Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.     

A New Spectrophotometric Method for the Detection of Lipase Activity Using 2,4-Dinitrophenyl Butyrate as a Substrate

A rapid and sensitive assay for the detection of lipase activity is described. The method is based upon the increase in absorbance at 360 nm due to the formation of the 2,4-dinitrophenolate anion during the enzymatic hydrolysis of 2,4-dinitrophenyl butyrate. The substrate is used in an emulsified form. Using a diode array spectrophotometer with internal referencing a correction can be made for absorbance changes due to clearance of the emulsion during hydrolysis. The small reaction volume and the high extinction coefficient of the product makes the method applicable for detection of both low substrate and low enzyme concentration.

Four lipases were tested: lipase from porcine pancreas, Candida cylindracea, Pseudomonas sp. and Aspergillus niger. All enzymes are readily able to catalyse the hydrolysis of 2,4-dinitrophenyl butyrate.     

Lipolytic activity of porcine pancreas lipase on fatty acid esters of dialkylglycerols: a structural basis for the design of new substrates for the assay of pancreatic lipases activity.

For the design of new synthetic substrates for the assay of pancreatic lipases activity, acyl dialkylglycerols of variable chain length were prepared. Titrimetric assay of these substrates showed the highest lipolytic activity of porcine pancreas lipase (pPL) with butanoyl dibutylglycerol. The activity is lower but comparable to that shown by pPL towards the classical substrate tributyrin. The 4-nitrophenylcarbonate of 1,2-di-O-butylglycerol, has been prepared and proposed as synthetic substrate for a new spectrophotometric assay of pancreatic lipases.     

Application of silver nanoparticles to the chemiluminescence determination of cefditoren pivoxil using the luminol–ferricyanide system

A new simple, accurate and sensitive sequential injection analysis chemiluminescence (CL) detection method for the determination of cefditoren pivoxil (CTP) has been developed. The developed method was based on the enhancement effect of silver nanoparticles on the CL signal arising from a luminol–potassium ferricyanide reaction in the presence of CTP. The optimum conditions relevant to the effect of luminol, potassium ferricyanide and silver nanoparticle concentrations were investigated. The proposed method showed linear relationships between relative CL intensity and the investigated drug concentration at the range 0.001–5000 ng/mL, (r = 0.9998, n = 12) with a detection limit of 0.5 pg/mL and quantification limit of 0.001 ng/mL. The relative standard deviation was 1.6%. The proposed method was employed for the determination of CTP in bulk drug, in its pharmaceutical dosage forms and biological fluids such as human serum and urine. The interference of some common additive compounds such as glucose, lactose, starch, talc and magnesium stearate was investigated. In addition, the interference of some related cephalosporins was tested. No interference was recorded. The obtained sequential injection analysis‐CL results were statistically compared with those from a reported method and did not show any significant differences. Copyright © 2014 John Wiley & Sons, Ltd.     

Isolation and chemical characterization of glicentin C-terminal hexapeptide in porcine pancreas

Using a radioimmunoassay specific for porcine glicentin C-terminal hexapeptide, we isolated a peptide from porcine pancreas and characterized it as the C-terminal 64-69 sequence of glicentin: H-Asn-Lys-Asn-Asn-Ile-Ala-OH. The purification steps included gel filtration, ion-exchange chromatography and HPLC. In each step, the recovery of the desired peptide, radioimmunologically estimated from the respective elution profile, was 71.4-91.7%. The final yield of the hexapeptide was 22 micrograms (4.3%) from 800 g pancreas. The pancreatic content of this peptide was estimated to be approximately equimolar to that of pancreatic glucagon. No hexapeptide-like component was detected in porcine intestinal extracts. The data confirmed that the processing of pancreatic proglucagon liberates the C-terminal hexapeptide of the intramolecular glicentin sequence in a tissue-specific manner during the production of glucagon.     

表面改性SBA-15材料固载猪胰脂肪酶

采用试剂y-氯丙基三乙氧基硅烷（cvrEs）对介孔硅材料SBA-15进行表面改性,并通过红外图谱（FT-IR）和N2吸附脱附等温图（BET）对其进行表征。结果表明：改性前原材料的比表面积为460．9m2／g,改性后材料比表面积提高到512．0m2／g。利用改性前和改性后的SBA-15对猪胰脂肪酶进行固载实验,并对实验结果进行比较,发现改性后的SBA-15在脂肪酶活性、pH环境适应性、热耐受性和可操作性都优于改性前的SBA-15,在最优条件下的酶活力提高超过60％。     

Development of an indirect method for measuring porcine pancreatic lipase in human duodenal fluid

Patients with exocrine pancreatic insufficiency are usually treated with porcine pancreatic enzymes but the bioavailability of these enzymes in the gut remains a matter of discussion. In order to determine the duodenal availability of porcine pancreatic lipase (PPL) present in pancreatic extracts (PE) taken orally, we developed a method for quantifying PPL in samples containing both PPL and human pancreatic lipase (HPL). Total pancreatic lipase activity measurements using the pH-stat technique and tributyrin as substrate were combined with an HPL-specific ELISA. Based on the known specific activity of the purified HPL, its activity was deduced from the ELISA measurements, and the PPL activity was obtained by subtracting the HPL activity from the total pancreatic lipase activity. This assay was established and validated using various samples containing pure PPL and recombinant HPL or PE, mixed or not with human duodenal juice. Samples collected in vivo from patients treated with PE were also tested. It was found that PPL did not affect the HPL ELISA, and the indirect PPL assay gave a measurement accuracy of 6.6% with the samples containing pure PPL and 10% with those containing PE. This assay was also used successfully to discriminate between PPL and the endogenous HPL present in the duodenal contents of patients with severe pancreatic insufficiency treated with PE. This method might provide a useful means of assessing the availability of PEs at their site of action, in the absence of a PPL-specific ELISA.     

Development of a sensitive and selective assay for the determination of procarboxypeptidase U (thrombin-activatable fibrinolysis inhibitor) in plasma

To date, several assays for procarboxypeptidase U (proCPU) determination exist, all having their own inherent disadvantages and advantages. A drawback of activity-based assays is the interference of the constitutively active carboxypeptidase N (CPN) in plasma. Recent screening of Bz-Xaa-Arg peptides with modified aromatic amino acids at the P1 position revealed a selective CPU substrate, N-benzoyl-ortho-cyano-phenylalanyl-arginine (Bz-o-cyano-Phe-Arg), which will allow straightforward determination of proCPU in plasma. Our assay shows an excellent linearity in the concentration range of 20-2600 U/L, with within- and between-run precision values of 2.7% and 4.6%, respectively. A good correlation with our high-performance liquid chromatography (HPLC)-assisted proCPU activity assay using hippuryl-l-arginine (HipArg) as substrate was found. Besides the major improvement regarding the selectivity, the assay is much easier to perform and far less time-consuming compared with the proCPU activity assay using HipArg as substrate.     

Detection of proteolytic activity by fluorescent zymogram in-gel assays

Proteases are involved in the regulation of many biological functions. This study describes a novel method for detecting protease activity by fluorescent zymogram in-gel protease assays, using SDS polyacrylamide gels copolymerized with a peptide-MCA (4-methyl-coumaryl-7-amide) substrate. This method allows simultaneous determination of protease cleavage specificity and molecular weight. Trypsin was electrophoresed in SDS polyacrylamide gels copolymerized with Boc-Gln-Ala-Arg-MCA, the gel was then incubated in assay buffer, and trypsin cleavage of the peptide-MCA substrate generated fluorescent AMC (7-amino-4-methyl-coumarin), which was subsequently detected under UV transillumination. Chymotrypsin activity was detected in gels copolymerized with Suc-Ala-Ala-Pro-Phe-MCA substrate. Selective detection of these proteases was demonstrated by the absence of trypsin activity in gels containing the chymotrypsin substrate, and the lack of chymotrypsin activity in gels containing the trypsin substrate. Detection of proteolytic activity from secretory vesicles of adrenal medulla (chromaffin granules) was observed with the trypsin substrate, Z-Phe-Arg-MCA, but not with the chymotrypsin substrate. Overall, this sensitive fluorescent zymogram in-gel protease assay method can be used for rapid determination of protease cleavage specificity and enzyme molecular weight in biological samples. This assay should be useful for many research disciplines investigating the role of the many proteases that control cellular functions.     

Cationic polyelectrolytes-lipases complexes formation as tool for recovery of these enzymes from their natural sources

In the present paper the formation of complexes between positively charge polyelectrolyte (polyethyleneimine and chitosan) and Candida rugosa lipase from a crude extract and porcine lipase from pancreas commercial homogenate preparations were analyzed. The solubility of lipases-cationic polyelectrolytes formation was dependent on: polyelectrolyte densities electrical charge, polyelectrolyte and enzyme concentrations and salts present in the solution. The lipase was recovered from the non-soluble complex by adding of NaCl at a given pH. Although the polyelectrolytes did not affect lipase biological activity, both of them produced good enzyme recovery (>90%); however, purification factors were low. This methodology appears to be a good previous prepurification and concentration method, using, low-cost polymers, allows the design of a purification method where the protein of interest is present in a large volume with respect to the small amount of polyelectrolyte added.     

Development of a highly sensitive chemiluminescent assay for hydrogen peroxide under neutral conditions using acridinium ester and its application to an enzyme immunoassay

We developed a highly sensitive chemiluminescent (CL) assay for hydrogen peroxide using 10‐methyl‐9‐(phenoxycarbonyl) acridinium fluorosulfonate (PMAC) that produced chemiluminescence under neutral conditions and applied it to an enzyme immunoassay (EIA). One picomole of hydrogen peroxide could be detected using the optimized PMAC‐CL method and 6.2 × 10^‐20 mol β‐d ‐galactosidase (β‐gal) could be detected by combining an indoxyl derivative substrate and the proposed PMAC‐CL method. This highly sensitive CL β‐gal assay was applied to an EIA for thyroid‐stimulating hormone (TSH) using β‐gal as a label enzyme; 0.02–100.0 μU/mL TSH in human serum could be assayed directly and with high reproducibility. Copyright © 2013 John Wiley & Sons, Ltd.     

Porcine pancreatic lipase related protein 2 has high triglyceride lipase activity in the absence of colipase

Efficient dietary fat digestion is essential for newborns who consume more dietary fat per body weight than at any other time of life. In many mammalian newborns, pancreatic lipase related protein 2 (PLRP2) is the predominant duodenal lipase. Pigs may be an exception since PLRP2 expression has been documented in the intestine but not in the pancreas. Because of the differences in tissue-specific expression, we hypothesized that the kinetic properties of porcine PLRP2 would differ from those of other mammals. To characterize its properties, recombinant porcine PLRP2 was expressed in HEK293T cells and purified to homogeneity. Porcine PLRP2 had activity against tributyrin, trioctanoin and triolein. The activity was not inhibited by bile salts and colipase, which is required for the activity of pancreatic triglyceride lipase (PTL), minimally stimulated PLRP2 activity. Similar to PLRP2 from other species, PLRP2 from pigs had activity against galactolipids and phospholipids. Importantly, porcine PLRP2 hydrolyzed a variety of dietary substrates including pasteurized human mother's milk and infant formula and its activity was comparable to that of PTL. In conclusion, porcine PLRP2 has broad substrate specificity and has high triglyceride lipase activity even in the absence of colipase. The data suggest that porcine PLRP2 would be a suitable lipase for inclusion in recombinant preparations for pancreatic enzyme replacement therapy.     

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.     

Carotenol fatty acid esters: easy substrates for digestive enzymes?

To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied. Digestions were carried out in phosphate buffer at pH 7.4 and 37 °C. As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; β-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; β-cryptoxanthin esters) as well as retinyl palmitate. These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase. As reference enzyme a yeast lipase from Candida rugosa was applied. A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut. Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed. The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (U/mg solid). Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility.     

A Fluorometric Assay for Detection of Lysyl Oxidase Enzyme Activity in Biological Samples

Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking in the biosynthesis of normal mature functional insoluble extracellular matrices. In addition, lysyl oxidase has been identified as a possible tumor suppressor. Lysyl oxidase activity in biological samples is traditionally and most reliably assessed by tritium release end-point assays using radiolabeled collagen or elastin substrates involving laborious vacuum distillation of the released tritiated water. In addition, a less sensitive fluorometric method exists that employs nonpeptidyl amine lysyl oxidase substrates and measures hydrogen peroxide production with horseradish peroxidase coupled to homovanillate oxidation. The present study describes a more sensitive fluorescent assay for lysyl oxidase activity that utilizes 1,5-diaminopentane as substrate, and released hydrogen peroxide is detected using Amplex red in horseradish peroxidase-coupled reactions. This method allows the detection of 40 ng of enzyme per 2 ml assay at 37 degrees C and is 7.5 times more sensitive than the currently available fluorometric assay for enzyme activity. This method eliminates the interference that occurs in some biological samples and can be successfully used to detect lysyl oxidase activity in cell culture experiments.     

微孔比色法在猪胰腺PLA_2制备中的应用

微孔比色法采用合成的磷脂类似物２－硫代十六酰乙基磷酸胆碱作底物,在多孔聚苯乙烯板的小孔中反应,并用酶联免疫检测器连续测定和记录吸收值．同时应用此法及滴定法检测酶活力,从猪胰腺中制备了一种分子量低（１４．３ｋＤ）,对热、酸稳定,活性依赖Ｃａ２＋的ＰＬＡ２．两种方法检测结果具有可比性,而微孔比色法同时可测多个样品,有节约样品,灵敏度较高等优点．微孔比色法特别适用于大量的样品测定,如拮抗剂筛选、临床样品及制备酶时层析级分的检测等．     

Egg yolk lipoproteins as substrates for lipases

Egg yolk emulsions containing phospholipids (about 31%, w/w) are classically used as substrates for measuring phospholipase A2 activity using the pH-stat method. Here we investigated the susceptibility of egg yolk lipoproteins to lipolysis by various highly purified lipases of animal or microbial origin. Egg yolk lipoproteins, which contain up to 65% triacylglycerols, were found to be effective substrates for all the lipases tested. The specific activities measured on egg yolk lipoproteins using the pH-stat technique were found to be 8000, 1000, 1250 and 1700 U/mg in the case of human pancreatic lipase, horse pancreatic lipase, porcine pancreatic lipase and Humicola lanuginosa lipase, respectively. No activity was detected in the absence of colipase with any of the pancreatic lipases tested. Consequently, the classical egg yolk assay cannot be considered as a specific phospholipase A2 assay.     

Enhanced chemiluminescence enzyme‐linked immunoassay for the determination of DNA methyltransferase 1 in human serum

The occurrence of many diseases is closely related to the high expression of DNA methyltransferase 1 (DNMT1). However, most studies are focused on the detection of DNMT1 activity, a few are concerned with the detection of DNMT1 content. In this study, we developed a simple and highly sensitive chemiluminescence (CL) assay for the detection of DNMT1 content. In this method, anti‐DNMT1 monoclonal antibody was coated on a polystyrene microplate to capture DNMT1. Then anti‐DNMT1 polyclonal antibody and goat anti‐rabbit immunoglobulin G with horseradish peroxidase (IgG‐HRP) were respectively added to combine with captured DNMT1 to form a sandwich structure. Finally, the HRP could catalyze CL substrate and achieve CL signal response. Based on this novel sensitive strategy, the recovery percents were in the ranges from 71.5% to 91.0%. The precision of intra‐assays and inter‐assays were 5.45%–11.29% and 7.03%–11.25%, respectively. The method was successfully applied for the determination of DNMT1 in human serum. The detection results of serum samples showed that the proposed assay had a high correlation with enzyme‐linked immunosorbent assay (ELISA) kit. Compared with the ELISA kit (limit of detection = 0.1 ng/mL), the method has a lower limit of detection of 0.042 ng/mL. Therefore, our method has the potential for the detection of DNMT1 content in clinical diagnosis.     

Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

A facile turn-on chemiluminescence probe for sensitive imaging on aminopeptidase N activity

Aminopeptidase N, as a target for drug discovery, shows marked relationships with many diseases, especially liver injury and cancer. Here, we explored a chemiluminescence (CL) probe for sensing APN by tethering the APN-specific substrate group to the ortho-acrylated phenoxy-dioxetane scaffold. In this way, two CL probes ( APN-CL and BAPN-CL ) were designed with noncapped leucine and butoxy-carbonyl capped leucine as the protecting group to preserve the chemiexcitation energy. The uncovered leucine was demonstrated to be essential for detection of APN activity by comparing the CL intensity of two CL probes. Probe APN - CL was turned on upon APN cleavage, resulting in a high chemiluminescent emission, whereas the chemiexcitation energy of probe BAPN-CL was still restrained even with the high-level APN. The result was further elucidated by molecular docking simulations. Probe APN - CL exhibited a fast response and high sensitivity with a detection limit of 0.068 U/L, and an excellent specificity for the discrimination of APN from biological ions, small molecules, and other proteases commonly found in living system. By virtue of good stability and cell viability, probe APN - CL imaged abnormal levels of APN in tumour cells and tumour-bearing mice. Moreover, this probe APN-CL could be easily used to evaluate APN inhibitors and APN levels in plasma samples from 20 patients. Overall, as a facile and cost-effective probe, APN - CL will be a promising alternative in the early diagnosis of pathologies and for cost-effective screening of inhibitors.