Enzymatic Reagent Research Articles

Improving the properties of chemically damaged wool fabrics with carbohydrate polymers

ABSTRACTWool is a natural composite material consisting of keratin and keratin‐associated proteins as the key molecular components. During wool product processing, a variety of chemical and enzymatic reagents are used, the side‐effects of which can include the removal of the outside layers of the fiber (cuticle) and damage within the internal protein matrix of the fiber. This can reduce the mechanical strength and durability of wool fabrics. We report the use of neutral, cationic, and anionic carbohydrate polymers, namely 2‐hydroxyethyl cellulose, chitosan and alginate, as repair agents to improve the mechanical properties and morphology of wool fabrics damaged under harsh alkaline conditions. Tensile strength, peel adhesion, scanning electron micrographs, and fabric wettability evaluation reveal the cationic polymer, chitosan, to be most effective at remedying the effects of the alkaline treatment. The improved mechanical properties observed after chitosan treatment may offer viable remediation routes for adding value to processing‐damaged wool textiles. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 3105–3111, 2013

Effect of Picric Acid and Enzymatic Creatinine on the Efficiency of the Glomerular Filtration Rate Predicator Formula

To investigate the impact of serum creatinine measurement on the applicability of glomerular filtration rate (GFR) evaluation equations. 99mTc-DTPA plasma clearance rate was used as GFR reference (rGFR) in patients with chronic kidney disease (CKD). Serum creatinine was measureded using enzymatic or picric acid creatinine reagent. The GFR of the patients were estimated using the Cockcroft-Gault equation corrected for body surface area, simplified Modification of Diet in Renal Disease (MDRD) equation, simplified MDRD equation corrected to isotopes dilution mass spectrometry, the CKD epidemiology collaborative research equation, and two Chinese simplified MDRD equations. Significant differences in the eGFR results estimated through enzymatic and picric acid methods were observed for the same evaluation equation. The intraclass correlation coefficient (ICC) of eGFR when the creatinine was measured by the picric acid method was significantly lower than that of the enzymatic method. The assessment accuracy of every equation using the enzymatic method to measure creatinine was significantly higher than that measured by the picric acid method when rGFR was > or = 60 mL/min/1.73m2. A significant difference was demonstrated in the same GFR evaluation equation using the picric acid and enzymatic methods. The enzymatic creatinine method was better than the picric acid method.

Influence of Concentration and Activation on Hydrogen Peroxide Diffusion through Dental Tissues In Vitro

This study evaluated the effect of physical and chemical activation on the diffusion time of different concentrations of hydrogen peroxide (HP) bleaching agents through enamel and dentin. One hundred and twenty bovine cylindrical specimens were divided into six groups (n = 20): 20% HP; 20% HP with light activation; 20% HP with manganese gluconate; 35% HP; 35% HP with light activation; and 35% HP with manganese gluconate. The specimens were fixed over transparent epoxy wells with internal cavities to simulate a pulpal chamber. This chamber was filled with an enzymatic reagent to simulate pulpal fluid. The bleaching gels were applied on enamel surface and the image of the pulpal fluid was captured by a video camera to monitor the time of peroxide penetration in each specimen. ANOVA analysis showed that concentration and type of activation of bleaching gel significantly influenced the diffusion time of HP (P < 0.05). 35% HP showed the lowest diffusion times compared to the groups with 20% HP gel. The light activation of HP decreased significantly the diffusion time compared to chemical activation. The highest diffusion time was obtained with 20% HP chemically activated. The diffusion time of HP was dependent on activation and concentration of HP. The higher concentration of HP diffused through dental tissues more quickly.

Analytical evaluation of two enzymatic methods for the measurement of total bile acid in intrahepatic cholestasis of pregnancy

The aim of this study was to estimate bilirubin interference and accuracy of six routine methods for measuring creatinine compared with isotope dilution–liquid chromatography mass spectrometry (ID–LC/MS).A total of 40 clinical serum samples from 31 patients with serum total bilirubin concentration > 68.4 μmol/L were collected. Serum creatinine was measured using two enzymatic reagents and four Jaffe reagents as well as ID–LC/MS. Correlations between bilirubin concentration and percent difference in creatinine compared with ID–LC/MS were analyzed to investigate bilirubin interference. Bias estimations between the six reagents and ID–LC/MS were performed. Recovery tests using National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 967a were also performed.Both the enzymatic methods showed no bilirubin interference. However, three of the four Jaffe methods demonstrated significant bilirubin concentration-dependent interference in samples with creatinine levels < 53 μmol/L, and two of them showed significant bilirubin interference in samples with creatinine levels ranging from 53.0 to 97.2 μmol/L. Comparison of these methods with ID–LC/MS using patients' samples with elevated bilirubin revealed that the tested methods failed to achieve the bias goal at especially low levels of creatinine. In addition, recovery test using NIST SRM 967a showed that bias in one Jaffe method and two enzymatic methods did not achieve the bias goal at either low or high level of creatinine, indicating they had calibration bias. One enzymatic method failed to achieve all the bias goals in both comparison experiment and recovery test.It is important to understand that both bilirubin interference and calibration traceability to ID–LC/MS should be considered to improve the accuracy of creatinine measurement.

Chaperone Probes and Bead-Based Enhancement To Improve the Direct Detection of mRNA Using Silicon Photonic Sensor Arrays

Herein, we describe the utility of chaperone probes and a bead-based signal enhancement strategy for the analysis of full length mRNA transcripts using arrays of silicon photonic microring resonators. Changes in the local refractive index near microring sensors associated with biomolecular binding events are transduced as a shift in the resonant wavelength supported by the cavity, enabling the sensitive analysis of numerous analytes of interest. We employ the sensing platform for both the direct and bead-enhanced detection of three different mRNA transcripts, achieving a dynamic range spanning over 4 orders of magnitude and demonstrating expression profiling capabilities in total RNA extracts from the HL-60 cell line. Small, dual-use DNA chaperone molecules were developed and found to both enhance the binding kinetics of mRNA transcripts by disrupting complex secondary structure and serve as sequence-specific linkers for subsequent bead amplification. Importantly, this approach does not require amplification of the mRNA transcript, thereby allowing for simplified analyses that do not require expensive enzymatic reagents or temperature ramping capabilities associated with RT-PCR-based methods.

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence Detector

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH).

EnzMet™: An Enzymatic Metallography Reagent for Accurately Delineating Neuronal Boundaries for Segmenting Gap Junction-Coupled Neurons in their Three-dimensional Space

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Dual Photonic-Electrochemical Lab on a Chip for Online Simultaneous Absorbance and Amperometric Measurements

A dual lab on a chip (DLOC) approach that enables simultaneous optical and electrochemical detection working in a continuous flow regime is presented. Both detection modes are integrated for the first time into a single detection volume and operate simultaneously with no evidence of cross-talk. The electrochemical cell was characterized amperometrically by measuring the current in ferrocyanide solutions at +0.4 V vs gold pseudoreference electrode, at a flow rate of 200 μL min(-1). The experimental results for ferrocyanide concentrations ranging from 0.005 to 2 mM were in good agreement with the values predicted by the Levich equation for a microelectrode inside a rectangular channel, with a sensitivity of 2.059 ± 0.004 μA mM(-1) and a limit of detection (LoD) of (2.303 ± 0.004) × 10(-3) mM. Besides, optical detection was evaluated by measuring the absorbance of ferricyanide solutions at 420 nm. The results obtained therein coincide with those predicted by the Beer-Lambert law for a range of ferricyanide concentrations from 0.005 to 0.3 mM and showed an estimated LoD of (0.553 ± 0.001) × 10(-3) mM. The DLOC was finally applied to the analysis of L-lactate via a bienzymatic reaction involving lactate oxidase (LOX) and horseradish peroxidase (HRP). Here, the consumption of the reagent of the reaction (ferrocyanide) was continuously monitored by amperometry whereas the product of the reaction (ferricyanide) was recorded by absorbance. The DLOC presented good performance in terms of sensitivity and limit of detection, comparable to other fluidic systems found in the literature. Additionally, the ability to simultaneously quantify enzymatic reagent consumption and product generation confers the DLOC a self-verifying capability which in turn enhances its robustness and reliability.

One‐Pot Oxidation–Hydrocyanation Sequence Coupled to Lipase‐Catalyzed Diastereoresolution in the Chemoenzymatic Synthesis of Sugar Cyanohydrin Esters

AbstractA three‐step, one‐pot synthesis and diastereoresolution sequence is described in anhydrous toluene starting from methyl α‐D‐2,3,4‐tri‐O‐acetylgalacto‐ (1a), ‐manno‐ (1b) and ‐glucopyranosides (1c). The reaction sequence, including consecutive transformations through the aldehyde [PhI(OAc)2, 2,2,6,6‐tetramethylpiperidine‐1‐oxyl (TEMPO)] and cyanohydrin [basic resin or (R)‐oxynitrilase] into the (6R)‐cyanohydrin ester (lipase) is shown to proceed in a one‐pot cascade, except that the basic resin (when used) should be removed before the addition of the enzymatic acylation reagents. We have shown that the effective transformation of 1a (75 % reaction yield) through labile intermediates gives the stable (6R)‐cyanohydrin butanoate (85 % de). Further diastereomeric purification by chromatography is possible, although the product is already of high diastereopurity. (6R)‐Cyanohydrin esters are obtained through acylation with Burkholderia cepacia lipase. The (6S)‐ester (de 99 %) is produced by Candida rugosa lipase when the sequence is started from 1c whereas the other sugar derivatives are less suited to the reaction with lipase.

Doubt and Proof: The Need to Verify The Cleaning Process

<i>Doubt and Proof:</i> The Need to Verify The Cleaning Process

Anion‐exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl‐ligated gel with a perchlorate‐containing eluent

Rabbits are often used as experimental animals in studies of atherosclerosis and hyperlipidemia. Although rabbit lipoproteins can be quantitated by sequential ultracentrifugation, a simpler and more reproducible method is desirable for detailed analyses. The current study describes a method to analyze rabbit lipoproteins in plasma by anion-exchange high-performance liquid chromatography using a column filled with nonporous, diethylaminoethyl-ligated polymers. A solution of NaClO₄ was used to adjust the ionic strength of the eluent. The method required only 15 μL of plasma and analysis was completed in 23 min. Five lipoprotein fractions (high-density lipoprotein, low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein and chylomicrons) were eluted with step-wise increases in a concentration of NaClO₄. The post-column eluate was reacted with an enzymatic reagent to determine total cholesterol, and the lipoprotein-cholesterol fraction was calculated according to relative peak areas in the chromatogram. The within-day and between-day assay coefficients of variation for lipoprotein cholesterol levels ranged between 0.436 and 7.143% and between 2.905 and 10.526%, respectively. Administering a high-fat diet increased lipoprotein-cholesterol concentrations by 6- to 77-fold. The method described here was nevertheless able to quantitate levels of lipoprotein-cholesterol in plasma samples from these rabbits. These results indicate that this method may be applied to lipoprotein studies using hyperlipidemic rabbit models.

SiRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoE⁻/⁻ and low density lipoprotein receptor (LDLr)⁻/⁻ mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETP⁺/⁻ hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETP⁺/⁻ mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.

Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) (Lp(a)), Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examination cycle 5 in subjects participating in the Framingham Offspring Study who were free of CHD. After a mean follow-up of 12.3 years, 98 men and 47 women developed new CHD events. In multivariate analysis, the hazard ratio of CHD was approximately two-fold greater in men in the upper tertile of plasma Lp(a) levels, relative to those in the bottom tertile (P < 0.002). The apo(a) isoform size contributed only modestly to the association between Lp(a) and CHD and was not an independent predictor of CHD. In multivariate analysis, Lp(a) cholesterol was not significantly associated with CHD risk in men. In women, no association between Lp(a) and CHD risk was observed. Elevated plasma Lp(a) levels are a significant and independent predictor of CHD risk in men. The assessment of apo(a) isoform size in this cohort does not add significant information about CHD risk. In addition, the cholesterol content in Lp(a) is not a significant predictor of CHD risk.

Micro-colorimetric determination of adenosinetriphosphatase activity in freeze-dried sections of rat diaphragm muscle.

Abstract A technique is described for determination of ATPase activity in 3–30 μg samples dissected from freeze-dried sections of rat diaphragm muscle. Tissue samples are incubated at room temperature with optimal concentrations of ATP (10 mM) and MgSO4 (5 mM) in 0·1 M tris/HCl buffer at pH 7·4. Inorganic phosphate is measured spectrophotometrically. Mean activity for 20 rats was 0·22 ± 0·06 (s.d.) mole-Pi/kg/wet tissue/15 min. Results did not depend on plane of section or section thickness. 5 mM Mg2+, 10 mM Ca2+ and 1 mM 2, 4-dinitrophenol produced maximal ATPase activation and inhibition was obtained with p-chloromercuribenzoic acid (pI503·9) but not chlorpromazine (0·01–0·1 mM). There was no significant evidence of Na+ plus K+ activated ATPase or inhibition with ouabain (5 mM). ATPase activity was uncharacterized and relative contributions of specified muscle ATPase systems were unknown. As an insoluble enzyme system was involved and photomicrographs showed characteristic muscle features in freeze-dried sections, ATPase activity appeared due to enzymic function in situ. Results are discussed in relation to mutual availability of enzymic sites and reagents.

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo1[S

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Study on the distribution of deltamethrin residues in stored wheat using sequential fractionation procedure

The distribution of deltamethrin residues in different fractions of stored wheat were investigated by sequential fractionation procedure, which modified the plant cell wall fractionation procedure established by Langebartels and Hams (1985) to conform to the property of deltamethrin. In this procedure, deltamethrin - treated wheat were firstly extracted with pH 7.0 buffers and organic solvents to remove the extractable deltamethrnin. Subsequently, the treated wheat was fractionated into six macromolecular components using various enzymatic or chemical reagents. With the quantification of deltamethrin by gas chromatography, a majority of the released deltamethrin residues was found in organic solvents, and the unextractable residues were mainly distributed in starch, protein and pectin components of grain. Control incubations in the absence of enzyme or chemical reagents were further performed, which indicated that the interactions between unextractable deltamethrin and six macromolecular components may be different.

Tissue Lipid Analysis with Enzymatic Reagents

Featured Article: Carr TP, Andresen CJ, Rudel LL. Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts. Clin Biochem 1993;26:39–42.1 The need to measure serum or plasma cholesterol prompted the development of enzyme-based colorimetric assays during the 1970s. These assays eventually replaced the traditional chemical methods. The advantages of the enzymatic procedures were speed and simplicity. On the other hand, because the enzymes naturally work in aqueous environments, the procedures were applicable only to aqueous samples. Enzymatic quantification was not possible for tissue or cellular lipids extracted into organic solvents. Our 1993 report provided several important improvements in this regard. First, we described a procedure for solubilizing tissue lipids in water with the aid of detergent while optimizing enzyme activity. Second, our procedure allowed quantification of all major lipid classes in a single sample preparation. Third, we adapted the procedure to microplates, substantially reducing the volume and expense of enzymatic reagents. Fourth, the microscale approach allowed quantification …

Pasting properties of hydroxypropylated starches before or after proteinase treatment

AbstractTo examine the effect of starch protein on hydroxypropylation, corn (normal and waxy) and potato starches were hydroxypropylated with propylene oxide at two levels (8.3 and 12.3% based on dry basis of starch weight) before or after proteinase (thermolysin) treatment, and then pasting properties of the starches were measured. For normal corn starch, protein appeared to be involved in hydroxypropylation, especially at the higher concentration of reagent (12.3%); the starch hydroxypropylated after proteinase treatment (Enz‐HP) showed less reaction efficiency than that only hydroxypropylated (HP) or proteinase treated starch after hydroxypropylation (HP‐Enz). This suggests a possible reaction between some of the reagent and the starch proteins that exist in the channel and surface of the starch granules. However, not much difference was found with the waxy corn starch, as it contains much lower amounts of amylose and protein than normal corn starch. The potato starch, which has no channels, showed complicated protein effects. For potato starch, a different access of the enzyme and chemical reagents to the granule matrix could be possible.

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

Peroxisome proliferator-activated receptor delta (PPARdelta) is involved in regulation of energy homeostasis. Activation of PPARdelta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPARdelta activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPARdelta agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion ( approximately 200%) and TICE ( approximately 100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPARdelta activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPARdelta stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis.

Enzymatic determination of cholesterol and triglycerides in serum lipoprotein profiles by asymmetrical flow field-flow fractionation with on-line, dual detection

Alterations of lipoproteins (LPs) and related lipid levels in blood serum are correlated to the risk of coronary artery disease (CAD). Fast, possibly automated methods to obtain complete, multi-parametric LP profiles are therefore welcome to be developed for routine, clinical analysis practice. In this work, asymmetrical flow field-flow fractionation (AF4) with on-line, dual post-fractionation reaction detection (PFRD) is applied to develop a method for single-run, simultaneous quantification of cholesterol (CHOL) and triglycerides (TGs) in each fractionated LP class. The enzymatic reagents used for the post-fractionation reaction are available as commercial kits for certified, standard clinical protocols for the analysis of CHOL and TGs in serum. Using CHOL and glycerol as reference standards, a new procedure is applied to optimize the experimental conditions for PFRD-based, quantitative analysis. Upon optimized PFRD and AF4 conditions, results obtained for the determination of total CHOL (TC), TGs, HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) in a set of serum samples from healthy donors are found in agreement with the values provided by a clinical laboratory. The intra-day and inter-day precisions of the method were found always lower than 10% (CV). When the method was applied to serum samples from patients affected by sepsis, differences in CHOL and TG profiles between patients and healthy donors were observed.