Importancia de la determinación de actividad enzimática en el diagnóstico del déficit de adenosina desaminasa 2 (DADA2) / Importance of the determination of enzymatic activity in the diagnosis of deficiency of adenosine deaminase 2 (DADA2)

Objetivo: Describir la utilidad de la determinación de la actividad enzimática de adenosina desaminasa 2 (ADA2) en los pacientes con sospecha de déficit de ADA2 (DADA2).MétodoEstudio retrospectivo multicéntrico con análisis de los datos clínicos, bioquímicos y genéticos de los pacientes a los que se ha determinado la actividad enzimática de ADA2 mediante método espectrofotométrico.ResultadoEn tres de los 20 pacientes se confirmó el diagnóstico de DADA2 mediante la combinación de actividad enzimática reducida y variantes patogénicas bialélicas en el gen CECR1. En dos pacientes portadores de variantes de significado incierto en CECR1, el estudio de actividad enzimática permitió descartar la enfermedad.ConclusionesLa actividad enzimática reducida de ADA2 confirma el diagnóstico de DADA2, de especial importancia en los portadores de variantes de significado incierto en CECR1. (AU)

Objective: To describe the usefulness of determining the enzymatic activity of adenosine deaminase 2 (ADA2) in patients with suspected ADA2 deficiency (DADA2).MethodRetrospective multicenter study. Review with analysis of the clinical, biochemical and genetic data of the patients in whom the enzymatic activity of ADA2 has been determined by spectrophotometric method.ResultIn 3 of the 20 patients, the diagnosis of DADA2 was confirmed by the combination of reduced enzyme activity and biallelic pathogenic variants in the CECR1 gene. In 2 patients with variants of uncertain significance in CECR1, the study of enzymatic activity allowed to rule out the disease.ConclusionsThe reduced enzymatic detection of ADA2 confirms the diagnosis of DADA2, particularly important in carriers of variants of uncertain significance in CECR1. (AU)

Importance of the determination of enzymatic activity in the diagnosis of deficiency of adenosine deaminase 2 (DADA2). / Importancia de la determinación de actividad enzimática en el diagnóstico del déficit de adenosina desaminasa 2 (DADA2).

OBJECTIVE: To describe the usefulness of determining the enzymatic activity of adenosine deaminase 2 (ADA2) in patients with suspected ADA2 deficiency (DADA2). METHOD: Retrospective multicenter study. Review with analysis of the clinical, biochemical and genetic data of the patients in whom the enzymatic activity of ADA2 has been determined by spectrophotometric method. RESULT: In 3 of the 20 patients, the diagnosis of DADA2 was confirmed by the combination of reduced enzyme activity and biallelic pathogenic variants in the CECR1 gene. In 2 patients with variants of uncertain significance in CECR1, the study of enzymatic activity allowed to rule out the disease. CONCLUSIONS: The reduced enzymatic detection of ADA2 confirms the diagnosis of DADA2, particularly important in carriers of variants of uncertain significance in CECR1.

Enzymatic determination of carnosine in meat and fish using ß-Ala-Xaa dipeptidase and histidine ammonia-lyase derived from Pseudomonas putida NBRC100650.

Carnosine is a naturally occurring dipeptide and a functional component in foods, while also showing health-promoting effects. Generally, food-derived carnosine is quantified via high-performance liquid chromatography (HPLC). We have developed a method for quantifying carnosine in foods using microbial enzymes, ß-Ala-Xaa dipeptidase (BapA) and histidine ammonia-lyase (HAL). The carnosine concentrations in extracts of chicken, pork, beef, bonito, and tuna were determined via both HPLC and enzymatic determination. The carnosine contents measured via enzymatic determination were in agreement with those determined via conventional HPLC analysis. Relative standard-deviation values of the conventional HPLC method and the enzymatic determination of carnosine in foods were 0.728-5.76% and 0.504-4.58%, respectively. The recovery of carnosine in food extracts via enzymatic determination was 97-103%. Therefore, the developed enzymatic determination technique using BapA and HAL can be used for the determination of carnosine in meats and fishes with comparable accuracy to that of conventional HPLC analysis.

Gold nanoparticle formation as an indicator of enzymatic methods: colorimetric L-phenylalanine determination.

An enzymatic-colorimetric method has been developed based on the reaction between L-phenylalanine (L-Phe) and the L-amino acid oxidase (LAAO) in the presence of Au(III), which has led to the formation of gold nanoparticles. The intensity of the localized surface plasmon resonance (LSPR) band of the generated nanoparticles (550 nm) can be related to the concentration of L-Phe in the sample. The mechanism of the LAAO-L-Phe enzyme reaction in the presence of Au(III) has been studied through the evaluation and optimization of experimental conditions. These studies have reinforced the hypothesis that the catalytic center of the enzyme helps the Au(III) reduction and, thanks to the protein, the Au0 form is stabilized as gold nanoparticles (AuNPs). In the calibration study, a sigmoidal relationship between the concentration of the substrate and the LSPR of the nanoparticles was observed. The linearization of the signal has allowed the determination of L-Phe in the range from 17 to 500 µM with an RSD% (150 µM) of 4.8% (n = 3). The method is free of other amino acid interference normally found in blood plasma. These highly competitive results open the possibility of further development of a rapid method for L-Phe determination based on colorimetry.

Copper nanoclusters@Al3+ complexes with strong and stable aggregation-induced emission for application in enzymatic determination of urea.

The aggregates of glutathione-capped CuNCs induced by Al3+ (named CuNCs@Al3+ complexes) show a stable aggregation-induced emission (AIE) for about 1 month. Their fluorescence maintains a high level in the pH range 4.0 to 7.0, while it quenches as pH increases from 7.0 to 7.7 or decreases from 4.0 to 3.0. Under urease-catalyzed hydrolysis, urea produces ammonia, which can be further hydrolyzed to yield OH-. This leads to a pH increase of the immediate environment. Hence, the CuNCs@Al3+ complexes are a suitable probe to determine urea. The fluorescence of CuNCs@Al3+ complexes quenches linearly at 585 nm with the excitation wavelength at 340 nm when the concentration of urea increases from 20 to 150 µM. The limit of detection (LOD) of urea is 5.86 µM. This sensitivity is superior to other reported works due to the narrow pH response range of CuNCs@Al3+ complexes. This method has been successfully applied for measuring urea in human urine samples with satisfactory recoveries. Graphic abstract Schematic representation of urea determination based on pH-responsive property of copper nanoclusters@Al3+ complexes. Ammonia is produced in the hydrolysis of urea by urease, and it is further hydrolyzed to yield OH-, leading to increasing pH of the environment.

Cobalt-copper bimetallic nanostructures prepared by glancing angle deposition for non-enzymatic voltammetric determination of glucose.

A bimetallic nanostructure of Co/Cu for the non-enzymatic determination of glucose is presented. The heterostructure includes cobalt thin film on a porous array of Cu nanocolumns. Glancing angle deposition (GLAD) method was used to grow Cu nanocolumns directly on a fluorine-doped tin oxide (FTO) substrate. Then a thin film of cobalt was electrodeposited on the Cu nanostructures. Various characterization studies were performed in order to define the optimum nanostructure for the determination of glucose. The results showed remarkable boosting of the electrocatalytic activity of Co/Cu bimetallic structure compare to the responses achieved by the monometallic structures of Co or Cu. The sensor showed two linear response ranges for the determination of glucose at 0.55 V in 0.1 M NaOH, from 5 µM-1 mM and 2-9 mM. The sensitivity was 1741 (µA mM-1 cm-2) and 626 (µA mM-1 cm-2), respectively, while the detection limit for a signal-to-noise ratio of 3 was found to be 0.4 µM. The sensor exhibited excellent selectivity and was successfully applied to the determination of glucose in real human blood serum samples. Graphical Abstract Schematic representation of fabrication process of the glucose sensor of Co (Cobalt)/Cu (Copper) on Fluorine doped Tin Oxide (FTO). The current voltage plots show higher electrooxidation activity of the bimetallic nanostructure of Co/Cu/FTO relative to the bare Co/FTO.

Highly specific sophorose ß-glucosidase from Sphingomonas elodea ATCC 31461 for the efficient conversion of stevioside to rubusoside.

Enzyme specificity and particularity is needed not only in enzymatic separation methods, but also in enzymatic determination methods for plant compound extraction. Stevioside, rubusoside, and rebaudioside A are natural sweet compounds from plants. These compounds have the same skeleton and only contain different side-chain glucosyl groups, making them difficult to separate. However, enzymes that target diterpenoid compounds and show specific activity for side-chain glucosyl groups are rare. Herein, we report the identification and characterization of an enzyme that can target both diterpenoid compounds and sophorose, namely, ß-glucosidase SPBGL1 from Sphingomonas elodea ATCC 31461. SPBGL1 displayed high specificity toward sophorose, and activity toward stevioside, but not rebaudioside A. The stevioside conversion rate was 98%. SPBGL1 also operated at high substrate concentrations, such as in 50% crude steviol glycoside extract. Glucose liberated from stevioside was easy to quantify using the glucose oxidase method, allowing the stevioside content to be determined.

Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination.

Our screening study yielded a copper amine oxidase (SrAOX) from Syncephalastrum racemosum, which showed much higher affinity and catalytic efficiency toward ethanolamine (EA) than any other amine oxidase (AOX). Following purification of the enzyme to electrophoretic homogeneity from a cell-free extract, the maximum activity toward EA was detected at pH 7.2-7.5 and 45 °C. The SrAOX complementary DNA (cDNA) was composed of a 2052-bp open reading frame encoding a 683-amino acid protein with a molecular mass of 77,162 Da. The enzyme functions as a homodimer. The deduced amino acid sequence of SrAOX showed 55.3 % identity to Rhizopus delemar AOX and contains two consensus sequences of Cu-AOX, NYDY, and HHQH, suggesting SrAOX is a type 1 Cu-AOX (i.e., a topaquinone enzyme). Structural homology modeling showed that residues (112)ML(113), (141)FADTWG(146) M158, and N318 are unique, and T144 possibly characterizes the substrate specificity of SrAOX. The recombinant enzyme (rSrAOX) was produced using Escherichia coli. Steady-state kinetic analysis of rSrAOX activity toward EA (pH 7.5 and 45 °C) gave K m and k cat values of 0.848 ± 0.009 mM and 9.11 ± 0.13 s(-1), respectively. The standard curves were linear between 0.1 and 2 mM EA, and 10 µg mL(-1)-2.5 mg mL(-1) (15 µM-3.6 mM) phosphatidylethanolamine using Streptomyces chromofuscus phospholipase D, respectively, was sufficiently sensitive for clinical use.

Determinación enzimática y metales pesados en cerebro e hígado del modelo ecotoxicológico Gambusia punctata (Poeciliidae) / Enzymatic determination and heavy metals in the brain and liver in ecotoxicological model Gambusia punctata (Poeciliidae)

Se realizó un estudio durante el 2011, con vistas a evaluar los niveles de actividad enzimática y metales pesados bioacumulados en cerebro e hígado de la especie Gambusia punctata, que habita en los ecosistemas San Juan y Filé de Santiago de Cuba, para lo cual se seleccionaron 3 estaciones, correspondientes a la parte alta, media y baja de ambos sistemas. Se escogieron ejemplares que midieron biométricamente de 2,1-3,0 cm de longitud total. Se determinó la enzima acetilcolinesterasa en el cerebro y la glutation-S-transferasa y catalasa, respectivamente, en el hígado. En ambos órganos se analizaron concentraciones de cobre, cinc, plomo y cadmio, tratados por vía húmeda y cuantificados por espectroscopia de plasma inductivamente acoplado con vista axial. En la especie del San Juan, los niveles enzimáticos variaron entre las estaciones, siendo mayores y estadísticamente diferentes para las hembras (p<0,05); sin embargo, en la del Filé, fueron similares entre las partes y sexos. En cuanto a los metales, no se detectaron concentraciones en el cerebro, ni plomo ni cadmio en el hígado. Las concentraciones de cobre y cinc fueron mayores en la parte baja y menores en la alta, de manera que hubo diferencias con respecto a la especie del Filé. Se concluyó que las enzimas variaron desde una estación ambientalmente diferente a otra y que los metales cobre y cinc, a pesar de ser esenciales, representan un riesgo ecotoxicológico en la especie del San Juan, debido a sus elevadas capacidades bioacumulativas.

A study during 2011 was carried out in order to evaluate the levels of enzymatic activity and heavy metals bioaccumulated in brain and liver of the species Gambusia punctata, inhabiting San Juan and Filé ecosystems in Santiago de Cuba. For this purpose 3 stations were selected, corresponding to the upper, middle and lower parts of both systems. Specimens biometrically measuring 2.1-3.0 cm in total length were chosen. Acetylcholinesterase enzyme in the brain and glutathione-S-transferase and catalase in the liver were determined, respectively. Copper, zinc, lead and cadmium concentrations were analyzed in both organs, wet processed and quantified by axial view inductively coupled plasma spectroscopy. In San Juan species the enzymatic levels varied between the stations, being higher and statistically different for females (p<0.05), but in that from Filé they were similar between the parts and genders. Regarding metals, concentrations were not detected in brain, neither lead nor cadmium in the liver. Copper and zinc concentrations were higher in the lower part and lower in the upper one, so that there were differences regarding the Filé species. It was concluded that the enzymes varied from an environmentally different station to another and copper and zinc metals, in spite of being essential, pose an ecotoxicological risk in the San Juan species, due to their high bioaccumulative capabilities.