A simple method for the separation of triacylglycerols from fatty acids released in lipase assays

A simple procedure for the partition of triacylglycerols from albumin-bound fatty acids is described. This procedure is based on the ability of fumed silicon dioxide to remove emulsified triacylglycerols from aqueous media. The method was developed for the assay of lipoprotein lipase activity but it may be used for the assay of other lipases.     

Validation of a "clearing" assay for milk lipoprotein lipase in agarose gel

We have developed a simplified method for the quantitative assay of lipoprotein lipase in cow's milk based on the hydrolysis of a glyceride emulsion in semisolid agarose gel. The area of clearing produced thereby is a function of enzyme concentration. Absolute molar rates for unknown samples may be calculated if standards of known activity are used concurrently. The precision of the simplified assay compared favorably with a method based on titrimetric determination of the rate of free fatty acid release. A modified assay has been used to assess the potency of lipoproteins in lipoprotein lipase activation.     

Role of lipoprotein lipase in regulating endogenous triacylglycerols in rat heart

Administration of glucagon (10 μg/rat) to the intact animal increased the levels of lipoprotein lipase activity by 92% in the heparin-non-releasable fraction of the heart. At the same time, cardiac levels of triacylglycerols were reduced 47% and free fatty acids were increased about 2-fold. In contrast, the administration of a lower dose of glucagon (0.5 μg/rat) resulted in an 80% reduction in lipoprotein lipase activity in the heparin perfused myocardium. At the same time, triacylglycerols were increased 44% and free fatty acids were decreased 69%. These results provide circumstantial evidence that lipoprotein lipase is involved in the regulation of endogenous triacylglycerols such that higher levels of enzyme activity result in cardiac lipolysis and, conversely, lower levels result in triacylglycerol production.     

Combined (n-3 and n-6) essential fatty deficiency is a potent modulator of plasma lipids, lipoprotein composition, and lipolytic enzymes

Essential fatty acids (EFA) are important structural and functional components of cell membranes. Their deficiency has been associated with several clinical and biochemical abnormalities. In the present study, the lipid profile as well as the concentration, composition, and metabolism of lipoproteins were examined in rats rendered EFA-deficient over a period of 12 weeks. Changes in plasma fatty acids mainly induced an increase of palmitoleic (16:1 n-7) and eicosatrienoic (20:3 n-9) acids, while linoleic (18:2 n-6), arachidonic (20:4 n-6), linolenic (18:3 n-3), and docosahexaenoic (22:6 n-3) acids were decreased. The results show increased concentrations of free fatty acids (FFA) (P less than 0.001), triglycerides (P less than 0.001), total cholesterol (P less than 0.02), free cholesterol (P less than 0.005), and phospholipids (P less than 0.05) when compared to pair-fed controls. Similar levels of cholesteryl esters were found in the two groups, and lecithin: cholesterol acyltransferase activity (nmol/100 microliters plasma per h) (8.98 +/- 1.44 vs 8.72 +/- 0.50) did not differ. On the other hand, postheparin extrahepatic lipoprotein lipase (LPL) activity was significantly (P less than 0.002) decreased (5.96 +/- 0.29 vs 7.29 +/- 0.68 mumol FFA/ml per h) and could account for the hypertriglyceridemia as well for the relative triglyceride enrichment of very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein particles. This enzymatic depletion of LPL was mainly due to the adipose tissue, since a higher level (P less than 0.001) of hepatic lipase (325.8 +/- 16.0 vs 130.8 +/- 9.5 nmol FFA/mg protein per h) was found in liver acetone powder extracts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver.

The subcellular distributions of acidic (pH 4.5) and neutral (pH 7.5) longchain triacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction;the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate 90-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 mM emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60 degrees C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.     

Simultaneous conversion of free fatty acids and triglycerides to biodiesel by immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase

The presence of high levels of free fatty acids (FFA) in oil is a barrier to one‐step biodiesel production. Undesirable soaps are formed during conventional chemical methods, and enzyme deactivation occurs when enzymatic methods are used. This work investigates an efficient technique to simultaneously convert a mixture of free fatty acids and triglycerides (TAG). A partial soybean hydrolysate containing 73.04% free fatty acids and 24.81% triglycerides was used as a substrate for the enzymatic production of fatty acid methyl ester (FAME). Whole‐cell Candida antarctica lipase B‐expressing Aspergillus oryzae, and Novozym 435 produced only 75.2 and 73.5% FAME, respectively. Fusarium heterosporum lipase‐expressing A. oryzae produced more than 93% FAME in 72 h using three molar equivalents of methanol. FFA and TAG were converted simultaneously in the presence of increasing water content that resulted from esterification. Therefore, F. heterosporum lipase with a noted high level of tolerance of water could be useful in the industrial production of biodiesel from feedstock that has high proportion of free fatty acids.     

Regulation of lipid flux between liver and adipose tissue during transient hepatic steatosis in carnitine-depleted rats

Rats with carnitine deficiency due to trimethylhydrazinium propionate (mildronate) administered at 80 mg/100 g body weight per day for 10 days developed liver steatosis only upon fasting. This study aimed to determine whether the transient steatosis resulted from triglyceride accumulation due to the amount of fatty acids preserved through impaired fatty acid oxidation and/or from up-regulation of lipid exchange between liver and adipose tissue. In liver, mildronate decreased the carnitine content by approximately 13-fold and, in fasted rats, lowered the palmitate oxidation rate by 50% in the perfused organ, increased 9-fold the triglyceride content, and doubled the hepatic very low density lipoprotein secretion rate. Concomitantly, triglyceridemia was 13-fold greater than in controls. Hepatic carnitine palmitoyltransferase I activity and palmitate oxidation capacities measured in vitro were increased after treatment. Gene expression of hepatic proteins involved in fatty acid oxidation, triglyceride formation, and lipid uptake were all increased and were associated with increased hepatic free fatty acid content in treated rats. In periepididymal adipose tissue, mildronate markedly increased lipoprotein lipase and hormone-sensitive lipase activities in fed and fasted rats, respectively. On refeeding, carnitine-depleted rats exhibited a rapid decrease in blood triglycerides and free fatty acids, then after approximately 2 h, a marked drop of liver triglycerides and a progressive decrease in liver free fatty acids. Data show that up-regulation of liver activities, peripheral lipolysis, and lipoprotein lipase activity were likely essential factors for excess fat deposit and release alternately occurring in liver and adipose tissue of carnitine-depleted rats during the fed/fasted transition.     

A stable, radioactive substrate emulsion for assay of lipoprotein lipase.

A method is described for the assay of lipoprotein lipase, using a stable, radioactive substrate emulsion. Fatty acid-labeled trioleoylglycerol was emulsified by homogenization in glycerol with lecithin as detergent. This anhydrous emulsion was stable for at least six weeks. Substrate solutions for enzyme assay were prepared by diluting the emulsion with buffer containing serum and albumin. The fatty acid produced on hydrolysis was isolated in a one-step liquid-liquid partition system. Incubations with extracts of acetone powders from adipose tissue displayed characteristics of lipoprotein lipase activity, i.e., serum dependence and inhibition by NaCl and protamine. The method is rapid (less than 1 hour), sensitive and reproducible, and suitable for routine use.     

Contribution of triglyceride-rich lipoproteins to plasma free fatty acids.

Free fatty acids are the major lipid fuel of the body. Dysregulation of adipose tissue lipolysis results in increased plasma free fatty acid concentrations, and via that mechanism contributes to insulin resistance in obesity and type 2 diabetes mellitus. Adipose tissue hormone sensitive lipase is thought to be responsible for the production of the majority of free fatty acids. However, a separate contribution comes from the action of endothelial lipases, especially lipoprotein lipase, on triglyceride-rich lipoproteins via a process known as spillover. The primary substrate for spillover appears to be chylomicrons derived from dietary fat. The spillover of fatty acids into the free fatty acid pool varies from one tissue to another. For example, spillover is low ( approximately 14%) in the forearm of healthy volunteers, suggesting that triglyceride fatty acid storage is relatively efficient in skeletal muscle. In contrast, spillover appears to be higher in adipose tissue and may also be higher in the splanchnic bed, based on preliminary data. If systemic spillover is increased in insulin resistant states such as diabetes, this could represent a mechanism contributing to the abnormal increases in plasma concentrations of free fatty acids in that condition.     

Lipoprotein lipase activity and its relationship to high milk fat transfer during lactation in grey seals

Lipoprotein lipase regulates the hydrolysis of circulating triglyceride and the uptake of fatty acids by most tissues, including the mammary gland and adipose tissue. Thus, lipoprotein lipase is critical for the uptake and secretion of the long-chain fatty acids in milk and for the assimilation of a high-fat milk diet by suckling young. In the lactating female, lipoprotein lipase appears to be regulated such that levels in adipose tissue are almost completely depressed while those in the mammary gland are high. Thus, circulating fatty acids are directed to the mammary gland for milk fat production. Phocid seals serve as excellent models in the study of lipoprotein lipase and fat transfer during lactation because mothers may fast completely while secreting large quantities of high fat milks and pups deposit large amounts of fat as blubber. We measured pup body composition and milk fat intake by isotope (deuterium oxide) dilution and plasma post-heparin lipoprotein lipase activity in six grey seal (Halichoerus grypus) mother-pup pairs at birth and again late in the 16-day laction period. Maternal post-heparin lipoprotein lipase activity increased by an average of four-fold by late lactation (P=0.027), which paralleled an increase in milk fat concentration (from 38 to 56%; P=0.043). Increasing lipoprotein lipase activity was correlated with increasing milk fat output (1.3–2.1 kg fat per day) over lactation (P=0.019). Maternal plasma triglyceride (during fasting) was inversely correlated to lipoprotein lipase activity (P=0.027) and may be associated with the direct incorporation of longchain fatty acids from blubber into milk. In pups, post-heparin lipoprotein lipase activity was already high at birth and increased as total body fat content (P=0.028) and the ratio of body fat: protein incrased (P=0.036) during lactation. Although pup plasma triglyceride increased with increasing daily milk fat intake (P=0.023), pups effectively cleared lipid from the circulation and deposited 70% of milk fat consumed throughout lactation. Lipoprotein lipase may play an important role in the mechanisms involved with the extraordinary rates of fat transfer in phocid seals.Abbreviations FFA free fatty acid - HL hepatic lipase - LPL lipoprotein lipase - PH-HL post-heparin hepatic lipase - PH-LPL post-heparin lipoprotein lipase - VLDL very low density lipoprotein     

Properties of triacylglycerol lipase in a mitochondrial fraction from baker's yeast (Saccharomyces cerevisiae).

A triacylglycerol lipase in a mitochondrial fraction isolated from yeast (Saccharomyces cerevisiae) has been characterized and the hydrolysis studied kinetically using an insoluble artificial triacylglycerol suspension. 1. The triacylglycerol was hydrolyzed almost completely to fatty acids and glycerol. The lipase activity was inhibited by potassium fluoride and the sodium salts of -chloride, -glycocholate and -pyrophosphate as well as by protamine sulfate but at concentrations much too high to indicate that the lipase is a non specific esterase or a lipoprotein lipase. Also parachloromercuribenzoate inhibited the lipase activity. Inhibitory effect of fatty acid was observed at concentrations above 1mM. This inhibition may provide a regulatory mechanism of the lipase in vivo. 2. On the day of isolation the lipase activity of intact mitochondria at pH 7.5 and 30 degrees C was 400 nmol free fatty acid -h-1 - mg-1 at a triacylglycerol concentration of 9.0 mM. Sonication of the mitochondria increased the activity 2-3 fold. Freezing of the mitochondria also activated the lipase and this activation was dependent upon the freezing method, the concentration of mitochondrial protein and the presence of bovine serum albumin. 3. The particulate nature of the assay system was illustrated by the observation that the apparent Km value of the lipase increased with the concentration of mitochondrial protein. For each protein concentration the lipase had two apparent Km values when the activity was assayed with intact mitochondria, but only one when assayed with submitochondrial particles. At the same protein concentration the Km value for the latter was identical with the "low affinity" Km for the lipase in intact mitochondria.     

Endothelium,the dynamic interface in cardiac lipid transport

Vascular endothelium is the dynamic interface in transport of lipid from blood to myocytes in heart and arteries. The luminal surface of endothelium is the site of action of lipoprotein lipase on chylomicrons and VLDL and the site of uptake of fatty acids from albumin. Fatty acids and monoacylglycerols are transported from the lumen in an interfacial continuum of endothelial and myocyte membranes. Lipoprotein lipase is transferred from myocytes to the vascular lumen, and is anchored there, by proteoheparan sulfate in cell membranes. Insulin, needed for synthesis of lipoprotein lipase and esterfication of fatty acids, is captured from the blood stream and delivered to myocytes by endothelial insulin receptors. Fatty acids, monoacylglycerols, lipoprotein lipase and insulin are transported along the same route, but by different mechanisms. The route involves the plasma membrane of endothelium and myocytes, the membrane lining transendothelial channels, and intercellular contacts. (Mol Cell Biochem116: 181–191, 1992)     

A method for the determination of lipoprotein lipase in postheparin plasma and body tissues utilizing a triolein-coated Celite substrate

A simple and specific method for assaying lipoprotein lipase activity is described. Postheparin plasma, heart homogenates, or extracts of acetone powder of adipose tissue were incubated with a triolein-coated Celite substrate, and enzyme activity was determined from the rate of free fatty acid (FFA) release in the incubation system. FFA release was linear for 30 min, and was proportional to protein concentration in the incubation system. FFA release was decreased by addition of deoxycholate or Triton X-100. Increasing the concentration of heparin in the incubation system caused a gradual decrease in FFA release by postheparin plasma and increases in activity of heart homogenates and adipose tissue lipoprotein lipase. The Celite substrate was found to be satisfactory for assaying pancreatic lipase activity as well.     

Genesis of fatty liver and hyperlipemia in the fetal guinea pig.

10 to 20% of [1-14C] palmitate injected into pregnant guinea pigs was recovered in lipids of their fetuses. From these data and the rate of transport of palmitate in maternal blood, it appears that placental transport of free fatty acids can account for the accumulation of lipids in late gestational fetuses. About 80% of the labeled palmitate in the fetus appeared initially in lipids of the liver. 14C appeared in plasma triglyceride fatty acids after a few minutes and subsequently accumulated in lipids of white and brown adipose tissue, suggesting that much of the palmitate deposited in adipose tissue were derived from hepatogenous triglyceride fatty acids. By contrast, 14C was usually maximal in heart and carcass lipids before it appeared in plasma triglyceride fatty acids. Lipoprotein lipase activity in fetal adipose tissue was low, and activity of cofactor protein of lipoprotein lipase in fetal blood plasma was much lower than that observed in other mammalian species. On the basis of these and earlier observations, it is concluded that the accumulation of triglycerides in liver and blood plasma of fetal guinea pigs during late gestation is at least partly the result of the large uptake of maternally derived free fatty acids by the fetal liver accompanied by rapid synthesis and secretion of triglyceride-rich very low density lipoproteins into the blood. However, limited uptake of triglyceride fatty acids in adipose tissue may contribute to the fatty liver and hyperlipemia.     

Regulation of lipoprotein lipase activity in cardiac myocytes from control and diabetic rat hearts by plasma lipids.

The objective of this investigation was to test the hypothesis that the diabetes-induced reduction in lipoprotein lipase activity in cardiac myocytes may be due to hypertriglyceridemia. Administration of 4-aminopyrazolopyrimidine (50 mg/kg) to control rats for 24 h reduced plasma triacylglycerol levels and increased the heparin-induced release of lipoprotein lipase into the incubation medium of cardiac myocytes. The acute (3-5 days) induction of diabetes by streptozotocin (100 mg/kg) produced hypertriglyceridemia and reduced heparin-releasable lipoprotein lipase activity in cardiac myocytes. Treatment of diabetic rats with 4-aminopyrazolopyrimidine resulted in a fall in plasma triacylglycerol content and increased heparin-releasable lipoprotein lipase activity. Administration of Triton WR-1339 also resulted in hypertriglyceridemia, but the heparin-induced release of lipoprotein lipase from control cardiac myocytes was not reduced in the absence of lipolysis of triacylglycerol-rich lipoproteins. Treatment with Triton WR-1339 did, however, increase the heparin-induced release of lipoprotein lipase from diabetic cardiac myocytes. Preparation of cardiac myocytes with 0.9 mM oleic acid resulted in a decrease in both total cellular and heparin-releasable lipoprotein lipase activities. These results suggest that the diabetes-induced reduction in heart lipoprotein lipase activity may, at least in part, be due to an inhibitory effect of free fatty acids, derived either from lipoprotein degradation or from adipose tissue lipolysis, on lipoprotein lipase activity in (and (or) release from) cardiac myocytes.     

A novel method for measurement of triglyceride lipase activity: suitable for microgram and nanogram quantities of tissue

The measurement of triglyceride lipase activity in microgram and nanogram quantities of tissue is reported. The method involves quantitation of glycerol released from a triglyceride substrate, which is shown to provide a value of approximately one-third of that obtained by quantitation of free fatty acid release. Influences on glycerol release, including pH optimum, NaCl inhibition, and activation by serum and heparin are characterized. Two separate assays are described for the measurement of glycerol that yield identical results with nanogram quantities of tissue. The advantage of one assay is its simplicity, while the advantage of the other is that it can be adjusted to measure very small tissue samples (nanogram) with the use of microanalytical procedures (i.e., enzymatic amplification of the NAD+ product of glycerol analysis). Sensitivity of the method is demonstrated by the analysis of triglyceride lipase activity in nanogram samples of single soleus muscle fibers. Measurement of picomole quantities of glycerol produced by lipase activity in single muscle fibers represents at least a 1,000-fold increase in sensitivity compared to currently available methods.     

Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase

The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and human hepatic lipase was examined in vitro. Chylomicrons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentaenoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the C14-C18 acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eicosatrienoic, docosatrienoic and docosahexanoic acids exhibited an intermediate lipolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 20:4 esters of both tri- and diacylglycerols. Addition of NaCl (final concentration 1 M) during the course of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylglycerol lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 20:4 and 20:5 esters thus exhibit a relative resistance to lipoprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids may accumulate at the surface of the remnant particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase.     

Characterization of enzymes involved in formation of ethyl esters of long-chain fatty acids in humans.

Elevated fatty acid ethyl ester (FAEE) concentrations have been detected in postmortem organs from alcoholics and patients acutely intoxicated by alcohol, and FAEE have been implicated as mediators of ethanol-induced organ damage. The formation of FAEE is catalyzed by acyl-coenzyme A:ethanol O-acyltransferase (AEAT) and by FAEE synthase, which utilize acyl-CoA and free fatty acids, respectively, as substrates. Because little is known about the capacity of various human tissues to synthesize and hydrolyze FAEE, we investigated formation of FAEE by AEAT and FAEE synthase in tissue homogenates from human gastric ventricular and duodenal mucosa, pancreas, liver, heart, lung, and adipose tissue, gallbladder mucosa, and in serum. Liver, duodenal mucosa, and pancreas were found to have the highest capacities to synthesize FAEE, mainly due to AEAT. FAEE hydrolyzing activity was highest in liver and pancreas, but hardly detectable in adipose tissue or heart. Because fatty acids and alcohol are absorbed by the intestinal mucosa, intestine may be a major site of FAEE synthesis, and FAEE may be delivered via the circulation to other organs and taken up by lipoprotein receptor-mediated uptake. A very low rate of FAEE hydrolysis was detected in heart and adipose tissue, which probably accounts for the previously observed accumulation of FAEE in these organs.     

Lipolysis products promote the formation of complexes of very-low-density and low-density lipoproteins

In the course of lipolysis, surface lipid products may accumulate on very-low-density lipoproteins (VLDL). To investigate potential lipoprotein interactions mediated by such products, radiolabeled low-density lipoproteins (LDL) were incubated with VLDL and bovine milk lipoprotein lipase in the presence of limited free fatty acid acceptor. With partial VLDL degradation, association of radiolabeled LDL with VLDL remnants or larger aggregates of VLDL density was demonstrated by gradient gel electrophoresis, agarose chromatography, and density gradient ultracentrifugation. VLDL-LDL complex formation was also observed in incubations with lipid extracts from lipolyzed VLDL or with purified palmitic acid in the absence of lipolysis. Complex formation was inhibited by addition of increasing amounts of albumin as free fatty acid acceptor, but could be detected at molar ratios of free fatty acids/albumin that occur in vivo. Composition analysis of LDL reisolated following incubation with VLDL and lipase under conditions favoring partial complex formation revealed enrichment in glycerides and depletion of cholesterol. We conclude that lipolysis products can promote the formation of stable complexes of LDL and VLDL, and that physical interactions of this nature may play a role in the transfer of lipids and apolipoproteins between lipoprotein particles.