Carboxyl esterases show limited use as catalytic labels in bioassays because of slow enzymatic reaction. We report that DT-diaphorase from Bacillus stearothermophilus (DT-D, EC 1.6.99.-) shows high carboxyl esterase-like activity in the presence of reduced β-nicotinamide adenine dinucleotide (NADH) and may be used as a better catalytic label than carboxyl esterases. DT-D is a redox enzyme and can participate in signal-amplifying redox cycling. Thus, an electrochemical immunosensor using a DT-D label allows for triple signal amplification based on (i) hydrolysis of a carboxyl ester, (ii) electrochemical–chemical (EC) redox cycling involving an electrode, a hydrolysis product, and NADH, and (iii) electrochemical–enzymatic (EN) redox cycling involving an electrode, a hydrolysis product, DT-D, and NADH. Ester hydrolysis by DT-D is confirmed via spectrophotometric measurement of a chromogenic substrate (4-nitrophenyl acetate) and 1H NMR spectra. Among two phenyl acetates and four naphthyl acetates considered, 4-aminonaphthalene-1-yl acetate (4-NH2-NAc) is chosen as the best acetyl ester substrate because 4-NH2-NAc is stable, its hydrolysis is slow in the absence of DT-D, its hydrolysis is very fast in the presence of DT-D, and EC and EN redox cycling involving the hydrolysis product (4-amino-1-naphthol) is rapid. However, hydrolysis of 4-NH2-NAc by esterase from porcine liver (EC 3.1.1.1.) is very slow. When DT-D is applied to sandwich-type detection of thyroid-stimulating hormone in artificial serum, the detection limit is ∼2 pg/mL, indicating that the developed immunosensor is highly sensitive because of triple signal amplification. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common alkaline phosphatase and horseradish peroxidase.
Vibrio harveyi is one of the pathogens that threaten the shrimp farming industry. However, metabolic changes induced by V. harveyi infection in shrimp remain unknown. In this study, we first conducted high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR)-based metabolomics studies on gill, hepatopancreas, and haemolymph of V. harveyi-infected white leg shrimp, Litopenaeus vannamei. Using multivariate statistical analysis, we observed a clear separation between the early (3 and 9 h post-injection (hpi)) and late phases (24, 72 and 144 hpi) of the infection in all tissues. Moreover, metabolic changes in response to V. harveyi infection were faster in the haemolymph in the early phase and significantly changed in the late phase of the infection in the gills. Extensive changes were observed in the hepatopancreas, with 24 hpi being the turning point of progression from early to late phase infection in the hepatopancreas. V. harveyi infection increased the energy demand in L. vannamei and the amino acid and carbohydrate metabolism pathways also exhibited significant changes depending on the tissue. Thus, each tissue displayed different metabolic changes, depending on the progress of the infection.
Continuous increases in water temperature disturb homeostasis and increase oxidative stress in fish. Glutathione (GSH) is an intracellular antioxidant that helps to relieve stress in animals. In this study, we observed the effect of GSH on olive flounder exposed to high temperature using serum parameters and NMR-based metabolomics. Based on the results from the first experiment, 20 mg of GSH was chosen as an effective dose with lower infection rates and mortality. Then, fish were divided into Control, Temp (PS injection), and GSH (glutathione injection) groups, and fish in Temp and GSH groups were exposed to temperature fluctuations (20 °C→24 °C→27 °C). In OPLS-DA score plots, Temp group was clearly distinguished from the other groups in the kidney. In the liver, the metabolic patterns of GSH group were close to the Temp group on day 4 and became similar to Control group from day 7. Serum parameters did not change significantly, but the deviation in Temp group was greater than that in GSH group. Metabolite levels that were significantly altered included GSH, lactate, O-phosphocholine, and betaine in the kidney and taurine, glucose, and several amino acids in the liver, which were related to antioxidant activity and energy system. Therefore, GSH supplements could relieve thermal stress influencing metabolic mechanisms in fish.
(1) Background: The ability to determine the age of ginseng is very important because the price of ginseng depends on the cultivation period. Since morphological observation is subjective, a new scientific and systematic method for determining the age of ginseng is required. (2) Methods: Three techniques were used for a metabolomics approach. High-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy was used to analyze powdered ginseng samples without extraction. Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and gas chromatography quadrupole time-of-fight mass spectrometry (GC-TOF/MS) were used to analyze the extracts of 4-, 5-, and 6-year-old ginseng. (3) Results: A metabolomics approach has the potential to discriminate the age of ginseng. Among the primary metabolites detected from NMR spectroscopy, the levels of fumarate and choline showed moderate prediction with an area under the curve (AUC) value of more than 0.7. As a result of UPLC-QTOF/MS-based profiling, 61 metabolites referring to the VIP (variable importance in the projection) score contributed to discriminating the age of ginseng. The results of GC×GC-TOF/MS showed clear discrimination of 4-, 5-, and 6-year-old ginseng using orthogonal partial least-squares discriminant analysis (OPLS-DA) to 100% of the discrimination rate. The results of receiver operating characteristic (ROC) analysis, 16 metabolites between 4- and 5-year-old ginseng, and 18 metabolites between 5- and 6-year-old ginseng contributed to age discrimination in all regions. (4) Conclusions: These results showed that metabolic profiling and multivariate statistical analyses can distinguish the age of ginseng. Especially, it is meaningful that ginseng samples from different areas had the same metabolites for age discrimination. In future studies, it will be necessary to identify the unknown variables and to collaboratively study with other fields the biochemistry of aging in ginseng.
Microcystins are one of the most frequently found hepatotoxic cyclic peptides in fresh-and brackishwater. Zebrafish (Danio rerio) were exposed to 20 ppb of microcystin-LR (MCLR) for 72 h in order to investigate the metabolic responses. Proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy was used to analyze the metabolic profiles. As a result, male and female zebrafish showed different patterns of metabolic perturbation in relation to their detoxification profiles. Glutathione was reduced for the MCLR excretion, and fumarate and creatine were increased for the energy demands required for detoxification in the male zebrafish. The correlation of metabolites in the metabolic pathway network was changed due to MCLR exposure. The correlations connected with myo-inositol, betaine, and NADPH considerably altered in the MCLR-exposed group.
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