A homogeneous assay to determine high-density lipoprotein subclass cholesterol in serum.

Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL2-C and HDL3-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL2, HDL3, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL3-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL2, LDL, and VLDL increased, but the size of HDL3 fraction particles remained constant, allowing only HDL3-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.

Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity.

Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

Integration of metal organic frameworks with enzymes as multifunctional solids for cascade catalysis.

Enzymes exhibit a large degree of compatibility with metal-organic frameworks (MOFs) which allows the development of multicomponent catalysts consisting of enzymes adsorbed or occluded by MOFs. The combination of enzymes and MOFs in a multicomponent catalyst can be used to promote cascade reactions in which two or more individual reactions are performed in a single step. Cascade reactions take place due to the cooperation of active sites present on the MOF with the enzyme. A survey of the available data establishes that often an enzyme undergoes stabilization by association with a MOF and the system exhibits notable recyclability. In addition, the existence of synergism is observed as a consequence of the close proximity of all the required active sites in the multicomponent catalyst. After an introductory section describing the specific features and properties of enzyme-MOF assemblies, the main part of the present review focuses on the description of the cascade reactions that have been reported with commercial enzymes associated with MOFs, paying special attention to the advantages derived from the multicomponent catalyst. Related to the catalytic activity to metabolize glucose, generating reactive oxygen species (ROS) and decreasing the solution pH, an independent section describes the recent use of enzyme-MOF catalysts in cancer therapy. The last paragraphs summarize the current state of the art and provide our view on future developments in this field.

Association of hormone-sensitive lipase (HSL) gene polymorphisms with the intramuscular fat content in two Chinese beef cattle breeds.

Hormone-sensitive lipase (HSL) was considered as an essential enzyme in glucolipid metabolism. It has been proposed to be a lead candidate gene for genetic markers of lipid deposition in livestock. The aim of this study was to identify sequence variants (SVs) of the bovine HSL gene and evaluate the relations to intramuscular fat in two indigenous Chinese beef cattle breeds. Expression analysis by quantitative real-time polymerase chain reactions (qPCR) indicated that expression levels of bovine HSL gene were highest in the perirenal fat and heart within two different age stage (adult and calf), respectively. Five SVs were identified by direct DNA sequencing, which included four missense mutations (g.16563C>T, g.16734G>A, g.16896A>G, g.17388G>T) in exon 8 and a synonymous mutation (g.17402C>T) in exon 9. Population genetic analysis showed that except for g.16563C>T and g.17402C>T, all the other detected SVs strongly affected the bovine intramuscular fat content (P < 0.01 or P < 0.05). The individuals with Hap5/5 diplotypes (CC-GG-GG-GG-CC) was highly significantly associated with intramuscular fat content than the other diplotypes (P < 0.01). The above results suggested that the HSL gene can used as potential candidate markers gene for the beef breed improvement through marker assisted selection in Chinese cattle breeds.

Enantioselective synthesis of a chiral intermediate of himbacine analogs by Burkholderia cepacia lipase A.

The enantiomers of (4R/S)-4-hydroxy-N, N-diphenyl-2-pentynamide are key chiral synthons for the synthesis of thrombin receptor antagonists such as vorapaxar. In this paper, we report the enzymatic preparation of enantiomerically enriched (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide using lipase A from Burkholderia cepacia ATCC 25416 as the catalyst. First, the lipase gene (lipA) and its chaperone gene (lipB) was cloned and expressed in Escherichia coli system. After purification, lipase A activation was performed with the assistance of foldase lipase B. Enzyme assay revealed that the activated lipase A showed the optimal catalytic activity at 60 ºC and pH 7. The effects of various metals on the activity were investigated and results demonstrated that most of the metals inhibited the activity. To further improve the catalytic outcome, two-phase reaction was studied, and n-hexane proved to be a good organic solvent for the combination system. Using the optimize conditions, (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide with 94.5% ee value and 48.93% conversion ratio was achieved. Our investigation on this lipase reveals lipase A as a promising biocatalyst for producing chiral propargyl alcohol for preparation of novel himbacine analogs.

Crystal structure of human lysosomal acid lipase and its implications in cholesteryl ester storage disease.

Lysosomal acid lipase (LAL) is a serine hydrolase that hydrolyzes cholesteryl ester (CE) and TGs delivered to the lysosomes into free cholesterol and fatty acids. LAL deficiency due to mutations in the LAL gene (LIPA) results in accumulation of TGs and cholesterol esters in various tissues of the body leading to pathological conditions such as Wolman's disease and CE storage disease (CESD). Here, we present the first crystal structure of recombinant human LAL (HLAL) to 2.6 Å resolution in its closed form. The crystal structure was enabled by mutating three of the six potential glycosylation sites. The overall structure of HLAL closely resembles that of the evolutionarily related human gastric lipase (HGL). It consists of a core domain belonging to the classical α/ß hydrolase-fold family with a classical catalytic triad (Ser-153, His-353, Asp-324), an oxyanion hole, and a "cap" domain, which regulates substrate entry to the catalytic site. Most significant structural differences between HLAL and HGL exist at the lid region. Deletion of the short helix, 238NLCFLLC244, at the lid region implied a possible role in regulating the highly hydrophobic substrate binding site from self-oligomerization during interfacial activation. We also performed molecular dynamic simulations of dog gastric lipase (lid-open form) and HLAL to gain insights and speculated a possible role of the human mutant, H274Y, leading to CESD.

Isolation and identification of cholesterol esterase and pancreatic lipase inhibitory peptides from brewer's spent grain by consecutive chromatography and mass spectrometry.

The isolation and identification of cholesterol esterase (CE) and pancreatic lipase (PL) inhibitory peptides obtained from the protein hydrolysate of brewer's spent grain (BSG) was performed. BSG peptides were fractionated and purified sequentially by anion exchange, gel filtration (FPLC), and reversed phase high-performance liquid chromatography (RP-HPLC). The fractions obtained from each chromatographic step were collected and the in vitro enzyme inhibitory activity was evaluated. The chromatographic purification process increased the in vitro activities. The most active fractions were evaluated using MALDI-TOF tandem mass spectrometry, which identified three peptides: a peptide with the highest CE inhibition capacity (WNIHMEHQDLTTME) and two peptides with PL inhibition capacity (DFGIASF and LAAVEALSTNG). These three peptides showed hydrophobic and acidic amino acid residues (Asp and Glu) and/or their amines (Asn and Gln), which could be a common feature among lipid-lowering peptides related to CE and PL enzyme inhibition. The in silico studies showed that the three peptides had high hydrophobicity and were susceptible to enzymatic hydrolysis performed by trypsin, pepsin, and pancreatin. The BSG byproduct was a good source of CE and PL inhibitory peptides, thus adding value to this byproduct of the beer industry. This is the first report to demonstrate that BSG peptides can inhibit CE and PL enzymes.

Effect of Cysteine Residue Substitution in the GCSAG Motif of the PMGL2 Esterase Active Site on the Enzyme Properties.

The gene coding for PMGL2 esterase, which belongs to the family of mammalian hormone-sensitive lipases (HSLs), was discovered by screening a metagenomic DNA library from a permafrost soil. The active site of PMGL2 contains conserved GXSXG motif which includes Cys173 residue next to the catalytic Ser174. In order to clarify the functional role of the cysteine residue in the GCSAG motif, we constructed a number of PMGL2 mutants with Cys173 substitutions and studied their properties. The specific activity of the C173D mutant exceeded the specific activity of the wild-type enzyme (wtPMGL2) by 60%, while the C173T/C202S mutant displayed reduced catalytic activity. The activity of the C173D mutant with p-nitrophenyl octanoate was 15% higher, while the activity of the C173T/C202S mutant was 17% lower compared to wtPMGL2. The C173D mutant was also characterized by a high activity at low temperatures (20-35°C) and significant loss of thermal stability. The kcat value for this protein was 56% higher than for the wild-type enzyme. The catalytic constants of the C173S mutant were close to those of wtPMGL2; this enzyme also demonstrated the highest thermal stability among the studied mutants. The obtained results demonstrate that substitutions of amino acid residues adjacent to the catalytic serine residue in the GXSXG motif can have a significant effect on the properties of PMGL2 esterase.

Functionalized magnetic nanomaterials for electrochemical biosensing of cholesterol and cholesteryl palmitate.

Synthesis and functionalization of magnetite nanoparticles (Fe3O4) was achieved with the view to covalently bind both cholesterol oxidase and cholesterol esterase biorecognition agents for the development of free and total cholesterol biosensors. Prior to enzyme attachment, Fe3O4 was functionalized with 3-aminopropyltriethoxysilane (APTES) and polyamidoamine (PAMAM) dendrimer. Characterization of the material was performed by FT-IR and UV spectroscopy, SEM/EDX surface analysis and electrochemical investigations. The response to cholesterol and its palmitate ester was examined using cyclic voltammetry. Optimum analytical performance for the free cholesterol biosensor was obtained using APTES-functionalized magnetite with a sensitivity of 101.9 µA mM-1 cm-2, linear range 0.1-1 mM and LOD of 80 µM when operated at 37 °C. In the case of the total cholesterol biosensor, the best analytical performance was obtained using PAMAM dendrimer-modified magnetite with sensitivity of 73.88 µA mM-1 cm-2 and linear range 0.1-1.5 mM, with LOD of 90 µM. A stability study indicated that the free cholesterol biosensors retained average activity of 98% after 25 days while the total cholesterol biosensors retained 85% activity upon storage over the same period. Graphical abstract Schematic representation of cholesterol esterase and oxidase loaded magnetic nanoparticles (Fe3O4@APTES or Fe3O4@APTES-PAMAM) generating hydrogen peroxide from cholesterol palmitate.

Orientational Switch of the Lipase A Enzyme at the Oil-Water Interface: An Order of Magnitude Increase in Turnover Rate with a Single Surfactant Tag Explained.

Interfacially active lipases can be immobilized at a biphasic interface to enhance turnover recyclability and to facilitate product separation. Extensive coarse-grained molecular dynamics simulations of lipase A (LipA) from Bacillus subtilis show a bimodal orientational distribution of the enzyme at an oil-water interface, arising from its ellipsoidal Janus particle-like character. The relative orientational preference can be tuned by pH. The simulations rationalize a rare experimental observation of an order of magnitude increase in the turnover rate of this lipase upon its noncovalent tagging by a single surfactant molecule at the interface, compared to its rate in bulk water. The adsorption free energy, the interfacial activation, a decrease in the number of orientational fluctuations, and an increased rate of translational diffusion, to all of which the Janus character of LipA contributes, are the factors responsible for this enhancement. This study can spur further investigations of the Janus behavior of enzymes to enhance their activity as well as to stabilize the biphasic emulsion needed for interfacial catalysis.

Reduced Enzyme Dynamics upon Multipoint Covalent Immobilization Leads to Stability-Activity Trade-off.

The successful incorporation of enzymes into materials through multipoint covalent immobilization (MPCI) has served as the foundation for numerous advances in diverse fields, including biocatalysis, biosensing, and chemical weapons defense. Despite this success, a mechanistic understanding of the impact of this approach on enzyme stability has remained elusive, which is critical for realizing the full potential of MPCI. Here, we showed that the stabilization of lipase upon MPCI to polymer brush surfaces resulted from the rigidification of the enzyme with an increase in the number of enzyme-brush attachments. This was evident by a 10-fold decrease in the rates of enzyme unfolding and refolding as well as a reduction of the intrinsic fluctuations of the folded and unfolded states, which was measured by single-molecule (SM) Förster Resonance Energy Transfer imaging. Moreover, our results illuminate an important trade-off between stability and activity as a function of this decrease in structural dynamics of the immobilized lipase. Notably, as the thermal stability of lipase increased, as indicated by the temperature optimum for activity of the enzyme, the specific activity of lipase decreased. This decrease in activity was attributed to a reduction in the essential motions of the folded state that are required for catalytic turnover of substrate. These results provide direct evidence of this effect, which has long been a matter of speculation. Furthermore, our findings suggest that the retention of activity and stabilization of an enzyme may be balanced by tuning the extent of enzyme attachment.

L-Carnitine Is Involved in Hyperbaric Oxygen-Mediated Therapeutic Effects in High Fat Diet-Induced Lipid Metabolism Dysfunction.

Lipid metabolism dysfunction and obesity are serious health issues to human beings. The current study investigated the effects of hyperbaric oxygen (HBO) against high fat diet (HFD)-induced lipid metabolism dysfunction and the roles of L-carnitine. C57/B6 mice were fed with HFD or normal chew diet, with or without HBO treatment. Histopathological methods were used to assess the adipose tissues, serum free fatty acid (FFA) levels were assessed with enzymatic methods, and the endogenous circulation and skeletal muscle L-carnitine levels were assessed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, western blotting was used to assess the expression levels of PPARα, CPT1b, pHSL/HSL, and UCP1. HFD treatment increased body/adipose tissue weight, serum FFA levels, circulation L-carnitines and decreased skeletal muscle L-carnitine levels, while HBO treatment alleviated such changes. Moreover, HFD treatment increased fatty acid deposition in adipose tissues and decreased the expression of HSL, while HBO treatment alleviated such changes. Additionally, HFD treatment decreased the expression levels of PPARα and increased those of CPT1b in skeletal muscle, while HBO treatment effectively reverted such changes as well. In brown adipose tissues, HFD increased the expression of UCP1 and the phosphorylation of HSL, which was abolished by HBO treatment as well. In summary, HBO treatment may alleviate HFD-induced fatty acid metabolism dysfunction in C57/B6 mice, which seems to be associated with circulation and skeletal muscle L-carnitine levels and PPARα expression.

Lysosomal acid lipase does not have a propeptide and should not be considered being a proprotein.

Lysosomal acid lipase (LAL) plays an important role in lipid metabolism by performing hydrolysis of triglycerides and cholesteryl esters in the lysosome. Based upon characteristics of LAL purified from human liver, it has been proposed that LAL is a proprotein with a 55 residue propeptide that may be essential for proper folding, intracellular transport, or enzymatic function. However, the biological significance of such a propeptide has not been fully elucidated. In this study, we have performed a series of studies in cultured HepG2 and HeLa cells to determine the role of the putative propeptide. However, by Western blot analysis and subcellular fractionation, we have not been able to identify a cleaved LAL lacking the N-terminal 55 residues. Moreover, mutating residues surrounding the putative cleavage site at Lys76 ↓ in order to disrupt a proteinase recognition sequence, did not affect LAL activity. Furthermore, forcing cleavage at Lys76 ↓ by introducing the optimal furin cleavage site RRRR↓EL between residues 76 and 77, did not affect LAL activity. These data, in addition to bioinformatics analyses, indicate that LAL is not a proprotein. Thus, it is possible that the previously reported cleavage at Lys76 ↓ could have resulted from exposure to proteolytic enzymes during the multistep purification procedure.

Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices.

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

Usefulness of magnetically-controlled MNPs-enzymes microreactors for the fluorimetric determination of total cholesterol in serum.

A new dynamic method containing a magnetically retained enzyme reactor (MRER) located in the reaction/detection zone of a flow injection (FI) system, has been used for the determination of total cholesterol in serum samples. The MRER was formed by a mixture ratio of 2/1 of immobilized enzymes cholesterol esterase (ChE) and cholesterol oxidase (COx) on magnetic nanoparticles (MNPs). The analytical signal is based on the fluorescence decreasing of the fluorophore naphtofluorescein (NF) due to its oxidation by the H2O2 formed in the enzymatic reactions. The dynamic range of the calibration graph was 1.55-100 mmol L-1 expressed as total cholesterol concentration (r2 = 0.9995, n = 5, r = 3), and the detection limit was 0.65 mmol L-1. The precision expressed as relative standard deviation (RSD %) was in the range of 4.7 and 0.6%. The method showed a sampling frequency of 10 h-1 and this method was applied to the determination of cholesterol in serum samples. The results were compared with those obtained using a previous automated clinical analyzer (ILab 600 analyzer). Also, recovery values ranging between 88.5 and 101.5% were achieved.

Molecular Characterization of a Novel Cold-Active Hormone-Sensitive Lipase (HaHSL) from Halocynthiibacter Arcticus.

Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the catalytic domains of human HSLs, have received great interest due to their uses in the preparation of highly valuable biochemicals, such as drug intermediates or chiral building blocks. Here, a novel cold-active HSL from Halocynthiibacter arcticus (HaHSL) was examined and its enzymatic properties were investigated using several biochemical and biophysical methods. Interestingly, HaHSL acted on a large variety of substrates including tertiary alcohol esters and fish oils. Additionally, this enzyme was highly tolerant to high concentrations of salt, detergents, and glycerol. Furthermore, immobilized HaHSL retained its activity for up to six cycles of use. Homology modeling suggested that aromatic amino acids (Trp23, Tyr74, Phe78, Trp83, and Phe245) in close proximity to the substrate-binding pocket were important for enzyme activity. Mutational analysis revealed that Tyr74 played an important role in substrate specificity, thermostability, and enantioselectivity. In summary, the current study provides an invaluable insight into the novel cold-active HaHSL from H. arcticus, which can be efficiently and sustainably used in a wide range of biotechnological applications.

Inhibitory Effect of Condensed Tannins from Banana Pulp on Cholesterol Esterase and Mechanisms of Interaction.

In the present study, the inhibitory effect of condensed tannins (CTs) on cholesterol esterase (CEase) was studied. The underlying mechanisms were evaluated by reaction kinetics, turbidity and particle size analyses, multispectroscopy methods, thermodynamics, and computer molecular simulations. CTs showed potent CEase inhibitory activity with an IC50 value of 64.19 µg/mL, and the CEase activity decreased with increasing CT content in a mixed-competitive manner, which was verified by molecular docking simulations. Fluorescence and UV-vis measurements revealed that complexes were formed from CEase and CTs by noncovalent interaction. Isothermal titration calorimetry indicated that the interaction between CEase and CTs occurred through hydrogen bonding and hydrophobic interactions. Circular dichroism analysis suggested that CTs inhibited the activity of CEase by altering the secondary structure of CEase. The inhibition of CTs on CEase in the gastrointestinal tract might be one mechanism for its cholesterol-lowering effect.

High-fat diet feeding and palmitic acid increase CRC growth in ß2AR-dependent manner.

Epidemiology studies indicate that consumption of high-fat diet (HFD) is directly associated with the development of colorectal cancer (CRC). However, the exact component in HFD and the mechanism underlying its effect on CRC growth remained unclear. Our study shows that HFD feeding increases ß2AR expression in the xenograft tissues of CRC-bearing mouse model; the elevated ß2AR expression is reduced when HFD is replaced by control diet, which strongly suggests an association between HFD feeding and ß2AR expression in CRC. HFD feeding increases palmitic acid and stearic acid levels in CRC; however, only palmitic acid increases ß2AR expression, which is dependent upon Sp1. ß2AR plays the dominant role in promoting CRC cell proliferation among all the ß-AR subtypes. More importantly, knockout of ß2AR or knockdown of Sp1 abolishes the palmitic acid increased CRC cell proliferation, suggesting palmitic acid increases CRC cell proliferation in ß2AR-dependent manner. HFD or palmitic acid-rich diet (PAD) also fails to increase the tumor growth in xenograft mouse models bearing ß2AR-knockout CRC cells. ß2AR promotes CRC growth by increasing the phosphorylation of HSL at the residue S552. The phosphorylated and activated HSL (S552) changes the metabolic phenotype of CRC and increases energy production, which promotes CRC growth. Our study has revealed the unique tumorigenic properties of palmitic acid in promoting CRC growth, and have delineated the underlying mechanism of action. We are also the first to report the linkage between HFD feeding and ß-adrenergic signaling pathway in relation to CRC growth.

Cyanidin-3-rutinoside acts as a natural inhibitor of intestinal lipid digestion and absorption.

BACKGROUND: Cyanidin-3-rutinoside (C3R), a naturally occurring anthocyanin, possesses anti-oxidant, anti-hyperglycemic, anti-glycation and cardioprotective properties. However, its mechanisms responsible for anti-hyperlipidemic activity have not been fully identified. The aim of the study was to investigate the lipid-lowering mechanisms of C3R through inhibition of lipid digestion and absorption in vitro. METHODS: The inhibitory activity of C3R against pancreatic lipase and cholesterol esterase was evaluated using enzymatic fluorometric and enzymatic colorimetric assays, respectively. An enzyme kinetic study using Michaelis-Menten and the derived Lineweaver-Burk plot was performed to understand the possible types of inhibition. The formation of cholesterol micelles was determined using the cholesterol assay kit. The bile acid binding was measured using the colorimetric assay. The NBD cholesterol uptake in Caco-2 cells was determined using fluorometric assay. The mRNA expression of cholesterol transporter (Niemann-Pick C1-like 1) was determined by RT-PCR. RESULTS: The results showed that C3R was a mixed-type competitive inhibitor of pancreatic lipase with the IC50 value of 59.4 ± 1.41 µM. Furthermore, C3R (0.125-1 mM) inhibited pancreatic cholesterol esterase about 5-18%. In addition, C3R inhibited the formation of cholesterol micelles and bound to primary and secondary bile acid. In Caco-2 cells, C3R (12.5-100 µM) exhibited a significant reduction in cholesterol uptake in both free cholesterol (17-41%) and mixed micelles (20-30%). Finally, C3R (100 µM) was able to suppress mRNA expression of NPC1L1 in Caco-2 cells after 24 h incubation. CONCLUSIONS: The present findings suggest that C3R acts as a lipid-lowering agent through inhibition of lipid digestion and absorption.

Enzymatic Reactions in a Lab-on-Valve System: Cholesterol Evaluations.

The micro sequential injection analysis / lab-on-valve (µSIA-LOV) system is a miniaturized SIA system resulting from the implementation of a lab-on-valve (LOV) atop of the selection valve. It integrates the detection cell and the sample processing channels into the same device, promoting the reduction of reagent consumption and waste generation, the improvement of the versatility, and the reduction of the time of analysis. All of these characteristics are really relevant to the implementation of enzymatic reactions. Additionally, the evaluation of cholesterol in serum samples is widely relevant in clinical diagnosis, since higher values of cholesterol in human blood are actually an important risk factor for cardiovascular problems. An automatic methodology was developed based on the µSIA-LOV system in order to evaluate its advantages in the implementation of enzymatic reactions performed by cholesterol esterase, cholesterol oxidase and peroxidase. Considering these reactions, the developed methodology was also used for the evaluation of cholesterol in human serum samples, showing reliable and accurate results. The developed methodology presented detection and quantification limits of 1.36 and 4.53 mg dL-1 and a linear range up to 40 mg dL-1. This work confirmed that this µSIA-LOV system is a simple, rapid, versatile, and robust analytical tool for the automatic implementation of enzymatic reactions performed by cholesterol esterase, cholesterol oxidase, and peroxidase. It is also a useful alternative methodology for the routine determinations of cholesterol in real samples, even when compared with other automatic methodologies.