saturated fatty acids, i.e., triglycerides (47.7%), diglycerides (86.7%), free fatty acids (70.6%), phosphatidylcholine (50.6%), phosphatidylethanolamine (50.8%), phosphatidylserine (35.3%), phosphatidylinositol (40.5%) and sphingomyelin (82.3%), respectively. The molecular distribution of fatty acids in the microsomal triglycerides and phosphatidylcholine was similar to that occurring in milk, i.e., the short chain and unsaturated fatty acids were concentrated in the primary positions (sn1 andsn3) of the triglycerides, and the unsaturated acids were preferentially located in positionsn2 of the phosphatidylcholine. The compositional data indicate that mammary microsomes are not the direct source of the phospholipids of the milk fat globule.     

Lipid composition of selected margarines

Results of analytical studies on the composition of 10 selected margarines representative of consumeravailable hard and soft types are presented. Paired hard and soft products from the same manufacturer were chosen where possible. All of the margarines were compared on the basis of total fatty acid composition, polyunsaturated to saturated fatty acid ratios, totaltrans and thetrans content of the monoene and diene fractions, location of the double bond in the monoene isomers, per cent conjugation, distribution of the fatty acids at the 2 position of the triglycerides, tocopherol content, and the ratios of α-tocopherol to polyunsaturated fatty acids. As expected the soft margarines contained more polyunsaturated fatty acids than their companion hard types, but all soft margarines did not contain more polyunsaturated fatty acids than all of the hard margarines. The one margarine containing safflower oil had the highest polyunsaturated to saturated ratio. Eight of the ten margarines contained more than 15%trans monoene and nine contained less than 5%trans diene. Positional isomers in the monoene fraction were Δ6 toΔ12 with thecis Δ9 isomer predominating. All of the margarines contained less than 1.9% conjugation. The percentage oftrans monoene at the 2 position was greater for some margarines than that in the total fatty acid. This was attributed to the preferential placement of polyunsaturated fatty acids at the 2 position in the original vegetable oils. The forms of tocopherol found were characteristic of the original vegetable oils. Ratios of α-tocopherol to PUFA varied from 0.1 to 0.5 mg/g. Determination of the relationship of the amount of tocopherol content to either source or hardness is not possible on the basis of our data.     

Lipid components and enzymatic hydrolysis of lipids in muscle of Chinese freshwater fish

The lipid and fatty acid composition of muscle of 10 species of freshwater fish obtained from a market of Shanghai City was examined. Total lipids (TL) ranged over 0.9–4.7% of muscle for all samples. The content of triacylglycerol (TG) in muscle ranged over 0.2–3.4% and that of polar lipids (PL) was 0.5–1.3%. Differences of TL content were dependent on TG contents. The predominant important fatty acids (>10% of the total fatty acids in TL) were 16∶0 and 18∶1n−9 with some 16∶1n−7, 18∶2n−6, and 22∶6n−3. The polyunsaturated fatty acids (PUFA) content was 10.2–43.4%, and especially Chinese sea bass contained above 20% of 22∶6n−3 in the total fatty acids. There were higher levels of PUFA such as 20∶5n−3 and 22∶6n−3 in PL than in neutral lipids. Muscle of the silver carp was stored at 20°C, and changes of lipid classes during storage were examined. Free fatty acids increased, and PL decreased during storage. This phenomenon was inhibited by heating the muscle, suggesting that lipid hydrolysis by phospholipase occurred in silver carp muscle.     

Composition of fatty acids and structure of triglycerides in medium and low erucic acid rapeseed

Total triglycerides in medium (MEAR) and low (LEAR) erucic acid cultivars of rapeseed were fractionated by argentation chromatography into twelve and ten fractions, respectively. Gas liquid chromatography of the fatty acids in the triglyceride fractions and their 2-monoglycerides was used to evaluate the structural characteristics of the individual fractions. Fractionation occurred on the basis of degree of unsaturation, molecular weight and positional characteristics. The most mobile fractions contained 34–50% of saturated fatty acids while the less mobile had 59–65% of polyunsaturated fatty acids. In the medium erucic acid oil, long chain fatty acids (C20–C22) were found in all fractions, but four fractions of low erucic acid oil were essentially free of long chain acids. Two of these fractions in the latter oil, which represented 44% of the total triglycerides, were glycerol trioleate and dioleoyllinoleoylglycerol. The majority of the 2-positions were occupied by unsaturated C18 fatty acids, generally in the order of linoleic ≥linolenic> oleic acids. The saturated and long chain fatty acids occurred predominantly in the 1-and 3-positions. The various fractions of medium and low erucic acid oils were similar in structural composition except that eicosenoic and erucic acids substituted for oleic acid in some external positions. Erucic acid did not appear to substitute directly for oleic acid in the 2-position.     

Composition of wax esters,triglycerides and diacyl glyceryl ethers in the jaw and blubber fats of the Amazon River dolphin (Inia geoffrensis)

The lower jaw fat of the Amazon River dolphinInia geoffrensis contains 52.8% wax ester, 44.7% triglyceride and 2.5% diacyl glyceryl ether, while its dorsal blubber fat is >98% triglyceride. Examination of the intact lipids, the derived fatty acids and the derived fatty alcohols by gas chromatography reveals that the blubber triglycerides show characteristics of freshwater fish fats, but the jaw fat lipids have several distinctive features. Jaw fat wax esters, triglycerides and diacyl glyceryl ethers are all rich in C₁₀, C₁₂ and C₁₄ fatty acids and contain no polyunsaturated acids. The fatty alcohols in the wax esters are over 90% saturated. The major carbon numbers in the jaw fat triglycerides (C₃₈–C₄₆) are considerably lower than those of the blubber triglycerides (C₄₈–C₅₄). The possible adaptation of the jaw lipids for use in the underwater echolocation process of this dolphin is discussed.     

Australian fish—An excellent source of both arachidonic acid and ω-3 polyunsaturated fatty acids

The fatty acid methyl esters obtained by the esterification of total lipids extracted from 24 species of fin fish and 4 species of invertebrates caught in the rivers and coastal waters of southern Australia were analyzed by gas chromatography. The lipids of most species contained significant levels of arachidonic acid (0.7–15.8%) as well as the more common marine polyunsaturate, eicosapentaenoic acid (0.7–15.9%). The major ω6 fatty acid present in most species was 20∶4; however, other fatty acids of this series, including 18∶2, 22∶4 and 22∶5, were present. The level of total ω6 fatty acids ranged from 3.9 to 22.3% of the total lipid. In general, the level of total ω3 polyunsaturates was higher than the total ω6 fatty acids with levels of ω3 fatty acids ranging from 9.6 to 48.2%. Only 2 fish (barramundi and gurnard perch) had ω6/ω3 ratios greater than 1.0. Most of the Australian species examined contained low levels of fat (0.5–7.8% of fresh weight). Two species examined, callop (freshwater) and blue groper (marine) contained sufficient quantities of both fat (7.7 and 7.8%) and arachidonic acid (4.8 and 9.3%) to warrant consideration for commercial exploitation.     

Comparison of lipid composition ofCandida guilliermondii grown on glucose,ethanol and methanol as the sole carbon source

The carbon and energy source for aerobically grown cultures ofCandida guilliermondii profoundly influenced the neutral lipid content and the fatty acid composition of the individual lipid components. Methanol (0.80%, w/v) grown cells cultivated at 30 C in presence of 0.025% ammonium sulfate contained 12% total lipids, 67% of which was neutral lipids. Glucose (0.74%, w/v) or ethanol (0.53%, w/v) grown cells contained 21–22% total lipids, 80% of which was neutral lipids, under the same conditions. Methanol-grown cells contained a decreased 18∶1 acid (52–54% of total fatty acids) and an increased 18∶2 acid (23–25%), as compared with glucose- or ethanol-grown cells which contained 57–66% 18∶1 acid and 8–14% 18∶2 acid, in both neutral and polar lipid fractions. The relationship between methanol metabolism and desaturation of fatty acid in yeast was discussed.     

Occurrence of 22∶3n−9 and 22∶4n−9 in the lipids of the topminnow (Poeciliopsis lucida) hepatic tumor cell line,PLHC-1

The lipids of the hepatic tumor cell line, PLHC-1, from the topminnow (Poeciliopsis lucida), were found to contain considerable amounts of a range of n−9 polyunsaturated fatty acids despite culture in serum containing significant amounts of essential fatty acids. The structural identity of all the n−9 polyunsaturated fatty acids was confirmed by gas chromatography/mass spectrometry. Of particular interest, PLHC-1 cell total lipid contained 1.9% of 22∶3n−9 and 3.3% of 22∶4n−9. As the culture medium contained virtually no n−9 polyunsaturated fatty acids, these fatty acids are all formed by the PLHC-1 cells, presumably form 18∶1n−9. The 22∶3n−9 and 22∶4n−9 are presumably formed by processes of elongation and “Δ4” desaturation of Mead acid, 20∶3n−9, present at over 11% in fatty acids of total lipid. Both 22∶3n−9 and 22∶4n−9 were primarily located in phosphatidylserine (4.1 and 8.5% respectively) and, to a lesser extent, in phosphatidylethanolamine (2.2 and 6.5%, respectively), in common with the C₂₂ derivatives of the n−3 and n−6 series, whereas 20∶3n−9 was preferentially located in phosphatidylinositol (31.2%). The results establish that long-chain polyunsaturated fatty acids of the n−9 series can be formed in vertebrate tissue other than in conditions of classical essential fatty acid deficiency.     

Quantitative and qualitative analyses of isolated lipid droplets from interstitial cells in renal papillae from various species

The lipid droplets of renal papillae homogenates from four different species were obtained by ultracentrifugation. Ca. 80–98% of the lipids (triglycerides, phospholipids, free fatty acids, and cholesterol esters) consist of triglycerides. The triglycerides were fractionated by argentation thin layer chromatography and each fraction characterized by gas liquid chromatography. No fraction contained any unique triglyceride. The fatty acid composition of the total triglycerides, as analyzed by gas liquid chromatography and ozonolysis, differed markedly from the fatty acid composition of the corresponding plasma triglycerides. The papillary triglycerides were characterized by higher concentrations of stearic acid, arachidic acid, and polyunsaturated acids with 20 or more carbon atoms. Particularly interesting was the presence in the lipid droplets of docosa-7,10,13,16-tetraenoic acid. This acid has been shown to be a major component in the cholesterol ester fraction of rat and canine adrenal lipids. In the papillary triglycerides, this acid accounted for 7%, 15%, and more than 20% of the total fatty acids in the dog, rat, and rabbit, respectively. The pig differs from these three species in having only ca. 1% of this acid. These observations suggest that the interstitial cells produce these triglycerides. This production could occur either by a transacylation from phospholipids and cholesterol esters and by a de novo synthesis from locally produced fatty acids. The possibility that the triglyceride production may be involved in a control of the prostaglandin production of the renal medulla is discussed.     

Nutritional Value of Seven Freshwater Fish Species From the Brazilian Pantanal

This study determined the proximate composition and fatty acid profile of the lipid fraction in muscle tissue (fillet) of seven fish species from the Miranda River, Brazil. Total lipid content had the largest coefficient of variation among species (73%), while protein content had the smallest (4.5%), allowing Pimelodus argenteus (mandi-prateado) to be categorized as lean fish; Pimelodus maculatus (mandi-amarelo), Hemisorubim platyrhynchos (jurupoca), and Pinirampus pirinampu (barbado) as species with medium fat content, and Paulicea luetkeni (jaú) and Surubim lima (jurupensém) as fatty fish. In all the species investigated, palmitic acid (23.76–25.99%) was the predominant saturated fatty acid. Oleic acid (16.09–32.90%) was the most abundant monounsaturated fatty acid. Total omega-6 polyunsaturated fatty acids (5.99–15.56%) were the predominant polyunsaturated fatty acids, except in Ageneiosus brevifilis (palmito), in which total omega-3 polyunsaturated fatty acids predominated (10.30%). All the species had favorable indices of nutritional quality for total lipids, with respect to human consumption.     

Total,polar and neutral lipids ofRhizopus arrhizus fischer

The changes in total lipid content, neutral and polar lipids, total fatty acids, and free fatty acids were investigated over a 4 day period in the zygomycete,Rhizopus arrhizus Fischer. The highest concentration of lipids occurred at the 72 hr period. The degree of unsaturation in the total fatty acid fraction increased during the growth period, whereas the degree of unsaturation decreased in the free fatty acid fraction during the same time period. The ratios of neutral to polar lipids over the 4 day period were: 0.75, 0.22, 1.94 and 0.94. The major components of polar lipids were phosphatidyl ethanolamine, lecithin, lysolecithin, and fatty acids. The fatty acids in the mono- and diglycerides were predominately saturated (67–96%). The fatty acids in the triglycerides shifted from a predominately unaturated (69%, 24 hr) to a more saturated pattern (62%, 96 hr).     

Composition of polar lipids in rapeseed

Phosphatidylcholine, -inositol and -ethanolamine represented 48, 18 and 8%, respectively, of the total phospholipids in medium and low erucic acid rapeseed. Oleic acid was the principal fatty acid in phosphatidylcholine and -ethanolamine but all components contained high levels of linoleic and saturated acids. There were eight components in the glycolipid fraction of which digalactosyl diglyceride, monogalactosyl diglyceride, sterol glycoside, esterified sterol glucoside and cerebrosides were identified. Defatted flours contained 2.0–3.5% of bound lipid which was composed primarily of phospholipids and glycolipids whereas neutral lipids were also a major component in the bound lipids of protein concentrates. The storage properties of these protein products would be adversely affected by the polyunsaturated fatty acids in these bound fractions.     

Temperature acclimation in the crayfish: Effects on phospholipid fatty acids

Acclimation to different temperatures by a poikilothermous animal must include modification of its membrane lipids to maintain the proper physical properties. The simplest way to achieve this acclimation would seem to be by modification of the phospholipid fatty acids. In a freshwater cray-fish,Procambarus clarkii, rapid changes in the degree of unsaturation of newly synthesized phospholipid fatty acids were correlated with changes in environmental temperature, both in whole animals and in slices of hepatopancreas tissue. At 5 C, the rate of fatty acid synthesis was about half that occurring at 23 C. Hepatopancreas tissue from animals acclimated to either 5 C or 23 C, when incubated for 2 hr at 5 C, incorporated a higher percentage of exogenous [1-¹⁴C] acetate into polyunsaturated acids (27–38% of the radioactivity in total fatty acids) than when incubated at 23 C (12–14%); conversely, more saturated fatty acids were synthesized at 23 C (73–80% vs 51–73%). The higher average unsaturation of the fatty acids biosynthesized at 5 C constitutes an effective response to the animal's need for modification of lipids to maintain adequate membrane function at the lower environmental temperature. Presented at the AOCS meeting in Mexico City, Mexico, April 27–May 2, 1974. Work done at the Laboratory of Nuclear Medicine and Radiation Biology, University of California, Los Angeles, CA 90024.