Nuclear Magnetic Resonance Assays Research Articles

Phlebotomy tube interference with nuclear magnetic resonance (NMR) lipoprotein subclass analysis

BackgroundLipoprotein subclass analysis by nuclear magnetic resonance (NMR) can be used in risk assessment of atherosclerotic cardiovascular disease (ASCVD). There is little information in the literature regarding phlebotomy tube interferences with NMR testing. MethodsPooled human serum was exposed to phlebotomy tubes manufactured by Becton Dickinson (BD), Greiner Bio-One, or Sarstedt. Serum was analyzed on the Axinon lipoFIT by NMR assay and by conventional lipid assays performed on a Roche Cobas 8000 system. The effect of incomplete fill volume was also assessed. ResultsAnalytical interference in NMR lipoprotein subclass results was observed across many different tube types. The 5 mL Greiner Bio-One Z Serum Sep Clot Activator tube correlated the best with non-gel containing serum tubes from BD and Greiner Bio-One. BD Serum Separator Tubes (SSTs) displayed strong interferences across several NMR analytes that were enhanced with decreased tube fill volumes. Interferences were also observed with different sizes of Greiner Bio-One Z Serum Sep Clot Activator tubes. Interference was generally not observed with conventional lipid testing, although minor interference was found for some tubes with lipoprotein(a) [Lp(a)]. ConclusionsNMR lipoprotein subclass analysis should be standardized by both tube type and tube size to prevent risk of analytical interference.

The Molecular Mechanism of Intermolecular Signal Transduction in Cystathionine‐β‐Synthase (CBS)

Human cystathionine‐β‐synthase (hCBS) is a pyridoxal phosphate‐dependent (PLP) enzyme that contains heme and catalyzes the β‐replacement reactions of serine (L‐Ser) by homocysteine (L‐Hcy) to produce cystathionine. Mutation in this key mammalian enzyme causes hyperhomocysteinemia, which leads to aggressive arterial diseases, a clinical phenotype observed in patients. Although the role of PLP is similar to other PLP dependent enzymes that catalyze the exchange of the hydroxyl group of serine to thiolate in homocysteine, little is known about the role that heme plays in regulating the catalytic process. My project seeks to develop a continuous, kinetic assay to measure CBS activity. Two approaches are being explored: 1) A coupled kinetic assay, in which the cystathionine produced by CBS is used by Cystathionine‐β‐lyase (hCBL) to generate pyruvate, and pyruvate production is measured phototrically; 2) A novel nuclear magnetic resonance assay, in which we will monitor the direct production of cystathionine over time. These methods will be compared to the standard, but discontinuous, radiometric assay. These assays will be used to understand the effect of small ligand binding to the heme and point mutations at the heme cofactor in relation to the enzyme activity. The long‐term goal of this work is to use time‐resolved absorption and magnetic circular dichroism spectroscopy to understand how small ligand binding to the heme induces conformational changes at the PLP active site in CBS and to measure activity of CBS with various mutated active site residues.Support or Funding InformationArnold and Mabel Beckman Foundation; NIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of alkyl bis(oxy)dibenzimidamide derivatives as novel protein arginine methyltransferase 1 (PRMT1) inhibitors.

Protein arginine methylation, a post-translational modification critical for a variety of biological processes, is catalyzed by protein arginine N-methyltransferases (PRMTs). In particular, PRMT1 is responsible for over 85% of the arginine methylation in mammalian cells. Dysregulation of PRMT1 is involved in diverse pathological diseases including cancers. However, most current PRMT1 inhibitors are lack of specificity, efficacy, and bioavailability. Herein, a series of alkyl bis(oxy)dibenzimidamide derivatives were identified as selective PRMT1 inhibitors. Among them, the most potent compound corresponds to hexamidine (IC50 =5.9±1.7μm), which is an antimicrobial agent. The binding between hexamidine and PRMT1 was further validated by thermal shift assays and nuclear magnetic resonance (NMR) experiments. Molecular docking and NMR assays indicated that hexamidine occupied the substrate binding pocket. Furthermore, hexamidine effectively blocked cell proliferation in cancer cell lines related to PRMT1 overexpression. Taken together, this study has provided a druggable scaffold targeting PRMT1 as well as a new way to repurpose old drugs which is a complementary tool for the discovery of new lead compounds.

NMR quantification of trimethylamine-N-oxide in human serum and plasma in the clinical laboratory setting

Background and objectivesTrimethylamine-N-oxide (TMAO) produced by gut microbiota metabolism of dietary choline and carnitine has been shown to be associated with increased risk of cardiovascular disease (CVD) and to provide incremental clinical prognostic utility beyond traditional risk factors for assessing a patient's CVD risk. The aim of this study was to develop an automated nuclear magnetic resonance (NMR) spectroscopy assay for quantification of TMAO concentration in serum and plasma using a high-throughput NMR clinical analyzer. MethodsKey steps in assay development included: (i) shifting the TMAO analyte peak to a less crowded region of the spectrum with a pH buffer/reagent, (ii) attenuating the broad protein background signal in the spectrum and (iii) using a non-negative least squares algorithm for peak deconvolution. Assay performance was evaluated according to Clinical and Laboratory Standards Institute guidelines. A method comparison study was performed to compare TMAO concentrations quantified by NMR and mass spectrometry (MS). ResultsThe within-run and within-lab imprecision ranged from 4.3 to 14.5%. Under the acquisition method employed, the NMR assay had a limit of blank, detection and quantitation of 1.6, 3.0 and 3.3μM, respectively. Linearity was demonstrated within the reportable range of 3.3 to 3000μM. TMAO measurements using the NMR assay, which involves minimal sample preparation, compared well with values obtained with the MS-based assay (R2=0.98). ConclusionsThe NMR based assay provides a simple and accurate measurement of circulating TMAO levels amenable to the high-throughput demands of the clinical chemistry laboratory. Moreover, assay performance enables the levels of TMAO to be quantified in serum or plasma at clinically actionable concentrations for the assessment of cardiovascular disease risks and individualized dietary monitoring.

Multi-platform metabolomics assays for human lung lavage fluids in an air pollution exposure study.

Metabolomics protocols are used to comprehensively characterize the metabolite content of biological samples by exploiting cutting-edge analytical platforms, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) assays, as well as nuclear magnetic resonance (NMR) assays. We have developed novel sample preparation procedures combined with GC-MS, LC-MS, and NMR metabolomics profiling for analyzing bronchial wash (BW) and bronchoalveolar lavage (BAL) fluid from 15 healthy volunteers following exposure to biodiesel exhaust and filtered air. Our aim was to investigate the responsiveness of metabolite profiles in the human lung to air pollution exposure derived from combustion of biofuels, such as rapeseed methyl ester biodiesel, which are increasingly being promoted as alternatives to conventional fossil fuels. Our multi-platform approach enabled us to detect the greatest number of unique metabolites yet reported in BW and BAL fluid (82 in total). All of the metabolomics assays indicated that the metabolite profiles of the BW and BAL fluids differed appreciably, with 46 metabolites showing significantly different levels in the corresponding lung compartments. Furthermore, the GC-MS assay revealed an effect of biodiesel exhaust exposure on the levels of 1-monostearylglycerol, sucrose, inosine, nonanoic acid, and ethanolamine (in BAL) and pentadecanoic acid (in BW), whereas the LC-MS assay indicated a shift in the levels of niacinamide (in BAL). The NMR assay only identified lactic acid (in BW) as being responsive to biodiesel exhaust exposure. Our findings demonstrate that the proposed multi-platform approach is useful for wide metabolomics screening of BW and BAL fluids and can facilitate elucidation of metabolites responsive to biodiesel exhaust exposure. Graphical Abstract Graphical abstract illustrating the study workflow. NMR Nuclear Magnetic Resonance, LC-TOFMS Liquid chromatography-Time Of Flight Mass Spectrometry, GC Gas Chromatography-Mass spectrometry.

Serum MSM Concentrations Following One Month of MSM Treatment in Healthy Men

Introduction: Methylsulfonylmethane (MSM) is a commonly used dietary supplement for the alleviation of joint and muscle pain. It is known primarily for its anti-inflammatory and antioxidant properties. While it is believed to have excellent bioavailability, little is known about its serum concentrations following chronic ingestion. Methods: 20 healthy men were supplemented with 3 grams of MSM daily for four weeks. Blood was collected at baseline and after two and four weeks of supplementation. Serum was analyzed for MSM concentration using Nuclear Magnetic Resonance (NMR) spectroscopy. Results: All baseline samples but one (0.028 mM) was below the limit of quantification for the NMR assay (0.002 mM). Serum MSM values increased across time (p 0.05). A total of 13 of the 20 men demonstrated higher serum MSM values at week 4 as compared to week 2. and eight of these men demonstrated an increase at week 4 of at least 25% above what was observed at week 2. Conclusions: Serum MSM concentrations increase following oral MSM supplementation, in somewhat of a timedependent manner in selected subjects. The pattern of increase varies somewhat from subject to subject, although all individuals experience an increase of approximately 1-3 mM after 2-4 weeks of supplementation.

Validation studies for multicomponent quantitative NMR analysis: the example of apple fruit juice

Laboratories intending to work as official laboratories for food control have to be accredited according to ISO/IEC 17025. This necessitates the use of validated analytical methods. In this study, we present validation results of the recently commercialized “JuiceScreener” based on nuclear magnetic resonance (NMR) spectroscopy with apple juice as application example. The quantitative analysis included 29 compounds such as major sugars, amino acids, organic acids, as well as acetoin, arbutin, benzaldehyde, hydroxymethylfurfural, acetaldehyde, methanol, and ethanol. Limit of detection (LOD), limit of quantification (LOQ), coefficients of variation (CV) for replicated measurements, repeatability, linear range, and recoveries were determined. The LOD and LOQ values varied in the 0.48–16 mg/L and in the 1.9–122 mg/L ranges with the lowest values for shikimic acid and highest for the principal sugars. The 1H NMR assays were linear in broad concentration ranges (R > 0.99), encompassed typical concentration in apple juices, and are sufficient to control the requirements of the code of practice of the European fruit juice association. Recoveries between 92 and 109 % on average for five separate standard additions were obtained. The average CVs were found to be 3.0 % (intraday) and 3.6 % (interday) excluding sample preparation (by measuring five time one solution) and 5.5 % (intraday) and 6.2 % (interday) including sample preparation (by preparing and analyzing five separate samples). The NMR method was judged as suitable for the simultaneous quantification of compounds in apple juice for official food control purposes. Our results show that multiparameter NMR methods can be successfully validated with standard instrumentarium and that they are fit for the purpose of official food control.

Genome-Wide Genetic Association Study Meets Systematic Metabolite Screening

To date, many genome-wide association studies (GWAS) have examined genetic variation in >100 individual blood metabolites or smaller panels of metabolites known to be related to cardiovascular diseases, such as lipids and glucose homeostasis markers. Emerging high-throughput technologies to determine components of the serum metabolome in combination with an unbiased GWAS approach may be capable of identifying genetic variation and unravel novel disease pathways beyond traditionally known blood metabolites in unselected population-based samples. Although less sensitive than mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy is one of the methods that has emerged to examine serum metabolites, with little blood sample preparation required. NMR can be applied at a larger scale for targeted analyte measurement. In 5 cohorts of >8000 unrelated individuals from the Finnish population and 561 twins, 117 fasting serum metabolites were measured and 99 additional variables derived.1 Metabolites chosen covered mostly lipoproteins and lipids but also small molecules such as pyruvic and amino acids. Derived variables, predominantly ratios of metabolites, were selected to represent enzymatic reactions of lipolysis, proteolysis, ketogenesis, glycolysis, and their substrates. Three molecular windows for metabolite quantification were applied on the metabolomics platform (ie, separate windows for lipoproteins, lipids, and low-molecular-weight metabolites). Genetic variation of the phenotypes was determined through GWAS applying a comprehensive imputation panel of 7.7 million single …

Determination of Neutralization Capacity and Stability of a Basic Methacrylate Monomer Using NMR

The durability of dental resin depends on the stability of the polymer. The neutralizing capacity of a basic methacrylate monomer and its chemical stability were measured using nuclear magnetic resonance (NMR) spectroscopy. Lactic acid solution was titrated with 2-(dimethylamino)ethylmethacrylate (DMAEMA) or 2-hydroxyethylmethacrylate (HEMA) and its chemical shifts monitored. Addition of DMAEMA alters the chemical shift proportionally to pH neutralization, whereas HEMA has no impact. Chemical shifts were used to quantify both the change in pH and monomer stability. The results demonstrate that neutralization by basic monomer can be achieved and that this can be measured using an NMR assay.

Genome-wide association study identifies multiple loci influencing human serum metabolite levels

Nuclear magnetic resonance assays allow for measurement of a wide range of metabolic phenotypes. We report here the results of a GWAS on 8,330 Finnish individuals genotyped and imputed at 7.7 million SNPs for a range of 216 serum metabolic phenotypes assessed by NMR of serum samples. We identified significant associations (P < 2.31 × 10(-10)) at 31 loci, including 11 for which there have not been previous reports of associations to a metabolic trait or disorder. Analyses of Finnish twin pairs suggested that the metabolic measures reported here show higher heritability than comparable conventional metabolic phenotypes. In accordance with our expectations, SNPs at the 31 loci associated with individual metabolites account for a greater proportion of the genetic component of trait variance (up to 40%) than is typically observed for conventional serum metabolic phenotypes. The identification of such associations may provide substantial insight into cardiometabolic disorders.

NMR evaluation of total statin content and HMG-CoA reductase inhibition in red yeast rice (Monascus spp.) food supplements

BackgroundRed yeast rice (i.e., rice fermented with Monascus spp.), as a food supplement, is claimed to be blood cholesterol-lowering. The red yeast rice constituent monacolin K, also known as lovastatin, is an inhibitor of the hydroxymethylglutaryl-CoA (HMG-CoA) reductase. This article aims to develop a sensitive nuclear magnetic resonance (NMR) method to determine the total statin content of red yeast rice products.MethodsThe total statin content was determined by a 400 MHz 1H NMR spectroscopic method, based on the integration of the multiplet at δ 5.37-5.32 ppm of a hydrogen at the hexahydronaphthalene moiety in comparison to an external calibration with lovastatin. The activity of HMG-CoA reductase was measured by a commercial spectrophotometric assay kit.ResultsThe NMR detection limit for total statins was 6 mg/L (equivalent to 0.3 mg/capsule, if two capsules are dissolved in 50 mL ethanol). The relative standard deviations were consistently lower than 11%. The total statin concentrations of five red yeast rice supplements were between 1.5 and 25.2 mg per specified daily dose. A dose-dependent inhibition of the HMG-CoA reductase enzyme activity by the red yeast rice products was demonstrated.ConclusionA simple and direct NMR assay was developed to determine the total statin content in red yeast rice. The assay can be applied for the determination of statin content for the regulatory control of red yeast rice products.

Frog skins keep redox homeostasis by antioxidant peptides with rapid radical scavenging ability

The question of how amphibians can protect themselves from reactive oxygen species when exposed to the sun in an oxygen-rich atmosphere is important and interesting, not only from an evolutionary viewpoint, but also as a primer for researchers interested in mammalian skin biology, in which such peptide systems for antioxidant defense are not well studied. The identification of an antioxidant peptide named antioxidin-RL from frog ( Odorrana livida) skin in this report supports the idea that a peptide antioxidant system may be a widespread antioxidant strategy among amphibian skins. Its ability to eliminate most of the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical tested within 2 s, which is much faster than the commercial antioxidant factor butylated hydroxytoluene, suggests that it has a potentially large impact on redox homeostasis in amphibian skins. Cys10 is proven to be responsible for its rapid radical scavenging function and tyrosines take part in the binding of antioxidin-RL to radicals according to our nuclear magnetic resonance assay.

2-5 核磁気共鳴(NMR)によるメタンハイドレート胚胎堆積物の評価 : 孔隙径分布と浸透率の関係((2)メタンハイドレード1,Session 2 天然ガス・メタンハイドレート等,研究発表(口頭発表))

2-5 核磁気共鳴(NMR)によるメタンハイドレート胚胎堆積物の評価 : 孔隙径分布と浸透率の関係((2)メタンハイドレード1,Session 2 天然ガス・メタンハイドレート等,研究発表(口頭発表))

Antioxidant properties of dihydroherbimycin A from a newly isolated Streptomyces sp.

During antioxidant screening using 1,1-diphenyl-picrylhydrazyl (DPPH) and a lipid peroxidation assay, a streptomycete strain was found to produce herbimycin A and dihydroherbimycin A as antioxidants in the culture filtrate. These molecules were identified by using spectral analyses, including infrared, ultraviolet, mass spectrum, and nuclear magnetic resonance assays. In the DPPH radical-scavenging assay, dihydroherbimycin A exhibited more potent antioxidant activity (IC(50), 1.3 microM) than alpha-tocopherol (IC(50), 2.7 microM) that was used as a reference compound. In the lipid peroxidation assay, both herbimycin A and dihydroherbimycin A demonstrated antioxidant activities of 61% and 72%, respectively, at 100 microg/ml, while alpha-tocopherol exhibited an activity of 93% at the same concentration. Therefore, dihydroherbimycin A might have the potential to be developed into a new therapeutic agent.

Phosphatidylserine Recruits Rac1

Rac1 is a member of the Rho family of guanosine triphosphatases (GTPases) and is involved in stimulating membrane ruffling and cell migration. Finkielstein et al . show, using liposome-binding assays, nuclear magnetic resonance assays, and lipid overlay assays, that purified Rac1, either prenylated or nonprenylated, GTP-bound or GDP-bound, interacts specifically with phosphatidylserine, an anionic lipid that is abundant on the inner leaflet of the plasma membrane. Surface plasmon resonance assays demonstrated that prenylated Rac1 had a higher affinity for phosphatidylserine than did nonprenylated Rac1. The interaction required the polybasic motif (PBM), and mutation of a lysine and an arginine (K186A/R187A) abolished Rac1 binding to phosphatidylserine. Cells expressing the K186A/R187A mutant showed an altered distribution of Rac1 that was located in cytosolic puncta, which may reflect binding to internal membranes, instead of the plasma membrane. Exposure of cells transfected with wild-type or various Rac1 mutants to phosphatidylserine caused membrane ruffling, formation of filopodia, and reduction in actin stress fibers and stimulated cell migration in a wound assay. Each response required Rac1 GTPase activity and an intact PBM. Exposure of the transfected cells to phosphatidylserine stimulated the translocation of Rac1 to the plasma membrane, Rac1 GTP loading, activation of Cdc42 (a Rho GTPase that is downstream of Rac1), and activation of the mitogen-activated protein kinase kinase (MEK1). The authors propose that, as with Src, Raf-1, and KRas4B, the PBM allows targeting of Rac1 to specific regions of the plasma membrane through the interactions with phosphatidylserine, thereby facilitating the interaction of Rac1 with its regulators and effectors. C. V. Finkielstein, M. Overduin, D. G. S. Capelluto, Cell migration and signaling specificity is determined by the phosphatidylserine recognition motif of Rac1. J. Biol. Chem. 281 , 27317-27326 (2006). [Abstract] [Full Text]

Quantitative nuclear magnetic resonance analysis and characterization of the derivatized Haemophilus influenzae type b polysaccharide intermediate for PedvaxHIB

PedvaxHIB is a pediatric vaccine that protects children from severe disease caused by the gram-negative bacterium Haemophilus influenzae type b (Hib). The vaccine is made by chemically conjugating Hib capsular polysaccharide to the outer membrane protein complex of Neisseria meningitidis. The protein-conjugated vaccine has proven to be extremely effective in preventing invasive Hib disease in infants and young children. This paper presents the nuclear magnetic resonance (NMR) methodology for the quantitative characterization of derivatized polysaccharide and its validation closely following ICH guidelines. The assay has been shown to be precise and accurate (relative standard deviation [RSD] ⩽1%), specific (no observable matrix interference), rugged (RSD <3% for day-to-day and operator-to-operator variations), and robust (to changes in temperature and polysaccharide concentration). Therefore, the quantitative NMR assay can be used as a reliable product release or process monitoring method and can replace several labor-intensive chromatographic and colorimetric methods.

Seafloor nuclear magnetic resonance assay of methane hydrate in sediment and rock

Proton nuclear magnetic resonance (NMR) of liquid water can be used to indirectly detect and assay methane hydrate in opaque earth materials. It is also potentially useful for quantifying pore size control of hydrate formation, and for estimating in situ hydraulic permeability of hydrate‐affected earth formations. The method is quantitative, nondestructive, and volumetrically averaging over a spatially selected region. In the first controlled validation of this measurement, we have used NMR to measure changes in the liquid water content of sediment and rock resulting from methane hydrate formation. Hydrates were artificially formed at the seafloor by introducing methane into tubes containing sediments or rock saturated with seawater; after several weeks the samples were visited by a remotely operated vehicle on which NMR equipment was mounted. Reduction of the NMR signal indicated hydrate had formed in the sample within a well‐defined measurement volume. Hydrate content determined by NMR quantitatively predicted the volume of methane gas evolved during subsequent dissociation.

Use and validation of NMR assays for the identity and O-acetyl content of capsular polysaccharides from Neisseria meningitidis used in vaccine manufacture

We describe a validated NMR (nuclear magnetic resonance) spectroscopic assay for the identity of the capsular polysaccharides (CPSs) from Neisseria meningitidis Groups A, C, W135 and Y used in vaccine manufacture, and to determine the proportion of residues carrying an O-acetyl substituent. Proof of structural identity and quantitation of the O-acetyl content are key control parameters for these vaccines. The meningococcal CPSs have variable levels of O-acetylation, present at multiple sites in the repeat unit, leading to complex NMR spectra. Base-catalysed de- O-acetylation of the Groups A, C, W135 and Y CPSs yields simplified and reproducible spectra suitable for comparison with reference data. The degree of O-acetylation of the original CPS can be determined by integration of the acetate anion resonance and a suitable resonance from the saccharide moiety. The assay was validated using 46 independent samples from five manufacturers, and is shown to be robust and reproducible.

Identification of 6-methylmercaptopurine derivative formed during acid hydrolysis of thiopurine nucleotides in erythrocytes, using liquid chromatography–mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance assay

6-Thioguanine and 6-methylmercaptopurine (Me6-MP) nucleotides are the two major thiopurine metabolites of azathioprine found in erythrocytes. During the acid hydrolysis required for the conversion of thiopurine nucleotides into their free bases, Me6-MP was converted into a compound that could be analyzed on a Purospher RP18-e column with dihydrogen phosphate-methanol buffer as eluent. The pH of the acid extract strongly influenced the conversion of Me6-MP into its derivative. The Me6-MP derivative was identified using liquid chromatography-mass spectrometry and infrared and nuclear magnetic resonance spectrometric methods. During the acid hydrolysis of thiopurine nucleotides in erythrocytes, Me6-MP undergoes degradation, leading to 4-amino-5-(methylthio)carbonyl imidazole.

4-Hydroxyanisole: The Most Suitable Monophenolic Substrate for Determining Spectrophotometrically the Monophenolase Activity of Polyphenol Oxidase from Fruits and Vegetables

A continuous spectrophotometric method for determining the monophenolase activity of polyphenol oxidase from several plant sources is described. This assay method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone and the quinone product of the oxidation of 4-hydroxyanisole in the presence of polyphenol oxidase. 4-Hydroxyanisole proved to be the best monophenol assayed to measure the monophenolase activity of polyphenol oxidase from apple, artichoke, avocado, medlar, pear, and strawberry. Kinetic constants of 4-hydroxyanisole were compared to those ofp-hydroxyphenyl propionic acid, a very sensitive monophenol previously reported to assay the monophenolase activity of polyphenol oxidase from apple, pear, and mushroom. The high values of the maximum steady state rate obtained for 4-hydroxyanisole suggest the existence of high catalytic constant toward this monophenol. These kinetic values were supported by nuclear magnetic resonance assays which predicted the highest reactivity of 4-hydroxyanisole. Therefore nuclear magnetic resonance assays proved to be a valuable and useful tool to predict the best monophenolic substrate for plant polyphenol oxidases. The 3-methyl-2-benzothiazlolinone-adduct for 4-hydroxyanisole was stable, with high molar absorptivity at the optimum pHs of the polyphenol oxidases assayed. All this together makes the use of 4-hydroxyanisol as monophenolic substrate and 3-methyl-2-benzothiazolinone as coupling reagent the most sensitive and precise assay method up to date reported in the literature to determine the monophenolase activity of polyphenol oxidase from fruits and vegetables.