Metabolism of individual fatty acids during infusion of a triacylglycerol emulsion

The triacylglycerol emulsion Intralipid was infused into six normal subjects to investigate the metabolism of individual fatty acids in subcutaneous adipose tissue and forearm muscle’ by measurement of arteriovenous differences. The composition of plasma nonesterified fatty acids changed steadily after passage through adipose tissue and became similar to that of the emulsion’ reflecting hydrolysis of the Intralipid-triacylglycerol by lipoprotein lipase’ since endogenous lipolysis (hormone-sensitive lipase activity plus lipoprotein lipase hydrolysis of verylow density lipoprotein triacylglycerol) was decreased. There was no significant net release of total or individual fatty acids from forearm muscle although there was a tendency for the composition of the fatty acids in forearm venous plasma to change during passage through the tissue to reflect the composition of the emulsion. This may reflect hydrolysis of emulsion particles by lipoprotein lipase situated in capillaries which drain into the forearm vein. The behavior of stearic acid in the plasma nonesterified fatty acid pool was consistently aberrant’ with arterialized concentrations considerably higher than predicted from adipose tissue release’ both before and during Intralipid infusion. We conclude that there are no significant differences in the metabolism of specific fatty acids’ with the exception of stearic acid.     

Increasing short-chain fatty acid yield during lipase hydrolysis of a butterfat fraction with periodic aqueous extraction

Factors affecting the release of short-chain fatty acids during hydrolysis of a butterfat fraction with a 1,3-positional and short-chain-specific Penicillium roqueforti lipase were investigated. When a short-chain triglyceride fraction was used as substrate, as opposed to whole butterfat, the ratio of desirable flavor short-chain free fatty acids (FFA) to undesirable medium-chain FFA in the FFA fraction increased from 0.75 to 1.80. However, with both substrates, FFA accumulation eventually led to lipase inhibition and limited the total amount of triglyceride hydrolysis. This inhibition phenomenon was principally due to product inhibition. Periodically extracting the FFA with a buffer solution minimized this inhibition phenomenon, thereby significantly increasing lipase activity and the degree of triglyceride hydrolysis. Thus, on-line extraction of FFA in lipase reactors has the potential of greatly increasing system productivity.     

Increasing short-chain fatty acid yield during lipase hydrolysis of a butterfat fraction with periodic aqueous extraction

Factors affecting the release of short-chain fatty acids during hydrolysis of a butterfat fraction with a 1,3-positional and short-chain-specific Penicillium roqueforti lipase were investigated. When a short-chain triglyceride fraction was used as substrate, as opposed to whole butterfat, the ratio of desirable flavor short-chain free fatty acids (FFA) to undesirable medium-chain FFA in the FFA fraction increased from 0.75 to 1.80. However, with both substrates, FFA accumulation eventually led to lipase inhibition and limited the total amount of triglyceride hydrolysis. This inhibition phenomenon was principally due to product inhibition. Periodically extracting the FFA with a buffer solution minimized this inhibition phenomenon, thereby significantly increasing lipase activity and the degree of triglyceride hydrolysis. Thus, on-line extraction of FFA in lipase reactors has the potential of greatly increasing system productivity.     

Isolation of tocopherol and sterol concentrate from sunflower oil deodorizer distillate

The isolation of tocopherols and sterols together as a concentrate from sunflower oil deodorizer distillate was investigated. The sunflower oil deodorizer distillate was composed of 24.9% unsaponifiable matter with 4.8% tocopherols and 9.7% sterols, 28.8% free fatty acid (FFA) and 46.3% neutral glycerides. The isolation technology included process steps such as biohydrolysis, bioesterification and fractional distillation. The neutral glycerides of the deodorizer distillates were hydrolyzed byCandida cylindracea lipase. The total fatty acids (initial FFA plus FFA from neutral glycerides) were converted into butyl esters withMucor miehei lipase. The esterified product was then fractionally distilled in a Claisen-vigreux flask. The first fraction, which was collected at 180–230°C at 1.00 mm of Hg for 45 min, contained mainly butyl esters, hydrocarbons, oxidized products and some amount of free fatty acids. The fraction collected at 230–260°C at 1.00 mm Hg for 15 min was rich in tocopherols (about 30%) and sterols (about 36%). The overall recovery of tocopherols and sterols after hydrolysis, esterification and distillation were around 70% and 42%, respectively, of the original content in sunflower oil deodorizer distillate.     

Positional specificity of adipose tissue lipoprotein lipase

The positional specificity of preparations of lipoprotein lipase derived from rat epididymal adipose tissue was investigated. The enzyme preparations were a crude extract of acetone powder of the whole tissue, partially purified lipoprotein lipase fractions a and b separated by gel chromatography from such an extract, and lipoprotein lipase activity eluted from adipose tissue into a medium by incubation with heparin in vitro. The enzyme preparations were incubated with triglyceride substrate labeled with³H in the glycerol moiety and with¹⁴C in the fatty acid esterified to the 2 position of the glycerol. The reaction products were separated by thin layer chromatography. All preparations preferentially hydrolyzed the 1(3) ester bonds of the tri- and diglycerides, indicating that, like lipoprotein lipase from other sources, the adipose tissue enzyme has a specificity for the 1(3) position.     

Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction

A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.     

Metabolism of fatty acid,glycerol and a monoglyceride analogue by rat cardiac myocytes and perfused hearts

Studies have been conducted on the uptake and metabolism of unesterified fatty acid, free glycerol and 1-hexadecyl glyceryl ether by rat cardiac myocytes, and of fatty acid, intact triglyceride and the glyceryl ether by perfused rat hearts. Cardiac myocytes efficiently extracted, oxidized and esterified oleic acid, but demonstrated little ability to utilize free glycerol. Although the glyceryl ether was efficiently extracted by myocytes, it was neither hydrolyzed or esterified. The perfused heart also extracted and metabolized unesterified fatty acid, and the fatty acid released during lipolysis of circulating lipoprotein triglyceride. The glyceride glycerol, however, was largely recovered (90%) in the perfusate suggesting inefficient myocardial utilization of either free glycerol or partial glycerides. Myocardial extraction of glyceryl monoether was demonstrated, but the monoglyceride analogue was also unmetabolized by intact heart tissue. The results suggest that if monoglycerides are produced by the action of lipoprotein lipase on circulating triglycerides, reutilization of intact monoglycerides for higher glyceride synthesis is not a major fate of these products.     

Hydrolysis of triglycerides in the isolated perfused rat lung

The purpose of this study was to investigate the hydrolysis of saturated and unsaturated triglycerides by lung lipoprotein lipase and to measure the incorporation of triglyceride fatty acids into lung tissue lipids. Lipolytic activity was studied in the isolated ventilated rat lung, perfused for 100 min in a recycling system with Krebs Ringer bicarbonate containing bovine serum albumin, 5.6 mM glucose, and 1.5 or 10 mM triglyceride. Saturated triglycerides were hydrolyzed at significantly (p<0.05) lower rates than unsaturated triglycerides; tricaprylin, trimyristin and tripalmitin were hydrolyzed at 8.1+1.8, 5.4+1.5 and 9.5+1.8 μmol free fatty acids/g dry wt/100 min, respectively, whereas triolein and trilinolein were hydrolyzed at 20.2+1.8 and 20.6+0.3 μmol free fatty acids/g dry wt/100 min, respectively. The polyunsaturated triglycerides, trilinolein and triarachidonin were hydrolyzed at even higher rates (44.3+3.0 and 50.9+5.4 μmol free fatty acids/g dry wt/100 min, respectively). Intralipid infused at a concentration of 10 mM triglyceride was hydrolyzed at a significantly higher rate than at 1.5 mM triglyceride (58+6.3 μmol free fatty acids/g dry wt/100 min vs 16.6+1.7 μmol free fatty acids/g dry wt/100 min, respectively). Labeled unsaturated triglycerides were broken down at significantly higher rates than labeled saturated triglycerides. Incorporation of triglyceride-fatty acid into lung lipid was greater into neutral lipids than into phospholipids. The data suggest that (a) the factors that appear to affect lung lipoprotein lipase activity are composition and concentration of circulating triglyceride, (b) uptake of fatty acids into the tissue was proportional to the rate of hydrolysis of the emulsion, and (c) triglyceride-fatty acids could therefore be used by the lung for metabolic needs. The data presented in part at the Annual Meetings of the American Physiological Society, Atlanta, GA, April 1981, and the American Thoracic Society, Detroit, MI, May 1981, and published in abstract form-Fed. Proc. 40, 621 (1981), andAm. Rev. Respir. Dis. 123, 219 (1981).     

Animal endogenous triglycerides: I. Swine adipose tissue

The objective of this study was to define the composition of endogenous glycerides of swine adipose tissues as a basis for further studies on the influences of diet and other environmental factors. It was also hoped that the endogenous triglyceride composition might suggest a fresh approach to the problem of the biosynthetic pathway of natural triglyceride mixtures. Swine were fed a fat-free diet from the age of 17 days to 5 months, and the triglycerides of their mesenteric, perirenal and inner and outer back adipose tissues analyzed by silver ion thin layer chromatography and gas liquid chromatography. Four silver ion fractions were found, S₃, S₂M, SM₂ and M₃ (S, saturated; M, monounsaturated fatty acids). Corresponding fractions from different adipose tissues had identical fatty acid composition. The fatty acid compositions of the 2 position of the fractions were also identical. However, the fatty acid composition of the unfractionated triglycerides varied from tissue to tissue. It is concluded therefore, that the various adipose tissues of swine contain the same triglycerides in varying concentrations. Stearic and oleic acids were located mainly in the combined 1 and 3 positions. Myristic, palmitoleic and palmitic were in the 2 position, with almost 90% of the saturated acids being palmitic. This specific distribution of the major fatty acids can explain the marked simplicity of swine endogenous triglycerides.     

Effect of dietary fat supplementation on the composition and positional distribution of fatty acids in ruminant and porcine glycerides

Dietary fats which were protected from ruminal metabolism were fed to ruminants, and the constituent fatty acids subsequently appeared in the glycerides of tissues and secretory products. These dietary fat induced alterations in tissue lipid composition were particularly apparent when the fat source was enriched with linoleic acid. Similarly, when pigs were fed linoleic-enriched fats, the linoleic acid was incorporated into the adipose tissue triglycerides. Stereospecific analyses were carried out on triglycerides from various tissues and secretory products obtained from animals fed control or linoleate-enriched diets. The analysis of adipose tissue triglycerides showed that linoleate and oleate were preferentially esterified to positions 2 and 3 (cattle and sheep), and positions 1 and 3 (pigs). Of the other major adipose tissue fatty acids, palmitate was preferentially esterified at position 1 (ruminants) and position 2 (pigs), and stearate was preferentially esterified at positions 1 and 3 (ruminants), and position 1 (pigs). Stereospecific analysis of high mol wt milk triglycerides showed that linoleate was either evenly distributed on all three positions (goats), or predominantly on position 3 (cows). Furthermore, the incorporation of this linoleate did not markedly alter the positional specificity of the other major milk triglyceride fatty acids. Of these fatty acids, the short and medium chain length acids (butyratelaurate) were mainly on position 3, myristate and palmitate on positions 1 and 2, and stearate and oleate evenly distributed. Thoracic duct lymph triglycerides from sheep tended to show preferential incorporation of linoleate at position 3, palmitate at position 2, and stearate at position 1 and 3; oleate, on the other hand, tended to be evenly distributed on all three positions of the lymph triglyceride. The stereospecific arrangement of fatty acids in sheep liver triglycerides was similar to that of lymph triglycerides, and this may reflect the uptake of intact or partially hydrolysed chylomicron and/or very low density lipoprotein triglycerides by the liver. There were also some analogies in the stereospecific arrangement of fatty acids on ruminant lymph and milk triglycerides and this may reflect an incomplete hydrolysis of chylomicron and/or very low density lipoprotein triglycerides prior to uptake by the mammary gland. An unusual feature of lymph from sheep fed linoleate was the presence of phospholipids which contained large amounts of linoleate in ca. equal proportion at both positions 1 and 2 of the phospholipid molecule.     

Glyceride distribution in adipose and liver glycerides of animals

The glyceride distribution in depot fats from a series of animals was determined by pancreatic lipase hydrolysis, isolation of hydrolytic products by thin-layer chromatography (TLC), and fatty acid analysis by gas-liquid chromatography (GLC). Distribution of the principal types of glycerides (S₃, S₂U, SU₂ U₃) in the internal and external adipose tissue fats from the same pig was nonrandom. The percentages of palmitic acid at the 2-position in these adipose fats were comparable. However, liver glycerides from this same animal differed strikingly from adipose glycerides, having, for example, only ca. 15% of its palmitic acid in the 2-position compared with > 80% for adipose fats. The liver glycerides of lamb, rabbit, and dog also differed considerably from adipose glycerides in glyceride distribution and in percentages of individual fatty acids in 2-position. The composition of adipose glycerides from Lamb, beef, deer, rabbit, chicken, and dog in terms of the four principal glyceride types approached closely the values calculated for random. When positional isomers were considered, however, only the adipose glycerides of the dog conformed to random distribution. Presented at the AOCS meeting, Chicago, October 11–14, 1964. E. Utiliz. Res. and Devel. Div., ARS, USDA.     

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.     

The hydrolysis of very low density lipoproteins and chylomicrons of intestinal origin by lipoprotein lipase in ruminants

The hydrolysis by lipoprotein lipase of a very low density lipoprotein/chylomicron fraction, obtained from the intestinal lymph of sheep, has been studied in vitro. Rapid hydrolysis of triacylglycerols, with an accumulation of free fatty acids, was observed. After an initial lag period, phosphatidylcholine also was hydrolyzed. No specificity for particular fatty acids in the triacylglycerols (or phosphatidylcholines) was observed.     

Effect of marginal zinc deficiency on lipoprotein lipase activities in postheparin plasma,skeletal muscle and adipose tissues in the rat

The activities of lipoprotein lipase in postheparin plasma, retroperitoneal adipose and gastrocnemius muscle tissues were determined in the rats fed 2.8 ppm of dietary zinc for eight weeks, as compared with pair-fed and ad libitumfed rats given 30.8 ppm of zinc. The postheparin lipoprotein lipase activity, as determined by using a lipid emulsion labeled with [³H]triolein as the substrate, was significantly lower in the first group of rats, relative to that in the second and third groups. Tissue lipoprotein lipase activities were compared using the lipid emulsion and activator serum obtained from the zinc-deficient rats and the ad libitum-fed rats. The activator sera were devoid of very low density and low density lipoproteins, but enriched in high density lipoproteins. Muscle lipoprotein lipase activities were significantly lower when assayed with the activator serum from the zinc-deficient compared with the activities determined with the activator serum from the ad libitum-fed. Similarly, muscle lipoprotein lipase activities were lower in all groups when [³H]-triolein-labeled chylomicrons from the zinc-deficient were used as the substrate, compared with the activities determined using the chylomicrons from the ad libitum-fed. Lipoprotein lipase activities in the adipose tissues were not affected by the different sources of the activator sera and chylomicrons. The results strongly suggest that the decrease in postheparin lipoprotein lipase activity in zinc deficiency is not due to changes in tissue lipoprotein lipase enzyme per se, but to compositional alterations in with regard to C apolipoproteins, modulators of lipoprotein lipase activity. Presented in abstract form at the 71st annual meeting of the Federation of American Societies for Experimental Biology.Fed. Proc. 46:1472, 1987.     

Isolation of erucic acid from rapeseed oil by lipase-catalyzed hydrolysis

Three lipases were compared for their ability to hydrolyze high erucic acid rapeseed oil, with the objective of concentrating the erucic acid in a single glyceride fraction. Lipase fromPseudomonas cepacia released all fatty acids rapidly and did not result in selective distribution of erucic acid.Geotrichum candidum lipase released C20 and C22 fatty acids extremely slowly, resulting in their accumulation in the di- and triglyceride fractions. Less than 2% of the total erucic acid was found in the free fatty acid (FFA) fraction. Lipase fromCandida rugosa released erucic acid more slowly than C20 and C18 fatty acids at 35°C but only resulted in a limited accumulation of the erucic acid in the di- and triglyceride fractions. However, when hydrolysis catalyzed byC. rugosa lipase was carried out below 20°C, the reaction mixture solidified and was composed solely of FFAs and diglycerides. The diglyceride fraction contained approximately 95% erucic acid while about 20% of the total erucic acid was found in the FFA fraction. It is concluded that hydrolysis at low temperature withC. rugosa lipase results in a higher purity of erucic acid in the glyceride fraction than can be obtained withG. candidum lipase, but with considerable loss of erucic acid to the FFA fraction.     

Enrichment of arachidonic acid: Selective hydrolysis of a single-cell oil fromMortierella withCandida cylindracea lipase

Three lipases, isolated previously in our laboratory, and a known lipase fromCandida cylindracea were screened for the enrichment of arachidonic acid (AA). The enzyme fromC. cylindracea was the most effective for the production of oil with high concentration of AA. When a single-cell oil fromMortierella alpina, containing 25% AA, was hydrolyzed with this lipase for 16 h at 35°C, the resulting glycerides contained 50% AA at 52% hydrolysis. After this, no further hydrolysis occurred, even with additional lipase. However, when the glycerides were extracted from the hydrolyzate and were hydrolyzed again with new lipase, the resulting oil contained 60% AA, with a recovery of 75% of its initial AA content. Triglycerides were the main components of the resulting oil. The release of each fatty acid from the oil depended on the hydrolysis rate of its ester. The fatty acid, whose ester is the poorest substrate for the enzyme, is concentrated in the glycerides.     

Studien zur Glyceridstruktur von Fetten VIII: Vergleichende Strukturuntersuchungen an ausgewählten natürlichen Triglyceriden mit Pankreaslipase,der Lipase von Geotrichum candidum und Äthylmagnesiumbromid

Studies on the Glyceride Structure of Fats VIII: Comparative Structural Studies of Selected Natural Triglycerides Using Pancreas Lipase, Lipase from Geotrichum candidum, and Ethyl Magnesiumbromide Studies on olive, poppyseed, linseed and rapeseed triglycerides using pancreas lipase, phospholipase A, ethyl magnesium-bromide, and lipase from Geotrichum candidum revealed that cis-9 unsaturated fatty acids preferentially occupy the position 2 of glycerides, whereas saturated fatty acids occupy preferentially the 1 and 3 positions. Erucic acid present in rapeseed oil is mainly located in the primary positions of glycerides. A method for calculating the individual classes of glycerides has been developed, which is based on the fatty acid composition of partial glycerides that are formed on hydrolysis of natural glycerides by the lipase from Geotrichum candidum. This method is applicable to glycerides which contain at least one cis-9 unsaturated fatty acid. The usefulness of this direct method for the determination of individual classes of glycerides was established by comparing the results with those reported in the literature that were obtained using other methods.     

Wide variations of plasma triglyceride concentrations in guinea pigs

Guinea pigs have varying plasma triglyceride concentrations ranging from 28 to 1392 mg/dl, with relatively uniform plasma cholesterol and phospholipid levels. To understand why the animals exhibit such wide variations of plasma triglyceride concentrations, we have explored the triglyceride hydrolyzing system by measuring tissue lipoprotein lipase activities and plasma activator for the enzyme. Lipoprotein lipase activities of epididymal adipose tissue of these animals were 759±117 (mean±SE) n moles FFA·min⁻¹·g wet tissue⁻¹, markedly low compared with those of rats. There were no relationships between plasma triglyceride concentrations and tissue lipase activities. Plasma activator for lipoprotein lipase was lacking in this animal. Guinea pigs with ascorbic acid deficiency for 2 weeks also showed marked variations of plasma triglyceride concentrations, without any changes in tissue lipoprotein lipase activities. Low adipose tissue lipoprotein lipase activities with deficient plasma activator for the enzyme suggest that the lipoprotein lipase-mediated triglyceride degradation could be impaired in this animal, and this may account for the marked variation of plasma triglyceride concentrations.     

Synthesis of docosahexaenoic acid-rich triglyceride with immobilizedChromobacterium viscosum lipase

Docosahexaenoic acid (DHA) in the free fatty acid (FFA) derived from enzymically hydrolyzed tuna oil was concentrated by partial titration and precipitation of other FFA as sodium salts with acetone. A triglyceride containing up to 46.2% DHA was synthesized from the DHA-rich glyceride mixture and FFA by use of an immobilizedChromobacterium viscosum lipase.     

Studies on positional specificity of the castor bean acid lipase

The acid lipase of castor bean endosperm catalyzed the hydrolysis of fatty acids from the 1 and 3 positions of synthetic glycerides immediately after achieving proper reaction conditions, but fatty acids from the 2 position were not detected in the reaction products until 7 to 10 min later. Results obtained with 2,3-butane dioleate, n-hexyl oleate and 2-hexyl oleate showed that the castor lipase does not cleave secondary ester linkages. These findings suggest that the acid lipase of the castor bean may catalyze hydrolysis of fatty acids from the 1 and 3 positions of triglycerides only; the steady appearance of 2 position fatty acids in the reaction products during lipolysis is probably the result of an apparent isomerization reaction. So. Utiliz. Res. Dev. Div., ARS, USDA.