Triglyceride Analogue Research Articles

Abstract 15518: The Effects of High Glucose and Lipoprotein Lipase on Macrophage Regulatory Function

Introduction: Atherosclerosis has an established inflammatory, macrophage(MФ)-mediated component which can be exacerbated by uncontrolled diabetes. However, there are also protective, regulatory MФ processes that can help quell atherosclerosis. Lipoprotein Lipase (LPL) on the MФ surface hydrolyzes circulating triglyceride-rich lipoproteins into glycerol and free fatty acids to provide a non-glucose energy source. Hypothesis: We hypothesized that regulatory MФs have high LPL activity, which could promote fatty acid metabolism to drive inflammation-resolving functions, and that this LPL activity is inhibited by ambient glucose. Methods and Results: MФs were cultured and differentiated from bone marrow harvested from wild-type mice aged 9-16 weeks. Two weeks after harvest, these MФs were polarized with (1) interferon-γ and lipopolysaccharide to stimulate classically-activated, inflammatory MФs or (2) interleukin-4 to stimulate alternatively-activated, regulatory MФs. The resulting phenotypes were validated by canonical transcript markers (Tnfa/Nos2 for inflammatory MФs and Ym1/Arg1 for regulatory MФs) and cell surface markers (CD86 and CD206, respectively). Using a triglyceride analog that fluoresces when cleaved by LPL, we found that regulatory MФs have far greater LPL activity than inflammatory macrophages at baseline (33 ± 2 vs 1 ± 0.2 a.u., p < 0.001, n=8). By varying glucose concentration during the polarization, this effect was most pronounced at normal, physiological concentrations of glucose (5 mM, ~90 mg/dL) and blunted at a high-glucose concentration that can be found in uncontrolled diabetes (25 mM). We also report on lipoxygenase expression and specialized pro-resolving mediator production as effector functions downstream of LPL activity in regulatory MФs. Conclusions: Taken together, our findings support a model in which regulatory MФs are programmed to utilize LPL to drive the inflammation-resolving phenotype and that this process works best at low, physiologic glucose concentrations. Future translational work will investigate whether a lack of LPL-mediated regulatory MФ function is a reason why patients with uncontrolled diabetes have higher atherosclerotic burden and plaque instability.

Abstract 14344: The Competing Roles of Glucose and Lipoprotein Lipase on Regulatory Macrophage Function

Background: Atherosclerosis causes more death than any other pathophysiologic process. It has a well-established inflammatory, macrophage(MФ)-mediated component which can be stoked by uncontrolled diabetes. However, there are also protective, regulatory MФ processes that can help quell atherosclerosis. Lipoprotein Lipase (LPL) on the MФ surface can hydrolyze circulating triglyceride-rich lipoproteins into glycerol and free fatty acids to provide a non-glucose energy source. We hypothesized that regulatory MФs have high LPL activity, which would promote fatty acid metabolism to drive inflammation-resolving functions, and that this LPL activity is inversely related to ambient glucose. Results: MФs were cultured and differentiated from bone marrow harvested from wild-type mice aged 9-16 weeks. Two weeks after harvest, these MФs were polarized with (1) interferon-γ and lipopolysaccharide to stimulate classically-activated, inflammatory MФs or (2) interleukin-4 to stimulate alternatively-activated, regulatory MФs. The resulting phenotypes were validated by canonical transcript markers ( Tnfa / Nos2 for inflammatory MФs and Ym1 / Arg1 for regulatory MФs) and cell surface markers (CD86 and CD206, respectively). Using a triglyceride analog that fluoresces when cleaved by LPL, we found that regulatory MФs have far greater LPL activity than inflammatory macrophages at baseline (33 ± 2 vs 1 ± 0.2 a.u., p < 0.001, n=8). By varying glucose concentration during the polarization, this effect was most pronounced at normal, physiological concentrations of glucose (5 mM, ~90 mg/dL) and blunted at a high-glucose concentration that can be found in uncontrolled diabetes (25 mM). Each experiment was repeated across multiple batches of MФs (n≥4) and from multiple mice (n=15), male and female. Conclusions: Taken together, our findings support a model in which regulatory MФs are programmed to utilize LPL to drive the inflammation-resolving phenotype and that this process works best at low, physiologic glucose concentrations. Future translational work will investigate whether a lack of LPL-mediated regulatory MФ function is a reason why patients with uncontrolled diabetes have higher atherosclerotic burden and plaque instability.

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens

It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis.

Abstract 537: Characterization of the Lipase Stimulating Domain for Apolipoprotein A-V and the Development of a Therapeutic Peptide for the Treatment of Hypertriglyceridemia

The impaired clearance of plasma triglycerides is a major cause of clinical hypertriglyceridemia, a condition which affects about one third of adults in the G7 countries and is a risk factor for a number of diseases, including cardiovascular disease, cancer and pancreatitis. The hydrolysis and removal of triglycerides from the plasma is dependent on cell surface lipoprotein lipase (LpL) and its enzymatic activity is in turn regulated by specific apolipoproteins; apoC-III, which acts as a potent inhibitor and apoC-II and apoA-V, which act as stimulators. Their importance in hypertriglyceridemia is underscored by GWAS analyses, which have identified the APOA1/C3/A4/A5 gene cluster (chromosome 11q23) as an important determinant of dylipidemia. ApoA-V is of particular interest since its physiological concentration (3.8 nM) is 10,000x lower than that of apoC-II and the other major apolipoproteins. To investigate the influence of different lipoproteins on lipase kinetics we have developed a fluorescence-based assay that uses a fluorogenic triglyceride analog (EnzChek®) in a microplate format. Here we report that a short alpha-helical amphipathic peptide (AV199-232) corresponding to apoA-V residues 199-232 exhibit; i) lipid-binding, ii) heparin-binding and iii) lipase-stimulating activities. Structural characterization and residue substitution studies have been employed to map these three activities to three separate discrete domains within AV199-232. Enzyme kinetic analysis with pure LpL indicates that AV199-232 can overcome the inhibition of LpL by apoC-III and stimulate enzyme activity by reducing its Km 8-fold. Similar increases in lipase activity are also observed with live THP-1 monocytes and HepG2 cells that express hepatic lipase. The influence of AV199-232 in cell culture is saturable, dependent on the presence of cell surface lipase, and enzymatic activation can be sustained for at least 2 hrs. We postulate that apoA-V and AV199-232 stimulate lipase activity through an allosteric mechanism by binding to lipases with high affinity near their catalytic site to affect enzyme kinetics.

Positional specificity of sardine digestive lipase in the hydrolysis of triacylglycerols and analogs

Using lipid emulsions as well as the monomolecular film technique, we studied some kinetic properties of sardine digestive lipase (SaDL). The pure lipase was able to hydrolyze a triolein emulsion in the absence of any additives and seems therefore to tolerate the accumulation of long free fatty acids at the interface. The kinetic behavior of SaDL can be explained by the high penetration capacity of this enzyme into a lipidic interface. SaDL hydrolyzes efficiently pure tributyrin, in the absence of additives. Subsequently, the sardine lipase is not denaturated at high interfacial energy. A kinetic study of the surface pressure dependency of the regioselectivity of SaDL was performed using an optically pure stereoisomer of diglyceride (1,2‐sn‐dicaprin) and a prochiral isomer (1,3‐sn‐dicaprin) that were spread as monomolecular films at the air‐water interface. At low surface pressure, SaDL acts preferentially on distal carboxylic ester groups of the diglyceride isomer (1,3‐sn‐dicaprin) but at high surface pressure, this enzyme prefers adjacent ester groups of the (1,2‐sn‐dicaprin) isomer. Interestingly, the SaDL was found to hydrolyze efficiently the secondary ester group of a triglyceride analog. This is in line with the fact that SaDL hydrolyzed triolein at the sn‐2 position, as shown using the thin‐layer chromatography technique. This property is sought for the synthesis of specific triacylglycerol‐based compounds, such as structured lipids.Practical applications: The sardine digestive lipase was found to efficiently hydrolyse the secondary ester group of a triglyceride analogue, a characteristic sought for the synthesis of structured lipids.The sardine digestive lipase (SaDL) showed an ability to hydrolyze the secondary ester group of a triglyceride analog (1,2‐DDG) with the R configuration (R3).

Abstract 31: A Novel Fluorescence-Based Assay is Used to Investigate the Triglyceride Hydrolytic Activity of Lipases and to Identify Synthetic Peptides With Strong Lipase-Stimulating Activities

Hypertriglyceridemia is estimated to affect about one third of Americans and is an independent risk factor for cardiovascular disease, pancreatitis and xanthomatosis. Lipoprotein lipase (LPL) is a key enzyme involved in the hydrolysis and clearance of triglycerides from plasma and its enzymatic activity is regulated by specific apolipoproteins; apoC-III, which acts as a potent inhibitor and apoC-II and apoA-V, which act as stimulators. Their importance in hypertriglyceridemia is underscored by GWAS analyses, which have identified the APOA1/C3/A4/A5 gene cluster (chromosome 11q23) as an important determinant of dylipidemia. We have developed a fluorescence-based lipase assay that utilizes a fluorogenic triglyceride analog (EnzChek®, Molecular Probes) and facilitates the rapid analysis of lipase enzyme kinetics in a microplate format. This assay has made it possible for us to undertake the screening of the Prestwick (1,200 FDA-approved drugs) and Natural Product (~3,000) libraries at the University of Illinois high-throughput screening facility, in order to identify lipase activating compounds. To validate the assay we have characterized the influence of heparin, different metals and apolipoproteins on the hydrolytic activities of LPL in vitro, and hepatic lipase in HepG2 cell culture. As expected, enzyme activities were supported by heparin and calcium, and both apoC-II and apoC-III when complexed with VLDL, stimulated and inhibited lipase activity, respectively. Interestingly, the heparin/Fe2+ complex was a strong inhibitor of lipase activity, possibly explaining the high incidence of hypertriglyceridemia associated with iron supplementation to treat iron-deficiency. Finally, using a synthetic peptide approach we have localized apoA-V’s lipase stimulating and heparin binding domains to a short amphipathic, alpha-helical peptide. When included in the assays this peptide could increase lipase hydrolytic rates by 3-6 fold both in vitro, and in cell culture. Given that on a molar basis apoA-V is about 1200-fold less abundant than apoC-II, we anticipate that apoA-V will provide valuable sequence information for the development of potent triglyceride lowering therapeutics.

Enhancement of liposomal stability and cellular drug uptake by incorporating tributyrin into celecoxib-loaded liposomes

Tributyrin, a triglyceride analog of short chain fatty acid butyrate, can act as a prodrug of an anticancer agent butyrate. In view of other reports that demonstrated the improved characteristics of conventional liposomes by incorporating small amount of non-phospholipids such as Tween 80, herein we sought to investigate whether the incorporation of tributyrin into the liposomal layers may provide additional advantages for liposomes as an anticancer drug carrier. Liposomes were prepared with dimyristoylphosphatidylcholine as a main phospholipid with or without addition of tributyrin. Celecoxib was loaded in liposomes as a model anticancer drug. Tween 80-incorporated liposomes were also prepared for comparison. Tributyrin-incorporated liposomes were ineffective in enhancing the skin permeation of celecoxib compared to Tween 80-incorporated ones. However, tributyrin-incorporated liposomes enhanced the entrapped celecoxib concentration to an extent comparable to Tween 80-incorporated ones. Furthermore, tributyrin-incorporated liposomes exhibited much higher stability compared to Tween 80-incorporated ones. Finally, the cellular uptake of celecoxib loaded in tributyrin-incorporated liposomes into mouse melanoma cells were more than 10-fold higher compared to that loaded in conventional- and Tween 80-incorporated liposomes. Taken together, the incorporation of tributyrin into conventional liposomes loaded with anticancer drugs may provide an advanced anticancer drug carrier delivering both drug and tributyrin.

Polymeric triglyceride analogs prepared by enzyme-catalyzed condensation polymerization

Unique crosslinkable unsaturated polyesters were prepared by a one-pot lipase-catalyzed copolymerization of crude linoleic acid (LA), glycerol (G) and 1,18-cis-9-octanedecenedioic acid (oleic diacid, OD). The lipase used is Candida antarctica Lipase B (CALB) immobilized on Lewatit (N435). Experiments were conducted to assess N435’s efficiency of forming poly(OD-co-G-co-LA) as a function of: (i) LA content in the co-monomer feed and (ii) reaction time. For comonomer feed ratio (OD:G:LA) 1:1:0.67, Mn reached ∼9500 in 8h, trisubstituted G-units increased to 64% and monomer was largely depleted. When LA and OD remained in products, a lower temperature thermal decomposition peak at about 280°C was observed in addition to a higher temperature decomposition peak at ∼475°C. By increasing LA content in the comonomer feed from 1:1:1 to 1:1:1.33, Mn decreased from 11,000 to 6300 while trisubstituted G-units increased from 92 to nearly 100%. Thus, by varying the feed ratio of LA, polymeric triglyceride-type structures were formed with control of chain length and trisubstituted G-unit content. Since G-units may be substituted in various ways with OD and LA, calculations were performed that determined the relative proportion of different substitution patterns in products. A hypothetical structure showing general characteristics of the substitution at glycerol units for one such product is presented.

Dual function of tributyrin emulsion: Solubilization and enhancement of anticancer effect of celecoxib

Tributyrin, a triglyceride analogue of butyrate, can act as a prodrug of an anticancer agent butyrate after being cleaved by intracellular enzymes. We recently demonstrated that the emulsion containing tributyrin as an inner oil phase possesses a potent anticancer activity. Herein we sought to develop tributyrin emulsion as a carrier of celecoxib, a poorly-water soluble drug with anticancer activity. Combined treatment of human HCT116 colon cancer cells with free celecoxib plus tributyrin emulsion inhibited the cellular proliferation more effectively than that of each drug alone, suggesting the possibility of tributyrin emulsion as a potential celecoxib carrier. The mean droplet size of emulsions tended to increase as the tributyrin content in emulsion increases and the concentration of celecoxib loaded in emulsions was affected by tributyrin content and the initial amount of celecoxib, but not by the total amount of surfactant mixture. The concentration of celecoxib required to inhibit the growth of HCT116 and B16-F10 cancer cells by 50% was 2.6- and 3.1-fold lowered by loading celecoxib in tributyrin emulsions, compared with free celecoxib. These data suggest that the anticancer activity of celecoxib was enhanced by loading in tributyrin emulsions, probably due to the solubilization capacity and anticancer activity of tributyrin emulsion.

Determination of lipoprotein lipase activity using a novel fluorescent lipase assay

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.

Preparation and evaluation of tributyrin emulsion as a potent anti-cancer agent against melanoma

Histone deacetylase inhibitors such as butyrate are known to exhibit anti-cancer activities in a wide range of cancer including melanoma. In spite of these potencies, butyrate is not practically used for cancer treatment due to its rapid metabolism and very short plasma half-life. Tributyrin, a triglyceride analog of butyrate, can act as a pro-drug of butyrate after being cleaved by intracellular enzymes. The present study sought to investigate a possibility to develop tributyrin emulsion as a potent anti-cancer agent against melanoma. Mixture of Tween80 and 1, 2-dimyristoyl-sn-glycero-3-phosphocholine as a surfactant to disperse tributyrin produced homogeneous emulsions with nanometer sizes, even without a harsh homogenization procedure. Tributyrin emulsion was more potent than butyrate in inhibiting the growth of B16-F10 melanoma cells. Accumulation of cells at sub G0/G1 phase and the DNA fragmentation induced by tributyrin emulsion treatment revealed that tributyrin emulsion inhibited the growth of B16-F10 cells by inducing apoptosis. Treatment with tributyrin emulsion suppressed the colony formation of melanoma cells in a dose-dependent manner. Furthermore, after intraperitoneal administration into mice, tributyrin emulsion inhibited the formation of tumor colonies in the lung following intravenous injection of melanoma cells. Taken together, our data suggests that tributyrin emulsion may be developed as a potent anti-cancer agent against melanoma.

The Kinetics of Phagosome Maturation as a Function of Phagosome/Lysosome Fusion and Acquisition of Hydrolytic Activity

Professional phagocytes function at the hinge of innate and acquired immune responses by internalizing particulate material that is digested and sampled within the phagosome of the cell. Despite intense interest, assays to measure phagosome maturation remain insensitive and few in number. In this current study, we describe three novel assays that quantify important biological properties of the phagosome as it matures. One assay exploits fluorescence resonance energy transfer to quantify mixing of phagocytosed particles carrying a donor fluor with an acceptor fluor loaded previously into the lysosomes as a fluid phase marker. Two additional assays describe the functional maturation of the phagosome as a hydrolytic compartment following the degradation of specifically designed peptide and triglyceride fluorogenic substrates. The peptide substrate is preferentially cleaved by cysteine proteinases, and its degradation reflects proteinase delivery and activation within the acidifying phagosome. The fluorescence emission of the triglyceride analogue profiles the kinetics of triglyceride lipase activity within the phagosome. The fluorescence profiles of all three assays are modulated by known inhibitors of phagosome maturation, demonstrating the veracity, sensitivity and versatility of the assays.

A study of fatty acid methyl esters with epoxy or alkyne functionalities

AbstractDescribed are the physical and chemical properties of the methyl esters of two uncommon fatty acids: vernolic acid, containing an epoxy group, and crepenynic acid, containing a triple bond. The incorporation of an epoxy or alkyne group into the fatty acid structure is shown to greatly affect the properties compared to conventional unsaturated fatty acids. The methyl esters have been characterized and compared with ordinary fatty acid methyl esters (i.e., methyl oleate and linoleate) with respect to spectroscopic characterization [1H nuclear magnetic resonance (NMR)], 13C NMR, and Fourier transform infrared), rheological properties, and oxidative reactivity (using chemiluminescence). Both methyl vernoleate and methyl crepenynate could successfully be produced by transesterification under basic conditions without reaction of the epoxy or alkyne groups. Rheological measurements showed that the methyl esters had a significantly lower viscosity compared to their triglyceride analogs. Smaller differences were seen when comparing the different methyl esters where methyl vernoleate had the highest viscosity due to the presence of the more polar oxirane group. Very large differences were found with respect to the oxidation rate of the different methyl esters. Methyl crepenynate was shown to oxidize extremely rapidly, whereas methyl vernoleate was very stable toward oxidation.

Design and synthesis of triglyceride analogue biotinylated suicide inhibitors for directed molecular evolution of lipolytic enzymes

The design, synthesis, and inhibition properties of two new triglyceride analogue biotinylated suicide inhibitors (2) and (3) for directed molecular evolution of lipolytic enzymes by phage-display is described.

Rational design of alpha-keto triglyceride analogues as inhibitors for Staphylococcus hyicus lipase.

We have synthesized a series of alpha-keto triglyceride analogues as inhibitors for the lipase from Staphylococcus hyicus (SHL). Hydrolysis at positions 1 and 2 was prevented by replacement of the ester bonds by nonhydrolyzable ether, carbamoyl, or amide bonds, and an alpha-keto fatty acid was introduced at position 3. Such compounds act as competitive inhibitors of SHL. Inhibition must be caused by the presence of the alpha-keto functions, since the compounds containing an ester or a hydroxyl group in position 3 did not inhibit the enzyme. We propose that our inhibitors react with the active site Ser of the lipase, hereby mimicking the tetrahedral intermediate occurring in substrate hydrolysis. We conclude that the localization of the alpha-keto triglycerides is very important for inhibition because only those compounds which are insoluble in water are lipase inhibitors. In addition, other specific protein-inhibitor interactions, probably with the carbonyl oxygen at position 1 and/or 2, improve inhibitor binding. This makes the compounds with amide or carbamoyl groups at positions 1 and 2 better inhibitors than their ether counterparts. The inhibitory power could be improved further by replacing the oxygen at position 3 by an amido group. The resulting inhibitor 1, 2-diethylcarbamoyl-3-amido-alpha-ketododecanoylglycerol has a Ki value of 0.008 mol %, indicating that it binds approximately 125-fold tighter than the substrate. These results illustrate that effective lipase inhibitors can be designed by combining an alpha-keto group with good micellar solubility and optimal protein-inhibitor interactions.

Structural investigations of the regio- and enantioselectivity of lipases

Although lipases are widely applied for the stereospecific resolution of racemic mixtures of esters, the atomic details of the factors that are responsible for their stereospecificity are largely obscure. We determined the X-ray structures of Pseudomonas cepacia lipase in complex with two enantiopure triglyceride analogues, that closely mimic natural substrates. This allowed an unambiguous view of how the two wings of the boomerang-shaped active site accommodate the acyl and alcohol parts of the triglyceride. The binding groove for the hydrophobic sn-3 fatty acid chain is large and hydrophobic. The cleft for the alcohol moiety is divided in two parts, one tightly binding the sn-2 acyl chain with hydrophilic and hydrophobic interactions, the other more weakly binding the sn-1 fatty acid. The enantioselectivity of Pseudomonas cepacia lipase seems therefore to be predominantly determined by the size and interactions of the sn-2 chain and by the size of the sn-3 chain.

Crystal structure of cutinase covalently inhibited by a triglyceride analogue.

Cutinase from Fusarium solani is a lipolytic enzyme that hydrolyses triglycerides efficiently. All the inhibited forms of lipolytic enzymes described so far are based on the use of small organophosphate and organophosphonate inhibitors, which bear little resemblance to a natural triglyceride substrate. In this article we describe the crystal structure of cutinase covalently inhibited by (R)-1,2-dibutyl-carbamoylglycero-3-O-p-nitrophenylbutyl-phos phonate, a triglyceride analogue mimicking the first tetrahedral intermediate along the reaction pathway. The structure, which has been solved at 2.3 A, reveals that in both the protein molecules of the asymmetric unit the inhibitor is almost completely embedded in the active site crevice. The overall shape of the inhibitor is that of a fork: the two dibutyl-carbamoyl chains point towards the surface of the protein, whereas the butyl chain bound to the phosphorous atom is roughly perpendicular to the sn-1 and sn-2 chains. The sn-3 chain is accommodated in a rather small pocket at the bottom of the active site crevice, thus providing a structural explanation for the preference of cutinase for short acyl chain substrates.

Computed tomography scanning of morris hepatomas with liver-specific polyiodinated triglycerides

We compared the computed tomography (CT) scanning characteristics of a polyiodinated triglyceride analog with those of a urographic agent to distinguish Morris-7777 hepatoma (MH) cells from normal hepatocytes in rats. Eighteen Buffalo rats were laparotomized and MH cells injected directly into the hepatic parenchyma or introduced via the portal vein to produce, respectively, focal or diffuse lesions in the liver. Baseline CT scans were obtained 21 days after implantation and prior to intravenous administration of either the polyiodinated triglyceride (45-100 mg I/kg) or the nonionic contrast agent, iohexol (560 mg I/kg). Images were obtained at 0-3 hr and 24 hr. Gross pathologic inspection was performed to validate the imaging results. Hepatomas were nearly isodense with normal liver parenchyma in many of the animals, rendering lesion detection difficult with no contrast agent. The bolus administration of iohexol improved lesion detection in many cases. Lesion conspicuity, however, was significantly improved with the polyiodinated triglyceride at less than one eighth the dose of iohexol. Because of their biochemical nature, polyiodinated triglyceride analogs are specifically cleared by the liver. Consequently, they offer several advantages over nonspecific urographic agents in their ability to enhance lesion conspicuity in this hepatoma model.

Formulation of polyiodinated triglyceride analogues in a chylomicron remnant-like liver-selective delivery vehicle.

A formulation methodology for the incorporation of polyiodinated triglyceride (ITG) analogues into a protein-free chylomicron remnant-like emulsion was developed to provide a vehicle for the selective hepatic delivery of these agents for contrast-enhanced X-ray computed tomography (CECT). Triglyceride emulsions (10% w/v) were prepared at various processing pressures, temperatures and times with a Microfluidizer 110-S using different emulsion component proportions to establish processing and compositional parameters in order to afford stable ITG emulsions (ITG-LE) approaching 200 nm mean diameter. Preliminary data indicated that with a formulation composed of 2.4% dioleoyl PC with a cholesterol:DOPC mole ratio of 0.4 emulsified at 14,700 psi, 35 degrees C for 10 min routinely afforded ITG-LE in the desired size range. The elimination of salt and amino acid from the bulk phase enhanced the stability of the ITG-LE. Incorporation of cholesterol into the monolayer was of critical importance in generating a stable emulsion near the targeted size, with a C:DOPC mole ratio of 0.4 producing a size minimum relative to higher or lower C:DOPC values. The ITG analogues can be readily incorporated into stable remnant-like emulsions of relatively uniform particle size. Combination of the unique ITG contrast agent with the remnant-like delivery vehicle demonstrates a high degree of hepatic selectivity in biodistribution studies and offers significant potential for selective hepatic CECT.

Cutinase from Fusarium solani pisi Hydrolyzing Triglyceride Analogs. Effect of Acyl Chain Length and Position in the Substrate Molecule on Activity and Enantioselectivity

Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomers of each compound were synthesized. These pseudo triglycerides contain only one hydrolyzable ester bond, and they are ideally suited to studying the influence of the chain length at the 1-, 2-, and 3-position on lipase activity and on stereopreference. These substrates were used to characterize cutinase from Fusarium solani pisi. Our results show that the activity of cutinase is very sensitive to the length and distribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atoms. The enzyme preferentially hydrolyzes the (R)-enantiomers, but this preference is strongly dependent on the acyl chain length distribution, with (R) over (S) activity ratios varying from about 30 to 1. This enantioselectivity was found in three different assay systems: a mixed micellar, a reverse micellar, and a monolayer study. Our data suggest that at least two alkyl chains of the pseudo triglycerides must be fixed during hydrolysis. Therefore, these substrates were used to characterize mutants of cutinase with mutations in putative lipid binding domains. Two mutants (A85F and A85W) have increased activities. The results obtained with these mutants suggest an interaction of the acyl chain of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.