Two-step Enzymatic Reaction Research Articles

High performance computational approach to study model describing reversible two-step enzymatic reaction with time fractional derivative

Enzyme reactions have numerous applications in diverse disciplines of science like chemistry, biology and biomechanics. In this study, we examine the role and act of enzymes in chemical reactions which is considered in the frame of fractional order model. The proposed model includes system of four equations which are studied via Caputo fractional operator. The systems of non-linear equations are evaluated by a semi-analytical approach called q\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$q$$\\end{document}-homotopy analysis transform method. The uniqueness and existence of the solutions has been investigated through fixed point theorem. The solutions of the proposed model are achieved through the considered method and the obtained outcomes are in the form of series which shows rapid convergence. The solutions are computed and graphs are plotted for the obtained results using mathematica software. The achieved results by the proposed method are unique and illustrate the significant dynamics of the considered model via 3D plots and graphs. The results of this study demonstrate the importance and effectiveness of projected derivative and technique in the analysis of time dependent fractional mathematical models. This study also gives an idea to extend the applications of enzymatic reactions in drug development, bio mechanics, and chemical reactions in various cellular metabolisms. Also, enzymatic reactions have a vital role in the fields of the food industry for processing food, in biotechnology for the manufacture of biofuels, and in metabolic engineering to design metabolic pathways.

Concentration of docosahexaenoic acid from tuna oil via a two lipase-catalyzed reaction

Docosahexaenoic acid (DHA) was concentrated successfully in the glyceride fractions from tuna oil via a two-step enzyme reaction involving hydrolysis and ethanolysis. In the first step, Candida rugosa lipase-catalyzed hydrolysis was carried out to concentrate DHA in the glyceride fractions. The DHA content in the glyceride fraction after hydrolysis increased from 30% in the initial tuna oil to 46%. In the second step, Lipozyme RM IM-catalyzed ethanolysis was conducted with the reaction mixture from the first step to further concentrate DHA in the glyceride fraction. In this step, the reaction mixture obtained from the first step was employed directly in Lipozyme RM IM-catalyzed ethanolysis without additional steps needed to remove free fatty acid. Finally, DHA was concentrated from an initial content of 30% in the tuna oil to 68.4% in the glyceride fractions via a novel two-step enzyme reaction strategy.

Self-assembled nanoparticle-mediated cascade chemotherapy and chemodynamic therapy for enhanced tumor therapy

Chemodynamic therapy (CDT) is a non-invasive treatment strategy that aims to combat tumor growth by efficiently generating reactive oxygen species (ROS). However, the effectiveness of CDT can be limited by factors such as the availability of endogenous hydrogen peroxide (H2O2). Here, we established an intelligent nano-based drug delivery system responsive to the tumor microenvironment by self-assembling Cu2+, cisplatin, and L-histidine to form nanoparticles (Pt/CuH NPs), which can achieve the therapeutic effect of specific tumor killing by cascading chemotherapy with CDT. The Pt/CuH NPs are triggered by the acidic conditions and high glutathione (GSH) levels present in the tumor microenvironment, leading to the release of cisplatin and Cu2+. Cisplatin facilitates the production of H2O2 through a two-step enzymatic reaction, subsequently triggering ROS cascade that is amplified by CDT. Concurrently, Cu2+ consumes GSH and is reduced to Cu+, which reacts with H2O2 in an efficient Fenton-like reaction, amplifying oxidative stress and ultimately enhancing the anti-tumor therapeutic efficacy. Pt/CuH NPs demonstrated a significant cytotoxic effect on tumor cells in vitro. In vivo studies in mice indicated minimal toxic side effects at the tested dosages. The Pt/CuH NPs effectively combine chemotherapy and CDT strategies, showcasing the potential of a chemotherapy-cascading CDT approach for anti-tumor treatment.

Self-cascade deoxynivalenol detoxification by an artificial enzyme with bifunctions of dehydrogenase and aldo/keto reductase from genome mining

Due to the severe health risks for human and animal caused by the intake of toxic deoxynivalenol (DON) derived from Fusarium species, elimination DON in food and feed has been initiated as a critical issue. Enzymatic cascade catalysis by dehydrogenase and aldo-keto reductase represents a fascinating strategy for DON detoxification. Here, one quinone-dpendent alcohol dehydrogenase DADH oxidized DON into less-toxic 3-keto-DON and NADPH-dependent aldo-keto reductase AKR13B3 reduced 3-keto-DON into relatively non-toxic 3-epi-DON were identified from Devosia strain A6-243, indicating that degradation of DON on C3 are two-step sequential cascade processes. To establish the bifunctions, fusion enzyme linking DADH and AKR13B3 was successfully assembled to promote one-step DON degradations with accelerated specific activity and efficiency, resulting 93.29 % of DON removal rate in wheat sample. Three-dimensional simulation analysis revealed that the bifunctional enzyme forms an artificial intramolecular channel to minimize the distance of intermediate from DADH to AKR13B3 for two-step enzymatic reactions, and thereby accelerates this enzymatic process. As the first report of directing single step DON detoxification by an interesting bifunctional artificial enzyme, this work revealed a facile and eco-friendly approach to detoxify DON with application potential and gave valuable insights into execute other mycotoxin detoxification for ensuring food safety.

Alanine aminotransferase electrochemical sensor based on graphene@MXene composite nanomaterials.

Graphene@MXene composite nanomaterials were utilized to construct anelectrochemical sensor for alanine aminotransferase (ALT) detection. The combination of graphene nanosheets with MXene avoids the self-stacking of MXene and graphene, and broadens the charge transfer channel. In addition, the composite nanomaterial provides increased loading sites for pyruvate oxidase. The principle of ALT detection is a two-step enzymatic reaction. L-Alanine was initially transferred to pyruvate catalyzed by ALT. The formed pyruvate was then oxidized by pyruvate oxidase, generating H2O2. Through the detection of the generated H2O2, ALT activity was measured. The linear range of the sensor to ALT was from 5 to 400 U·L-1 with a detection limit of 0.16 U·L-1 (S/N = 3). For real sample analysis, the spiked recovery test results of ALT in serum samples were between 96.89 and 103.93% with RSD < 5%, confirming the reliability of the sensor testing results and potential clinical application of the sensor.

Portable Near-Infrared Fluorometer for a Liposomal Blood Lactate Assay.

In sepsis, plasma lactate is a key biomarker of disease severity, prognosis, and treatment success. However, the median time to result for clinical lactate tests is 3 h. We recently reported a near-infrared fluorescent (NIRF) blood lactate assay that relies on a two-step enzymatic reaction in a liposomal reaction compartment. This assay was optimized in human blood and was capable of quantifying lactate in fresh capillary blood from human volunteers at clinically relevant concentrations in 2 min. However, these studies were performed with a tabletop fluorescence plate reader. For translation to the point of care, the liposomal lactate assay needs to be combined with a small portable NIR fluorometer. Portable NIR fluorometers were successfully used for the analysis of skin and soil samples, but reports for blood metabolite assays are scarce. We aimed at testing the performance of the liposomal lactate assay in combination with a commercial small portable NIR fluorometer. First, we tested the fluorophore of the liposomal lactate assay using the NIR dye sulfo-cyanine 7; we observed strong fluorescence signals and high linearity. Second, we performed the liposomal lactate assay in lactate-spiked human arterial blood using the portable fluorometer as the detector and observed strong and highly linear lactate sensing at clinically relevant lactate concentrations after 2 min. Finally, spiking fresh mouse blood with three clinically relevant lactate concentrations led to a significantly different response to all three concentrations after 5 min. These results highlight the usefulness of the tested portable NIR fluorometer for the liposomal lactate assay and motivate a clinical evaluation of this rapid and easy-to-use lactate assay.

Machine Learning-Assisted Nanoenzyme/Bioenzyme Dual-Coupled Array for Rapid Detection of Amyloids.

Array-based sensing methods offer significant advantages in the simultaneous detection of multiple amyloid biomarkers and thus have great potential for diagnosing early-stage Alzheimer's disease. Yet, detecting low concentrations of amyloids remains exceptionally challenging. Here, we have developed a fluorescent sensor array based on the dual coupling of a nanoenzyme (AuNPs) and bioenzyme (horseradish peroxidase) to detect amyloids. Various ss-DNAs were bound to the nanoenzyme for regulating enzymatic activity and recognizing amyloids. A simplified sensor array was generated from a screening model via machine learning algorithms and achieved signal amplification through a two-step enzymatic reaction. As a result, our sensing system could discriminate the aggregation species and aggregation kinetics at 200 nM with 100% accuracy. Moreover, AD model mice and healthy mice were distinguished with 100% accuracy through the sensor array, providing a powerful sensing platform for diagnosing AD.

Immobilization of Rhizomucor miehei lipase on magnetic multiwalled carbon nanotubes towards the synthesis of structured lipids rich in sn-2 palmitic acid and sn-1,3 oleic acid (OPO) for infant formula use

Nowadays, breast milk is considered as the ideal food for infants owing to the most common oleic acid-palmitic acid-oleic acid (OA-PA-OA) fatty acid distribution of the human milk fat (HMF). This study reports the synthesis of 1,3-dioleoyl-2-palmotoylglycerol (OPO)-rich human milk fat substitutes in a two-step enzymatic acidolysis reaction with Rhizomucor miehei lipase (RML) immobilized on magnetic multi-walled carbon nanotubes(mMWCNTs). The immobilized RML (RML-mMWCNTs) showed better thermal and pH stability, convenient recovery and reusability than the free soluble form. Under optimized reaction conditions (1:8 tripalmitin (PPP)/OA, 10%wt. enzyme, 50 °C, 5 h), PA content at the sn-2 position and OA incorporation at the sn-1,3 positions reached 93.46% and 59.54%, respectively. Comparison tests have also showed that RML-mMWCNTs has better catalytic activity and reusability than the commercial lipase Lipozyme RM IM. The results suggest that RML-mMWCNTs is a promising biocatalyst for the synthesis of OPO-rich TAGs with potential use in infant formulas.

Generation and identification of kokumi compounds and their validation by taste-receptor assay: An example with dry-cured lamb meat

Kokumi tastants are small γ-glutamyl peptides (GGP) that enhance flavour in foods. We sought to generate GGP from the meat crusts of dry-cured lamb, an underutilised protein resource, identify these using mass spectrometry, and validate their functional activity using a kokumi-calcium sensing receptor (CaSR) assay. The water-soluble extract (WSE) of meat crust was hydrolysed by protease A (PA) and treated with glutaminase (GA). Fifteen GGP were identified, with 14 being significantly increased in PA and GA groups compared to WSE, as were along with free amino acid levels. The GA extract activated CaSR with higher potency and efficacy than PA and WSE suggesting the generation of potent kokumi tastants. The in vitro receptor assay might be an expedient tool for screening kokumi tastants prior to conducting human sensory analysis. Collectively, our findings indicate that the meat crust can be a valuable source to generate kokumi tastants via a two-step enzymatic reaction.

Synthesis of palmitoleic acid-enriched triacylglycerol via a two-step enzyme reaction

Palmitoleic acid (POA) is known to have various biological activities in animal models. POA-enriched triacylglycerol (TAG) was successfully synthesized using a two-step enzyme reaction. First, the POA-enriched fatty acid obtained from macadamia nut oil by Candida rugosa lipase-catalyzed selective hydrolysis was employed as a substrate for the synthesis of POA-enriched TAG. Two parameters such as temperature and enzyme loading, were optimized to enrich POA by the selective hydrolysis. For the TAG synthesis, liquid Eversa lipase was immobilized in house on Lewatit VP OC 1600 as a carrier and the protein content in the carrier was 130 mg/ g carrier. The degree of synthesis of POA-enriched TAG using Eversa immobilized lipase prepared in house was investigated in comparison to three commercial lipases and Eversa immobilized lipase demonstrated a superior activity in TAG conversion. The effects of temperature, enzyme loading, and vacuum on the synthesis of POA-enriched TAG using the Eversa immobilized lipase were investigated. The optimum conditions were a temperature of 60 °C, an enzyme loading of 12.5 %, and a vacuum of 100 torr. The maximum TAG conversion was ca. 89 % after 12 h under the optimum conditions.

Efficient Synthesis of Stearidonic Acid Enriched Triacylglycerol from Ahiflower Seed Oil via a Two-Step Enzyme Reaction.

Stearidonic acid (SDA) is a plant-based n-3 polyunsaturated fatty acid with multiple biological activities. The enrichment of SDA and synthesis of triacylglycerol (TAG) were carried out consecutively via two lipase-catalyzed reactions, hydrolysis, and esterification. First, SDA was enriched into a glyceride fraction from ahiflower seed oil by Candida rugosa lipase-catalyzed hydrolysis. Under the optimum conditions of 35°C, 0.1% lipase powder of Lipase OF, and 50% buffer solution (based on the weight of total substrate), SDA was enriched from 21.6 to 40.7 wt% in glyceride fraction. SDA-enriched TAG was then synthesized from the SDA-enriched glyceride and the SDA-enriched fatty acid via esterification using an in-house immobilized lipase as a biocatalyst. The SDA-enriched fatty acid was obtained from part of the SDA-enriched glyceride by saponification and the in-house immobilized lipase was prepared from Eversa® Transform 2.0 using Lewatit VP OC 1600 as a carrier. The optimum reaction conditions for the synthesis of TAG were a temperature of 50°C, an enzyme loading of 10%, and a vacuum of 10 mmHg. A maximum conversion to TAG of ca. 94% was achieved after 12 h under the optimum conditions.

Epimerization of Deoxynivalenol by the Devosia Strain A6-243 Assisted by Pyrroloquinoline Quinone

Deoxynivalenol (DON) is a secondary metabolite produced by several Fusarium species that is hazardous to humans and animals after entering food chains. In this study, by adding cofactors, the Devosia strain A6-243 is identified as the DON-transforming bacteria from a bacterial consortium with the ability to biotransform DON of Pseudomonas sp. B6-24 and Devosia strain A6-243, and its effect on the biotransformation process of DON is studied. The Devosia strain A6-243 completely biotransformed 100 μg/mL of DON with the assistance of the exogenous addition of PQQ (pyrroloquinoline quinone) within 48 h and produced non-toxic 3-epi-DON (3-epi-deoxynivalenol), while Pseudomonas sp. B6-24 was not able to biotransform DON, but it had the ability to generate PQQ. Moreover, the Devosia strain A6-243 not only degraded DON, but also exhibited the ability to degrade 3-keto-DON (3-keto-deoxynivalenol) with the same product 3-epi-DON, indicating that DON epimerization by the Devosia strain A6-243 is a two-step enzymatic reaction. The most suitable conditions for the biodegradation process of the Devosia strain A6-243 were a temperature of 16–37 °C and pH 7.0–10, with 15–30 μM PQQ. In addition, the Devosia strain A6-243 was found to completely remove DON (6.7 μg/g) from DON-contaminated wheat. The results presented a reference for screening microorganisms with the ability of biotransform DON and laid a foundation for the development of enzymes for the detoxification of mycotoxins in grain and its products.

High-performance, two-step/Bi-enzyme lactate biofuel cell with lactate oxidase and pyruvate oxidase

The performance of biofuel cells (BFCs) depends on the number of electrons that can be harvested from one fuel molecule by the bioanode, as well as many other factors. Regular, one-enzyme bioanodes can usually harvest two electrons per fuel molecule, lowering the conversion efficiency of chemical energy stored in the fuel to electrical energy. In this study, we successfully develop a high-performance BFC with a two-enzyme bioanode that can harvest four electrons by oxidizing lactate to acetylphosphate through a two-step enzymatic reaction that uses lactate oxidase (LOx) and pyruvate oxidase (POx). A current of 1 mA is obtained from the BFC for 2500 s, with only a slight decrease in the cell voltage, reaching 0 V at 3010 s. During this time, a total of 1253 mJ is harvested. This is approximately 1.8-fold the time and double the energy that is obtained from a corresponding one-enzyme-bioanode BFC employing only LOx, thus confirming that twice the number of electrons are harvested per lactate molecule.

The effects of different plant extracts on bile salt hydrolase activity of Lactobacillus strains isolated from the gastrointestinal tract of poultry

The bile salt hydrolysis (BSH) enzyme weakens fat metabolism through bile salt deconjugation and reduces poultry performance, in order to cope with the antibacterial properties of the bile. Therefore, reducing the activity of this enzyme through the use of feed additives is probably a promising alternative to antibiotics for improving poultry performance. Plant extracts have long been used as feed additives for promoting poultry growth. In the current experiment, five Lactobacillus strains including Lactobacillus animalis, Lactobacillus acidophillus, Lactobacillus gallinarum, Lactobacillus lactis, and Lactobacillus returi were obtained from the poultry hindgut and were used as the probiotic application. A plate test and two-step enzymatic reaction method were used for deconjugation activity determination of the Lactobacillus strains. Further, four plant extracts (i.e., the aerial parts of Rosemary (Rosmarinus officinalis), Roselle calyx (Hibiscus sabdariffa), Berberis vulgaris root, and Green tea) were examined in terms of BSH enzyme inhibitors using the cell-free extracts as the potential antibiotic alternative. Furthermore, the gallbladders of the broilers were freshly collected from the poultry slaughterhouses, and their contents were extracted. The results showed that all Lactobacillus strains could hydrolyze the taurocholate acid (TCA) and chicken bile salt mixture (CBSM) to unconjugated bile acid. Moreover, ethanolic extracts of B. vulgaris root and Green tea relatively reduced the activity of the BSH enzyme that could potentially be investigated as an appropriate alternative in poultry feed in vivo. In conclusion, all five Lactobacillus strains were resistant to bile salts (i.e. TCA and CBSM) by BSH activity, and the addition of Green tea and B. vulgaris root extracts to the bacterial medium demonstrated inhibitory effects against the BSH enzyme.

Site-Specific Antibody Conjugation Strategy to Functionalize Virus-Based Nanoparticles.

Amine/thiol-reactive chemistries are commonly used to conjugate antibodies to pharmaceuticals or nanoparticles. Yet, these conjugation strategies often result in unfavorable outcomes such as heterogeneous antibody display with hindered biological activity or aggregation due to multivalent interactions of the antibody and nanoparticles. Here, we report the application of a site-specific and enzymatically driven antibody conjugation strategy to functionalize virus-based nanoparticles (VNPs). Specifically, an azide-handle was introduced into the Fc region of a set of immunoglobulins using a two-step enzymatic reaction: (1) cleavage of N-linked glycan in the Fc region by a glycosidase and (2) conjugation of a chemically reactive linker (containing an azide functional handle) using a microbial transglutaminase. Conjugation of the azide-functional antibodies to several VNPs was achieved by making use of strain-promoted azide-alkyne cycloaddition. We report the conjugation of three immunoglobulin (IgG) isotypes (human IgG from sera, anti-CD47 Rat IgG2a, κ, and Trastuzumab recombinant humanized IgG1, κ) to the plant virus cowpea mosaic virus (CPMV) and the lysine mutant of tobacco mosaic virus (TMVlys) as well as bacteriophage Qβ. Site-specific conjugation resulted in stable and functional antibody-VNP conjugates. In stark contrast, the use of heterobifunctional linkers targeting thiols and amines on the antibodies and VNPs, respectively, led to aggregation due to nonspecific and multivalent coupling between the antibodies and VNPs. We demonstrate that antibody-VNP conjugates were functional, and Trastuzumab-displaying VNPs targeted HER2-positive SKOV-3 human ovarian cancer cells. This bioconjugation strategy adds to the portfolio of methods that can be used for designing functional antibody-VNP conjugates.

Chemical Information Exchange in Organized Protocells and Natural Cell Assemblies with Controllable Spatial Positions.

An ultrasound-based platform is established to prepare homogenous arrays of giant unilamellar vesicles (GUVs) or red blood cell (RBCs), or hybrid assemblies of GUV/RBCs. Due to different responses to the modulation of the acoustic standing wave pressure field between the GUVs and RBCs, various types of protocell/natural cell hybrid assemblies are prepared with the ability to undergo reversible dynamic reconfigurations from vertical to horizontal alignments, or from 1D to 2D arrangements. A two-step enzymatic cascade reaction between transmitter glucose oxidase-containing GUVs and peroxidase-active receiver RBCs is used to implement chemical signal transduction in the different hybrid micro-arrays. Taken together, the obtained results suggest that the ultrasound-based micro-array technology can be used as an alternative platform to explore chemical communication pathways between protocells and natural cells, providing new opportunities for bottom-up synthetic biology.

Electrochemical luminescence sensor based on double suppression for highly sensitive detection of glyphosate

An electrochemical luminescence sensor based on a double inhibition strategy for the detection of glyphosate was constructed. In this method, the luminol-gold nanoparticles-L-cysteine-Cu(II) composites (Lu-Au-Lcys-Cu(II)) were used as luminescent reagents, and H2O2 obtained from two-step enzymatic hydrolysis reaction was used as the co-reactive agent of Lu-Au-Lcys-Cu(II). Glyphosate could inhibit the enzymatic reaction, and inhibit catalytic action of the peroxidase-mimicking by competing with L-cysteine to form a complex of glyphosate-Cu(II). The electrochemical luminescence intensity decreased with the increase of glyphosate concentration, under the optimal conditions, the linear range of glyphosate concentration was 0.001 ∼ 1.0 μM, and the limit of detection (LOD) could be as low as 0.5 nM. Importantly, the Electrochemiluminescence sensor also showed acceptable stability and selectivity.

A substrate tagging and two-step enzymatic reaction strategy for large-scale synthesis of 2,7-anhydro-sialic acid

A sialyltransferase acceptor tagging and two-step enzymatic reaction strategy has been developed for multigram-scale chemoenzymatic synthesis of 2,7-anhydro-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac), a compound that can serve as a sole carbon source for the growth of Ruminococcus gnavus, a common human gut commensal. Different approaches of introducing hydrophobic UV-active tags to lactose as well-suited sialyltransferase acceptors have been explored and a simple two-step high-yield chemical synthetic procedure has been identified. The UV-active hydrophobic tag facilitates monitoring reaction progress and allows facile product purification by C18-cartridges. A two-step enzyme-catalyzed reaction procedure has been established to combine with C18 cartridge-based purification process for high-yield production of the desired product in multigram scales with the recycled use of chromophore-tagged lactoside starting material and sialoside intermediate. This study demonstrated an environmentally friendly highly-efficient synthetic and purification strategy for the production of 2,7-anhydro-Neu5Ac to explore its potential functions.

Expression and Characterization of the Human Intestinal Bacterial Enzyme Which Cleaves the C-Glycosidic Bond in 3″-Oxo-puerarin.

Puerarin (daidzein 8-C-glucoside) is an isoflavone C-glucoside contained in the roots of Pueraria lobata OHWI. We have previously isolated the human intestinal bacterium, strain PUE, which metabolizes puerarin to daidzein, though the enzyme which cleaves C-glycosidic bond has not been clarified. Here, we identified one of the intermediates of enzymatic puerarin C-deglycosylation reaction as 3″-oxo-puerarin (1): C-3 in the glucose moiety connecting to hydroxyl is oxidized to ketone group. 1 was easily isomerized to the mixture of 1, 2″-oxo-puerarin (2a) and cyclic acetal (2b) of 2a in non-enzymatic condition. We identified the putative puerarin-metabolizing operon of strain PUE composed of 8 genes (dgpA-H). Among them, DgpB-C complex was expressed in Escherichia coli, which cleaved the C-glycosidic bond in 1 but not puerarin. These results suggested that the puerarin C-deglycosylation reaction is a two-step enzymatic reaction, including the oxidation reaction at C-3″ in puerarin to give 1, and the subsequent C-deglycosylation of 1 to provide daidzein.

The nanomolar sensing of nicotinamide adenine dinucleotide in human plasma using a cycling assay in albumin modified simulated body fluids

Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. In particular, extracellular NAD+ has recently been demonstrated to moderate pathogenesis of multiple systemic diseases as well as aging. Herein we present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. An albumin modified revised simulated body fluid was employed as standard matrix in order to optimise enzymatic activity and enhance the linear behaviour and sensitivity of the method. In addition, we evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. In summary, our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies.