Study on immunocapture-chemiluminescence assay of lipase activity in a biological sample.

A new approach for the determination of lipase (triacylglycerol lipase, EC.3.1.1.3) activity in a biological sample was investigated by combining an immunocapture technique with a chemiluminescence (CL) assay method in order to eliminate interference with CL detection. The proposed method consists of an immunocapture step to trap lipase and a subsequent step for CL detection of the activity of the captured lipase. The CL detection is based on the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) reaction and utilizes a proenhancer substrate [a lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI)] which liberates an active enhancer, HDI, by enzymatic hydrolysis. A polyclonal antibody prepared with porcine pancreas lipase was used for the immunocapture. The proposed immunocapture-CL method effectively eliminated the interference with the CL reaction from biological components and enabled the determination of spiked porcine pancreas lipase activity in serum samples in the range 0.41-1.1 U(HDI) (1 U(HDI) corresponds to the amount which liberates 1 pmol HDI/min at 37 degrees C from the substrate). The method was further applied to the assay of the activity for human pancreas lipase in serum and the results showed good correlation (r = 0.871) with those by the conventional colorimetric method.     

Human monocytes in culture synthesize and secrete lipoprotein lipase

Within the first day in culture, human monocytes begin to synthesize and secrete a triglyceride lipase. The designation of this activity as lipoprotein lipase is based upon: 1) a requirement of serum or apolipoprotein C-II for full activity; 2) inhibition by 1M NaCl or apolipoprotein C-III₂; 3) a pH optimum of 8; and 4) binding to endothelial cells that is releasable by heparin. The enzyme also exhibits immunological cross reactivity with antibody to purified bovine milk lipoprotein lipase as does human postheparin plasma lipoprotein lipase. Lymphocytes and polymorphonuclear leukocytes do not appear to contain this enzyme.     

Measurement of lipoprotein lipase and hepatic triacylglycerol lipase in post-heparin plasma of the cynomolgus monkey

Conditions for measurement of the lipolytic activities, lipoprotein lipase and hepatic triacylglycerol lipase in cynomolgus monkey postheparin plasma are described. The two activities are separable by heparin-Sepharose chromatography. Goat anti-human hepatic triacylglycerol lipase serum inhibits monkey hepatic triacylglycerol lipase activity and allows direct measurement of lipoprotein lipase in post-heparin plasma. While both human and homologous serum can be used as a source of activator apolipoprotein, homologous serum produces a much greater activation.     

Purification and characterization of lipoprotein lipase from pig myocardium.

1. Lipoprotein lipase was purified from pig myocardium by a two-step purification procedure involving (a) the formation of an enzyme-substrate complex and (b) affinity chromatography on Sepharose which contained covalently linked heparin. The purified enzyme gave in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis one main band with an apparent molecular weight of 73 000. The enzyme, which was purified 70 000-fold, had a specific activity of 860 mumol of unesterified fatty acid liberated/h per mg of protein. 2. The purified enzyme hydrolysed [14C]triolein emulsions in the absence of added cofactors but its activity was increased fivefold by adding normal human serum. Of the low-density lipoprotein apoproteins only apolipoprotein CII could be substituted for serum in activating the enzyme. This lipase had maximum activity at 0.05-0.15 M-NaCl. Heparin increased the activity of the purified enzyme twofold at low concentrations, but high concentrations inhibited. The triglyceride lipase of pig myocardium thus resembles lipoprotein lipase purified from adipose tissue and from plasma, but is clearly different from pig hepatic triglyceride lipase.     

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.     

Human milk lipases. I. Serum-stimulated lipase

Lipase activity has previously been demonstrated in human milk. This study shows that there are two separate triglyceride lipases in human milk. One is mainly in the skim milk and is stimulated by bile salts; the other is mainly in the cream and is inhibited by bile salts but stimulated by serum. The serum-stimulated lipase was purified by affinity chromatography on heparin-substituted Sepharose 4B. This gave a 9500-fold purification over whole milk. Although polyacrylamide gel electrophoresis showed that the enzyme was not purified to homogeneity, it had the highest specific activity so far reported for a human serum-stimulated lipase. The purified enzyme was free from bile salt-stimulated lipase activity and had the characteristics of other serum-stimulated or so-called lipoprotein lipases. Thus, it was almost completely inhibited by 1 M NaCl. The purified enzyme was active against tributyrylglycerol also in the absence of exogenous serum factors.     

Polarographic determination of esterase and lipase activities using esters of -naphthols

The polarographic method of the determination of esterase and lipase activities using esters of β-naphthol has been elaborated. The activity of esterase of pig liver, house-fly heads, lyophilized horse serum, and human serum; and lipase activity of pig liver and pancreas was determined. Advantages of the polarographic method lie in using the same calibration curve of nitrosated β-naphthol for determinations of activities of several enzyme sources and in the stability of nitrosated β-naphthol in comparison to the colored reaction product of β-naphthol and tetraazotized o-dianisidin needed for spectrophotometry.     

Lipoprotein lipase in cultured heart cells: characteristics and cellular location.

Lipase activity extracted from cultured neonatal rat heart cells was characterized and identified as lipoprotein lipase. Enzyme activity was stimulated by human apoC-II and rat serum; serum stimulation was prevented by human apoC-I and by apoC-II. Lipolysis was maximal at pH 8.0 and was inhibited by protamine sulfate, NaCl, and high concentrations of heparin. About 50% of heart cell lipase activity applied to heparin-Sepharose bound to the gel and was eluted with a NaCl gradient. A peak of lipase activity was observed at 0.84 M NaCl. Neonatal rat heart cells in culture are a mixture of muscle and non-muscle cells. To determine the cellular location of the lipoprotein lipase, enzyme activity and muscle cell content of the cultures were determined. Myosin ATPase was used as an index of muscle cell content since ATPase specific activity correlated (r = +0.97) with muscle cell content determined immunofluorescently. When muscle cell content of cultures was decreased or increased by differential plating, lipase specific activity was constant. Moreover, lipase specific activity was constant during culture growth despite a decrease in muscle cell content. It was concluded that lipoprotein lipase activity of cultured heart cells is not associated solely with either muscle or non-muslce cells.     

Characterization of the lipolytic activity of endothelial lipase

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

Lipoprotein lipase in heart and myocytes: characteristics with intralipid as substrate.

1. Intralipid is a suitable substrate for measuring lipoprotein lipase activity in the presence of other triacylglycerol lipases in heart and myocytes. 2. Triacylglycerol lipase activity in heart and myocytes was increased 10-fold in the presence of serum at pH 7.4 and 8.1. The serum-stimulated activity in myocytes was 95% inhibited by saturating concentrations of antiserum to lipoprotein lipase. 3. Both heparin-releasable and non-releasable lipoprotein lipase fractions had similar Km values for Intralipid and a similar pattern of inhibition by high density lipoprotein but different responses to heparin. 4. Isoproterenol did not alter lipoprotein lipase activity in cardiac myocytes.     

A Spectrophotometric Lipase Activity Assay by Coupled Lipase-Lipoxygenase in Reverse Micelles

A new, continuous spectrophotometric method is described for determining lipase activity using a reverse micelle system, in which lipase (EC 3.1.1.3) and lipoxygenase (EC 1.13.11.12) are dissolved. The reverse micelle system consists of 2-ethyl hexyl sodium sulfosuccinate (AOT)-isooctane and water. Trilinolein is used as the lipase substrate; linoleate hydroperoxide is the end product of the oxidation catalyzed by lipoxygenase, which acts as an auxiliary coupled-enzyme of lipase. The method appears useful both for detailed kinetic studies of lipase and for serial analyses using sunflower oil, a cheaper substrate. This assay offers the typical advantages of the continuous direct photometric methods in that it is rapid, reproducible and sufficiently sensitive for measuring lipase activity even in some crude commercial preparations.     

A Spectrophotometric Lipase Activity Assay by Coupled Lipase-Lipoxygenase in Reverse Micelles

A new, continuous spectrophotometric method is described for determining lipase activity using a reverse micelle system, in which lipase (EC 3.1.1.3) and lipoxygenase (EC 1.13.11.12) are dissolved. The reverse micelle system consists of 2-ethyl hexyl sodium sulfosuccinate (AOT)-isooctane and water. Trilinolein is used as the lipase substrate; linoleate hydroperoxide is the end product of the oxidation catalyzed by lipoxygenase, which acts as an auxiliary coupled-enzyme of lipase. The method appears useful both for detailed kinetic studies of lipase and for serial analyses using sunflower oil, a cheaper substrate. This assay offers the typical advantages of the continuous direct photometric methods in that it is rapid, reproducible and sufficiently sensitive for measuring lipase activity even in some crude commercial preparations.     

Human gastric lipase: a sulfhydryl enzyme

One sulfhydryl group was modified per mol of native human gastric lipase after incubation at pH 8.0 with 5,5'-dithiobis(2-nitrobenzoic acid) for 18 h or with 4,4'-dithiopyridine for 100 min. With both reagents a direct correlation was found between the modification of one sulfhydryl group and the loss of human gastric lipase activity. Incubation of human gastric lipase with a new hydrophobic sulfhydryl reagent dodecyldithio-5-(2-nitrobenzoic acid) in 30-fold molar excess, at pH 3.0, 5.0, and 8.0, induced immediate and complete human gastric lipase inactivation. Unlike 5,5'-dithiobis(2-nitrobenzoic acid) and 4,4'-dithiopyridine, dodecyldithio-5-(2-nitrobenzoic acid) almost instantaneously stopped the course of tributyrin hydrolysis by human gastric lipase. Human gastric lipase can thus be said to be a sulfhydryl enzyme.     

Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.     

Studies of lipoprotein lipase during the adipose conversion of 3T3 cells.

Lipoprotein lipase activity is negligible in exponentially growing 3T3-L1 cells and 3T3-F442A cells, but develops in both lines when they reach a confluent state and undergo adipose conversion. 3T3-C2 cells, which undergo adipose conversion with extremely low frequency, do not develop the enzyme. The lipase activity of 3T3-L1 and 3T3-F442A is greatly enhanced by insulin and increases 80–180 fold during the adipose conversion. The lipase has the following characteristics in common with lipoprotein lipase from adipose and other tissues: it is dependent upon serum, is inhibited by 0.5–1.0 M sodium chloride, is recovered from acetone powders, has an alkaline pH optimum and is released from the cells by heparin. Like the lipoprotein lipase of tissue adipose cells, the enzyme of 3T3-L1 decays in the presence of cycloheximide with a half-time of about 25 min at 37°C.The ability of 3T3-F442A and 3T3-L1 to take up triglyceride from the medium depends almost completely upon lipoprotein lipase. They incorporate the fatty acids of a large fraction of a triglyceride emulsion added to the medium, and this utilization is stimulated by heparin. Very little of the glycerol portion of the triglyceride is incorporated. 3T3-C2, which lacks lipoprotein lipase, utilizes very little of either the fatty acid or the glycerol portion of triglyceride.The relevance of external lipid or lipoprotein to both the adipose conversion and the appearance of lipoprotein lipase was tested using confluent cultures in medium depleted of these components. In the presence of serum whose lipoproteins have been removed by flotation, lines 3T3-F442A and 3T3-L1 undergo adipose conversion as completely as in the presence of untreated serum, and lipoprotein lipase activity appears at essentially the same rate. In medium whose serum supplement has been extracted with acetone:ethanol, 3T3-F442A cells undergo adipose conversion to nearly the same extent as in untreated serum, and develop nearly the same increase in lipoprotein lipase activity.Unless even very low concentrations of lipids or lipoprotein are saturating it can be concluded that the adipose conversion does not depend upon external lipids or lipoproteins for its induction; rather the differentiation program is built into the cell type and comes into operation when growth is arrested even in their absence. The source of fatty acids utilized for triglyceride synthesis, however, may be affected by the amount of lipid provided to the cells.     

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.     

Arachidonic acid release from diacylglycerol in human neutrophils. Translocation of diacylglycerol-deacylating enzyme activities from an intracellular pool to plasma membrane upon cell activation

We have studied the capacity of human neutrophils to release arachidonic acid from diacylglycerol, employing 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol and 1-[1-14C]stearoyl-2-arachidonoyl-sn-glycerol as exogenous substrates. We have found that arachidonic acid is removed from diacylglycerol by the sequential action of two enzymes. First, the sn-1 position is split by 1-diacylglycerol lipase activity, and then, arachidonic acid is released from the resulting 2-monoacylglycerol by a 2-monoacylglycerol lipase. The specific activity of the 2-monoacylglycerol lipase, using 2-[1-14C]arachidonoyl-sn-glycerol as exogenous substrate, was at least 9-fold higher than that of 1-diacylglycerol lipase, indicating that the action of the 1-diacylglycerol lipase is the rate-limiting step in arachidonic acid release from diacylglycerol. Postnuclear supernatants from A23187-treated cells showed a 2.5-fold increase in both lipase activities. The arachidonic acid-releasing diacylglycerol lipase system showed an optimum pH of 4.5 and was not inhibited by EGTA or stimulated by Ca2+, Mg2+, Mn2+, Zn2+, or Co2+. However, arachidonic acid release was inhibited by Hg2+, suggesting the involvement of sulfhydryl groups in catalytic activity. The subcellular distribution of both 1-diacylglycerol lipase and 2-monoacylglycerol lipase activities was examined in resting and A23187-treated human neutrophils by fractionation of postnuclear supernatants on continuous sucrose gradients. Both lipases were localized mainly in the membrane of gelatinase-containing granules, which were resolved from cytosol, plasma membrane, phosphasomes, and specific and azurophilic granules. When neutrophils were stimulated by the calcium ionophore A23187, a drastic shift of the 1-diacylglycerol lipase and 2-monoacylglycerol lipase toward the plasma membrane was detected. This shift was due to fusion of gelatinase-containing granules with the plasma membrane upon neutrophil stimulation. As a result of the membrane fusion process, the capacity to release arachidonic acid from diacylglycerol was increased. This translocation from the membrane of gelatinase-containing granules to the plasma membrane may play an important role in regulating the diacylglycerol level in stimulated human neutrophils.     

On hepatic and extrahepatic postheparin serum lipase activities and the influence of experimental hypercortisolism and diabetes on these activities.

This paper shows that the palmitoyl-CoA hydrolase activity of postheparin serum of the rat is mainly derived from the liver. The identity of this activity with the heparin-releasable hepatic triacylglycerol hydrolase activity is established. The consequences of the different substrate specificities of the hepatic and extrahepatic enzymes for the measurement of the overall postheparin serum lipase activity are discussed. Treatment of the rats with either a corticosteroid or with streptozotocin was found to lower the lipolytic activity from the liver and to enhance the extrahepatic activity. Also in human postheparin serum, palmitoyl-CoA hydrolase activity is shown to behave identical with hepatic triacylglycerol hydrolase activity. The possible function of the liver in the serum triacylglycerol metabolism is discussed in connection with the proposed mechanism for the role of extrahepatic lipoprotein lipase in atherogenesis.     

Guinea pig very low density lipoproteins are a good substrate for lipoprotein lipase.

In contrast to plasma from most other animals, guinea pig plasma causes little or no stimulation of lipoprotein lipase activity. Very low density lipoproteins (VLDL) isolated by ultracentrifugation of guinea pig serum caused a definite stimulation of lipase activity, whereas the infranatant inhibited the activity. Gel filtration in 5 M guanidinium hydrochloride of delipidated VLDL demonstrated that the activation was caused by a low molecular weight protein. The VLDL themselves were hydrolized at similar rates as human VLDL both by guinea pig and by bovine lipoprotein lipases. Thus, guinea pig VLDL contain an activator for lipoprotein lipase analogous to that in other animals and there is enough of the activator to support rapid hydrolysis of the VLDL lipids by the lipase.