Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases

In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.     

Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward chemically alike, but sterically nonequivalent ester groups within one single triglyceride molecule was investigated. Lipolysis reactions were carried out on synthetic trioctanoin or triolein, which are homogenous, prochiral triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further separated by high performance liquid chromatography. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The stereoselectivity of human and rabbit gastric lipases, expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% for triolein, respectively. The corresponding values with porcine pancreatic lipase were 3% in the case of trioctanoin and 8% in that of triolein. It is worth noting that rabbit gastric lipase, unlike human gastric lipase, became more stereoselective for the triglyceride with shorter acyl chains (trioctanoin). This is one of the most striking catalytic differences observed between these two gastric lipases.     

Stereoselectivity of lipases: esterification reactions of octadecylglycerol.

Stereoselectivity of several triacylglycerol lipases (EC 3.1.1.3) has been investigated in the enzymatic esterification of rac-1-O-octadecylglycerol with oleic acid in the presence of organic solvents, such as hexane. X-1(3)-O-Octadecylmonooleoylglycerols were the only products formed with most lipases; considerable proportions of X-1(3)-O-octadecyldioleoylglycerols were also formed with the lipase from Candida cylindracea. The mixtures of unesterified enantiomeric substrates, i.e., X-1(3)-O-octadecylglycerols were converted to their 3,5-dinitrophenylurethane derivatives and subsequently resolved into sn-1 and sn-3 enantiomers by HPLC on a chiral stationary phase (Sumichiral OA 2100). The data on enantiomeric excess (ee) and enantiomeric ratio (E) in the unesterified substrate revealed for the lipases from porcine pancreas, Rhizopus sp., Pseudomonas sp., Candida cylindracea, Chromobacterium viscosum and Penicillium cyclopium a distinct preference for 1-O-octadecyl-sn-glycerol over its enantiomer indicating stereoselectivity for the sn-3 position. For the lipase from Rhizomucor miehei a slight stereoselectivity for the sn-1 position was observed. Solvents, such as diethyl ether and dichloromethane, strongly inhibited the esterification reaction, but the enzymatic activity could be restored upon removal of such solvents by washing with hexane indicating reversible inhibition.     

Stereoselectivity of Mucorales lipases toward triradylglycerols--a simple solution to a complex problem

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn-2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'-C1' ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N'-C1' amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn-1 and sn-3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C-terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Stereospeific 1H-NMR analysis of trimethylsilyl derivatives of glycerides with a chiral shift reagent

A proton magnetic resonance procedure with tri(3-heptafluorobutyryl-d-camphorato)praseodymium (III) as a chiral shift eagent has been developed to determine the enantimeric purity of monoglycerides 1,2-diglycerides and triglycerides with one mono-unsaturated fatty acid at position $\text{sn-1}$ or $\text{sn-3}$ and two saturated fatty acids at the two other glycerol positions. A model compound, 1-oleoyl-2,3-dipalmitoyl-sn-glycerol, was converted ito the trimethylsilyl either of 2,3-dipalmitoyl-an-glycerol by epoxidation of the double bond, followed by pancreatic hydrolysis and separation and trimethylsilylation of the resulting $\text{sn-1,2}$, and $\text{sn-2,3-}\text{diglycerides}$. This separation becomes feasible by the contribution of the epoxy group to the polarity of the diglyceride. The protons of the trimethysilyl ether group were used for determining the enantiomeric ratio. The addition of a chira shift reagent induces a useful enantiomeric splitting which allows the accurate determination of the ratio of both enantiomers. The trimethylsilyl emers of 1,2-diglycerides are better suited for this purpose than the acetyl compounds. For monoglycetides, the earlier published method with the diaceltates gives a better line separation in ¹H-NMR spectra.     

Lipase regio- and stereoselectivities toward three enantiomeric pairs of didecanoyl-deoxyamino-O methyl glycerol: a kinetic study by the monomolecular film technique

A kinetic study was carried out on the regio- and stereoselectivities of 12 lipases of animal and microbial origin. For this purpose, monomolecular films consisting of three pairs of enantiomers (didecanoyl-deoxyamino-O methyl glycerol, DDG) containing a single hydrolyzable decanoyl ester bond and two lipase-resistant groups were spread at the air-water interface. Each of the lipases tested displayed a particular type of behavior, on the basis of which they were classified in two groups, depending on their ability to hydrolyze the sn-2 position. From the qualitative point of view, the sn-2 preference measured on triacylglycerides and DDG were in good agreement. The inductive chemical effect might explain why a greater level of hydrolytic activity was observed with the diglycerides than with DDG. With most of the lipases tested, it was observed that the enantiomeric pair having two distal acyl chains was more clearly differentiated stereochemically than the two homologous pairs with two adjacent acyl chains. This finding is consistent with the hypothesis that during the chiral recognition process two of the three attachment points may be the external (distal) hydrophobic chains, which is in line with the hypothesis of a tuning fork conformation of a triglyceride in the lipase active site.     

Substrate specificity of Staphylococcus aureus (TEN5) lipases with isomeric oleoyl-sn-glycerol ethers as substrates

For the first time fully protected substrates with only one hydrolyzable ester bond have been used to analyze the substrate specificity of microbial lipases. In these substrates the ester is attached to the glycerol molecule in a precisely defined position. The use of three different substituents generates chirality and thus allows the analysis of positional specificities of individual lipases. Therefore, these new substrates have been used to study the enzymatic activities of two closely related lipases isolated from Staphylococcus aureus (TEN5) designated the 44 and 43 kDa lipase. The lipases, especially the 44 kDa molecule, show a high specificity for the hydrolysis of the ester in the sn-1 position (S-configuration), which is hydrolyzed by a factor of ten faster than that in the sn-3 position. In addition, the study demonstrates for the first time that the rate of hydrolysis of a fatty acid ester attached to the sn-2 position of glycerol by microbial lipases depends on the configuration of the substrate molecule.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.     

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters

Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Kinetic properties of turkey pancreatic lipase: a comparative study with emulsified tributyrin and monomolecular dicaprin

Using the classical emulsified system and the monomolecular film technique, we compared several interfacial properties of turkey pancreatic lipase (TPL) and human pancreatic lipase (HPL). TPL, like HPL, presented the interfacial activation phenomenon when vinyl ester was used as substrate. In the absence of colipase and bile salts, using tributyrin emulsion or monomolecular films of dicaprin at low surface pressure, TPL, unlike HPL, hydrolyzes pure tributyrin emulsion as well as dicaprin films maintained at low surface pressures. TPL was also able to hydrolyze triolein emulsion in the absence of any additive and despite the accumulation of long-chain free fatty acids at the interface. The difference of behaviors between TPL and HPL can be explained by the penetration power of each enzyme. The enzyme that presents the maximal pi(c) (TPL) interacts more efficiently with interfaces, and it is not denaturated at high interfacial energy. Turkey pancreatic lipase is more active on rac-dicaprin than HPL; a maximal ratio of 9 was found between the catalytic activities of the two lipases measured at their surface pressure optima (20 mN m(-1)). A kinetic study on the surface pressure dependency, stereospecificity, and regioselectivity of TPL was performed using enantiopure diglyceride (1,2-sn-dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-dicaprin) that were spread as monomolecular films at the air-water interface. At low surface pressure (15 mN m(-1)), TPL acts preferentially on primary carboxylic ester groups of the diglyceride isomers (1,3-dicaprin), but at high surface pressure (23 mN m(-1)), this enzyme prefers both adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). HPL prefers adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). Furthermore, TPL was found to be markedly stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin at low surface pressure (15 mN m(-1)), while at high surface pressure (23 mN m(-1)), this lipase presents a stereopreference for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. HPL is stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin both at 15 and 23 mN m(-1).     

Kinetic resolution of amino acid esters catalyzed by lipases

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D ,L -homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids. © 1996 Wiley-Liss, Inc.     

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

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

The His gap motif in microbial lipases: a determinant of stereoselectivity toward triacylglycerols and analogs

Lipases preferably hydrolyze the sn-1 and sn-3 acyl chain of triacylglycerols and sn-2 substituted analogs. Molecular modeling studies of the stereopreference of microbial lipases from Rhizopus oryzae, Rhizomucor miehei, Candida rugosa, and lipase B from Candida antarctica toward the hydrolysis of triacylglycerols and analogs revealed that sterical interactions occurring between the sn-2 substituent and the His gap affect substrate geometry, which can be monitored by a single torsion angle. This torsion angle correlates to the experimentally determined stereopreference and is, therefore, suitable to predict stereopreference by molecular modeling. For a given microbial lipase, stereopreference can be estimated by measuring the distance between the side chains of the His gap residues: a narrow His gap cleft implies sn-3 stereopreference for all investigated substrates; a medium-sized His gap discriminates by flexibility of the substrates: flexible substrates are hydrolyzed in sn-1, while rigid substrates are hydrolyzed in sn-3. A wide open His gap implies sn-1 stereopreference for all substrates. This rule holds for all investigated microbial wild type lipases and mutants.     

Stereochemical and positional specificity of the lipase/acyltransferase produced by Aeromonas hydrophila.

Aeromonas species secrete a glycerophospholipid-cholesterol acyltransferase (GCAT) which shares many properties with mammalian plasma lecithin-cholesterol acetyltransferase (LCAT). We have studied the stereochemical and positional specificity of GCAT against a variety of lipid substrates using NMR spectroscopy as well as other assay methods. The results show that both the primary and secondary acyl ester bonds of L-phosphatidylcholine can be hydrolyzed but only the sn-2 fatty acid can be transferred to cholesterol. The enzyme has an absolute requirement for the L configuration at the sn-2 position of phosphatidylcholine. The secondary ester bond of D-phosphatidylcholine cannot be hydrolyzed, and this lipid is not a substrate for acyl transfer. In contrast to the phospholipases, but similar to LCAT, the enzyme does not interact stereochemically with the phosphorus of phosphatidylcholine. In fact, the phosphorus is not required for enzyme activity, as GCAT will also hydrolyze monolayers of diglyceride, although at much lower rates.     

Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester

A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68–93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50–55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4 days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids).     

Hydrolysis of diacylglycerols by lipoprotein lipase.

Enantiomeric diacylglycerols were emulsified, mole for mole, with lyso(1-acyl) lecithin and were hydrolyzed with lipoprotein lipase in NH4Cl-beef serum albumin buffer at pH 8.6 after a brief incubation with delipidated rat serum. The enzyme was prepared from lyophilized and dialyzed bovine skim milk in a 4 percent solution. The course of hydrolysis for each set of enantiomers was determined by gas-liquid chromatography of the masses of the diacylglycerols remaining or monoacylglycerols released in the medium between 0 and 15 min. The majority of sets of sn-1,2- and 2,3-diacylglycerols, including an isotope-labeled true enantiomeric set which was assessed by mass spectrometry, demonstrated preference by the enzyme for lipolysis at position 1 but with less specificity than previously was shown in sn-triacylglycerol hydrolysis. The results preclude the possibility that the predominance of sn-2,3-diacylglycerol intermediates during triacylglycerol hydrolysis is due solely to a preferential breakdown of the 1,2-isomers and reinforce the conclusion that lipoprotein lipase is specific for position 1.     

Lipolysis of the semi-solid self-emulsifying excipient Gelucire 44/14 by digestive lipases

Gelucire 44/14 is a semi-solid self-emulsifying excipient used for the oral delivery of poorly water-soluble drugs. It is composed of C8-C18 acylglycerols and PEG-32 esters, all of which are potential substrates for digestive lipases. Here we studied the lipolysis of Gelucire 44/14 by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases. Human pancreatic lipase (HPL), the main lipase involved in the digestion of triacylglycerols, did not show any significant activity on Gelucire 44/14 or on either of its individual compounds, C8-C18 acylglycerols and PEG-32 esters. Other pancreatic lipases such as human pancreatic lipase-related protein 2 (HPLRP2) showed low activity on Gelucire 44/14 although the highest activity of HPLRP2 was that observed on the C8-C18 acylglycerol fraction, which accounts for 20% (w/w) of Gelucire 44/14. In addition, HPLRP2 showed low activities on the PEG-32 esters, whether these were tested individually or mixed together. Carboxyl ester hydrolase (CEH) showed high activity on Gelucire 44/14, and the highest activities of CEH were those recorded on the total PEG-32 ester fraction and on each individual PEG-32 ester, except for PEG-32 monostearate. The highest activity of all the enzymes tested was that of dog gastric lipase (DGL) on Gelucire 44/14, although DGL showed low activity on the PEG-32 ester fraction and on each individual PEG-32 ester. We compared the lipolysis of Gelucire 44/14 with that of Labrasol, another self-emulsifying excipient, which is liquid at room temperature. Human pancreatic juice showed similar rates of activity on both Gelucire 44/14 and Labrasol. This finding means that these excipients are hydrolyzed in vivo during pancreatic digestion, mainly by CEH in the case of Gelucire 44/14 and by both HPLRP2 and CEH in that of Labrasol, whereas HPL showed very low activities on each of these two excipients. This is the first time the effects of PEG and acyl chain length on the lipolytic activity of digestive lipases on PEG esters have been investigated.     

Diacylglycerol metabolism and arachidonic acid release in human fetal membranes and decidua vera

Diacylglycerol lipase activity has been demonstrated in human fetal membranes and decidua vera tissues. The specific activity of the enzyme is highest in the microsomal fraction of decidua vera tissue. The acylester bond at the sn-1 position of 1,2-diacyl-sn-glycerol is hydrolyzed followed by release of the fatty acid at the sn-2 position. The diacylglycerol lipase activity present in the microsomal fraction of decidua vera tissue hydrolyzes preferentially a diacylglycerol containing an arachidonoyl group in the sn-2 position. Monoacylglycerol lipase activity was also demonstrated in these tissues. The specific activity of monoacylglycerol lipase was significantly greater than that of diacylglycerol lipase and catalyzed preferentially the hydrolysis of monoacylglycerols containing an arachidonyl group in the sn-2 position. Based on the subcellular distribution and the differential effects of various inhibitors, we suggest that the monoacylglycerol lipase and diacylglycerol lipase in decidua vera tissue are 2 distinct enzymes. Diacylglycerol kinase specific activity was examined also and was found to be 4-5 times greater in amnion than in either chorion laeve or decidua vera. The importance of diacylglycerol metabolism in the mechanism of arachidonic acid release and prostaglandin biosynthesis is discussed.