NADH Concentration Research Articles

The relationship between resistance evolution and carbon metabolism in Staphylococcus xylosus under ceftiofur sodium stress.

Staphylococcus xylosus has emerged as a bovine mastitis pathogen with increasing drug resistance, resulting in substantial economic impacts. This study utilized iTRAQ analysis to investigate the mechanisms driving resistance evolution in S. xylosus under ceftiofur sodium stress. Findings revealed notable variations in the expression of 143 proteins, particularly glycolysis-related proteins (TpiA, Eno, GlpD, Ldh) and peptidoglycan (PG) hydrolase Atl. Following the induction of ceftiofur sodium resistance in S. xylosus, the emergence of resistant strains displaying characteristics of small colony variants (SCVs) was observed. The transcript levels of TpiA, Eno, GlpD and Ldh were up-regulated, TCA cycle proteins (ICDH, MDH) and Atl were down-regulated, lactate content was increased, and NADH concentration was decreased in SCV compared to the wild strain. That indicates a potential role of carbon metabolism, specifically PG hydrolysis, glycolysis, and the TCA cycle, in the development of resistance to ceftiofur sodium in S. xylosus.

Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity

Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd–EtfAB (BCD) complex was overexpressed, the resulting strain PRH–B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45–0.51 g/L/h and 0.46–0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin–NAD+ reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production.

Enzyme mimicking activity of the Ru(pic)3 complex in the oxidation NADH coenzymes: kinetic and mechanistic studies

Mimicking the enzymatic oxidation of NADH and obtaining mechanistic insight into the electron transfer reaction pertinent to the redox inter-conversion of NADH/NAD+ couple is a long-term challenge in biological and energy-related chemistry. The kinetics and mechanism of the oxidation of NADH by [RuIII(pic)3] (pic– = picolinate anion) has been studied spectrophotometrically and kinetically in aqueous solution at pH 7.0 (MES buffer). The reaction was followed over time at 466 nm (metal to ligand charge transfer band of [RuII(pic)3]–). The rate of NADH oxidation was found to be first-order with respect to Ru(III)-complex concentration. The values of the observed rate constant (k obs) increased linearly with increase in the concentration of NADH. The rate of the reaction was independent of the ionic strength of the reacting solution. Activation parameters (ΔH≠ = 56 ± 1 kJ mol−1 and ΔS≠ = −65 ± 3 J.deg−1 mol−1) were determined from the temperature dependence studies (15-35 °C) of the reaction. Kinetic data and activation parameters are interpreted in terms of a mechanism involving outer-sphere electron transfer.

Shewanella oneidensis MR-1 coupled with biomass-derived carbon dots for promoted bioelectrochemical CO2 reduction: Mechanism elucidation of intensified energy metabolism

Bioelectrochemical CO2 reduction reaction (CO2RR) has been touted as one of the promising strategies to convert CO2 to high-value chemicals via the efficient microbial electrosynthesis (MES). However, the sluggish electron transfer efficiency between microbes and cathodes hampers the CO2 reduction efficiency. Herein, biomass-derived carbon dots (CDs) with high electrical conductivity were coupled with Shewanella oneidensis (S. oneidensis) MR-1 to construct a nanomaterial-bacterial biohybrid system to promote CO2-to-formate formation. Specifically, the corncob-derived CDs (cc-CDs) were highly-biocompatible with S. oneidensis MR-1, with such a constructed biohybrid system showing a marked formate titer of 0.50 mmol/L. Upon doping with Fe element, the resultant Fe-cc-CDs exhibited a considerably boosted formate titer of 1.22 mmol/L, which was 7.7 times higher than the pure S. oneidensis MR-1 system. Such significant CO2-to-formate conversion enhancement was contributed by the internalized CDs in S. oneidensis MR-1 stimulating extracellular electron transfer efficiency, therefore accelerating energy metabolism for formate bioelectrosynthesis. The intensified energy metabolism in S. oneidensis MR-1 was corroborated by the increased concentrations of ATP, NADH and overexpression of energy-related genes. This work demonstrates an intensified energy metabolism exemplification for CO2 reduction, which can be expanded to a host of different nanomaterials-internalized MES systems for bioelectrochemical implications.

Quantitative Optical Redox Imaging of Melanoma Xenografts with Different Metastatic Potentials.

To develop imaging biomarkers for tumors aggressiveness, our previous optical redox imaging (ORI) studies of the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp, containing flavin adenine dinucleotide, i.e., FAD) in tumor xenografts of human melanoma associated the high optical redox ratio (ORR = Fp/(Fp + NADH)) and its heterogeneity to the high invasive/metastatic potential, without having reported quantitative results for NADH and Fp. Here, we implemented a calibration procedure to facilitate imaging the nominal concentrations of tissue NADH and Fp in the mouse xenografts of two human melanoma lines, an indolent less metastatic A375P and a more metastatic C8161. Images of the redox indices (NADH, Fp, ORR) revealed the existence of more oxidized areas (OAs) and more reduced areas (RAs) within individual tumors. ORR was found to be higher and NADH lower in C8161 compared to that of A375P xenografts, both globally for the whole tumors and locally in OAs. The ORR in the OA can differentiate xenografts with a higher statistical significance than the global averaged ORR. H&E staining of the tumors indicated that the redox differences we identified were more likely due to intrinsically different cell metabolism, rather than variations in cell density.

Estimation of the kinetic parameters (Km & Vmax) and the optimization of yeast alcohol dehydrogenase (ADH) assay.

Alcohol dehydrogenases (ADHs) are zinc-containing enzymes that catalyze the oxidation of alcohols to aldehydes or ketones. The enzymes also play a critical role in the metabolism of a number of drugs and metabolites containing alcohol functional groups. Kinetic parameters of yeast alcohol dehydrogenase (ADH) was estimated using spectrophotometer, optimum temperature and pH for ethanol, as well as ADH specificity to other substrates was determined. Results from the assay showed value for the initial velocity of reaction of the enzyme estimated as 0.297. Change in concentration of NADH which correspond to ADH activity for ethanol was calculated as 48nanokatals (4.8 X 10-5 mol-1l), the results also showed average values of the Km and Vmax of ADH for ethanol as estimated from Michaelis-Menten and Line weaver-Burk double reciprocal plots, to be 21.5mM and 0.426 respectively. Optimum temperature and pH of ADH for ethanol were estimated to be 25 °C and 8 respectively. Although, reaction specificity of ADH to other substrates showed gradation between propan-2-ol and ethanol with ethanol showing highest activity and methanol least. 2-propene-1-ol also presented high enzyme activity relative to ethanol. In most of enzyme assays, enzyme activity is determined by measuring the rate of conversion of substrate or rate of production of products within a given period of time. In this experiment, the rate of oxidation of NADH was monitored since NADH has a known maximum light absorbance at 340nm.

Metabolic perturbation of Streptomyces albulus by introducing NADP-dependent glyceraldehyde 3-phosphate dehydrogenase.

The available resources of Streptomyces represent a valuable repository of bioactive natural products that warrant exploration. Streptomyces albulus is primarily utilized in the industrial synthesis of ε-poly-L-lysine (ε-PL). In this study, the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans was heterologously expressed in S. albulus CICC11022, leading to elevated intracellular NADPH levels and reduced NADH and ATP concentrations. The resulting perturbation of S. albulus metabolism was comprehensively analyzed using transcriptomic and metabolomic methodologies. A decrease in production of ε-PL was observed. The expression of gapN significantly impacted on 23 gene clusters responsible for the biosynthesis of secondary metabolites. A comprehensive analysis revealed a total of 21 metabolites exhibiting elevated levels both intracellularly and extracellularly in the gapN expressing strain compared to those in the control strain. These findings underscore the potential of S. albulus to generate diverse bioactive natural products, thus offering valuable insights for the utilization of known Streptomyces resources through genetic manipulation.

Biochemical and metabolomics analyses reveal the mechanisms underlying ascorbic acid and chitosan coating mediated energy homeostasis in postharvest papaya fruit

Papaya is a climacteric fruit that undergoes rapid ripening and quality deterioration during postharvest storage, resulting in significant economic losses. This study employed biochemical techniques and targeted metabolomics to investigate the impact of exogenous AsA + CTS application on the energy metabolism regulation of papaya fruit during postharvest storage. We found that AsA + CTS treatment significantly increased the levels of key metabolic compounds and enzymes, such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and the energy charge, as well as the succinic acid content and the activities of succinic dehydrogenase (SDH), cytochrome c oxidase (CCO), H+-ATPase, and Ca2+-ATPase. Moreover, AsA + CTS coating augmented the nicotinamide adenine dinucleotide kinase (NADK) activity and increased the NADH and NADPH concentrations. Regarding sugar metabolism, it increased the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase and raised d-glucose-6-phosphate levels. These findings suggest that AsA + CTS coating application can mitigate the metabolic deterioration and sustain a primary metabolism homeostasis in papaya fruit by enhancing the tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP), thereby preserving their quality attributes during postharvest storage.

Loss-of-function mutations in ndh do not confer delamanid, ethionamide, isoniazid, or pretomanid resistance in Mycobacterium tuberculosis.

Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that ndh (Rv1854c) is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis. This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration.

Smart Ratiometric SERS Nanoprobe for Real-Time Monitoring Hydrogen Peroxide in Living Cells during NADH Treatment Associated with Ferroptosis.

Studying the oxidative stress, especially the reactive oxygen species (ROS) response of ferroptosis, is crucial for the diagnosis and treatment of cancer based on ferroptosis. However, reliable quantitative analysis of intracellular ROS in cancer treatment for drug screening is still a challenge. Herein, a superior ratiometric SERS nanoprobe was developed for in situ, real-time, and highly sensitive detection of content variation of H2O2 within living cells. The SERS nanoprobe was prepared by coassembly of the internal standard molecule p-mercaptobenzonitrile and the reporter molecule p-mercaptophenylboronic acid on the surface of gold nanoparticles, used for synergistic calibration and detection of H2O2, which enables reliable detection of the true content of intracellular H2O2 without the interference of other substances in cells. Based on the nanoprobe, we found that the level of intracellular H2O2 of cancer cells was increased after the nicotinamide adenine dinucleotide (NADH) treatment, with a dose-dependence to the concentration of NADH. High doses of NADH (above 20 mM) can induce cell death by means of ferroptosis associated with the level elevation of intracellular lipid hydroperoxides. This study highlights the potential of the SERS nanoprobe for tracking content variation of cellular H2O2 and understanding its roles in screening new anticancer drugs.

Near-infrared absorption and emission probes with optimal connection bridges for live monitoring of NAD(P)H dynamics in living systems

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Probe solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 µM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe A in sensing changes in NAD(P)H levels within a whole organism.

NADH-based kinetic model for acetone-butanol-ethanol production by Clostridium

We present in this work a kinetic model of the acetone-butanol-ethanol (ABE) fermentation based on enzyme kinetics expressions. The model includes the effect of the co-substrate NADH as a modulating factor of cellular metabolism. The simulations obtained with the model showed an adequate fit to the experimental data reported by several authors, matching or improving the results observed with previous models. In addition, this model does not require artificial mathematical strategies such as on-off functions to achieve a satisfactory fit of the ABE fermentation dynamics. The parametric sensitivity allowed to identify the direct glucose → acetyl-CoA → butyryl-CoA pathway as being more significant for butanol production than the acid re-assimilation pathway. Likewise, model simulations showed that the increase in NADH, due to glucose concentration, favors butanol production and selectivity, finding a maximum selectivity of 3.6, at NADH concentrations above 55 mM and glucose concentration of 126 mM. The introduction of NADH in the model would allow its use for the analysis of electrofermentation processes with Clostridium, since the model establishes a basis for representing changes in the intracellular redox potential from extracellular variables.

Quantitative Galactose Colorimetric Competitive Assay Based on Galactose Dehydrogenase and Plasmonic Gold Nanostars.

We describe a competitive colorimetric assay that enables rapid and sensitive detection of galactose and reduced nicotinamide adenine dinucleotide (NADH) via colorimetric readouts and demonstrate its usefulness for monitoring NAD+-driven enzymatic reactions. We present a sensitive plasmonic sensing approach for assessing galactose concentration and the presence of NADH using galactose dehydrogenase-immobilized gold nanostars (AuNS-PVP-GalDH). The AuNS-PVP-GalDH assay remains turquoise blue in the absence of galactose and NADH; however, as galactose and NADH concentrations grow, the reaction well color changes to a characteristic red color in the presence of an alkaline environment and a metal ion catalyst (detection solution). As a result, when galactose is sensed in the presence of H2O2, the colored response of the AuNS-PVP-GalDH assay transforms from turquoise blue to light pink, and then to wine red in a concentration-dependent manner discernible to the human eye. This competitive AuNS-PVP-GalDH assay could be a viable analytical tool for rapid and convenient galactose quantification in resource-limited areas.

Alkaline arginine promotes the gentamicin-mediated killing of drug-resistant Salmonella by increasing NADH concentration and proton motive force.

Antimicrobial resistance, especially the development of multidrug-resistant strains, is an urgent public health threat. Antibiotic adjuvants have been shown to improve the treatment of resistant bacterial infections. We verified that exogenous L-arginine promoted the killing effect of gentamicin against Salmonella in vitro and in vivo, and measured intracellular ATP, NADH, and PMF of bacteria. Gene expression was determined using real-time quantitative PCR. This study found that alkaline arginine significantly increased gentamicin, tobramycin, kanamycin, and apramycin-mediated killing of drug-resistant Salmonella, including multidrug-resistant strains. Mechanistic studies showed that exogenous arginine was shown to increase the proton motive force, increasing the uptake of gentamicin and ultimately inducing bacterial cell death. Furthermore, in mouse infection model, arginine effectively improved gentamicin activity against Salmonella typhimurium. These findings confirm that arginine is a highly effective and harmless aminoglycoside adjuvant and provide important evidence for its use in combination with antimicrobial agents to treat drug-resistant bacterial infections.

Development and Application of an Electrochemical Sensor with 1,10-Phenanthroline-5,6-dione-Modified Electrode for the Detection of Escherichia coli in Water

The routine monitoring of bacterial populations is crucial for ensuring water quality and safeguarding public health. Thus, an electrochemical sensor based on a 1,10-phenanthroline-5,6-dione-modified electrode was developed and explored for the detection of E. coli. The modified electrode exhibited enhanced NADH oxidation ability at a low potential of 0.1 V, which effectively eliminated the interference from other redox compounds in bacteria. The sensitivity for NADH was 0.222 μA/μM, and the limit of detection was 0.0357 μM. Upon cell lysis, the intracellular NADH was released, and the concentration of E. coli was determined through establishing the relationship between the oxidation current signal and NADH concentration. The performance of the electrochemical sensor in the detection of NADH and E. coli suspensions was validated using the WST-8 colorimetric method. The blank recovery experiment in real water samples exhibited good accuracy, with recovery rates ranging from 89.12% to 93.26% and relative standard deviations of less than 10%. The proposed electrochemical sensor realized the detection of E. coli without the usage of biomarkers, which provides a promising approach for the broad-spectrum detection of microbial contents in complex water environments.

Exploring influence mechanism of small-molecule carbon source on heterotrophic nitrification-aerobic denitrification process from carbon metabolism, nitrogen metabolism and electron transport process

The heterotrophic nitrification-aerobic denitrification (HNAD) process can remove nitrogen and organic carbon under aerobic conditions. To get the in-depth mechanism of the HAND process, a strain named Acinetobacter johnsonii ZHL01 was isolated, and enzyme activity, electron transport, energy production, and gene expression of the strain were studied with small-molecule carbon sources, including sodium citrate, sodium acetate, sodium fumarate, and sodium succinate. The HNAD pathway of ZHL01 was NH4+→NH2OH → NO, and nitrogen balance analysis shows that ZHL01 could assimilate and denitrify 58.29 ± 1.05 % and 16.58 ± 1.07 % of nitrogen, respectively. The assimilation, the nitrification/denitrification, and the respiration processes were regulated by the concentration of reduced nicotinamide adenine dinucleotide (NADH) produced from the different metabolic pathways of small-molecule carbon sources. The HNAD process occurs to reduce intracellular redox levels related to NADH concentrations. This discovery provides a theoretical basis for the practical application of HAND bacteria.

Abstract P2121: Relative Importance Of Reactive Oxygen Species From Reverse Electron Transfer At Mitochondrial Complex I In Reperfusion Injury

The metabolite succinate accumulates during tissue ischemia. In early reperfusion, succinate is rapidly metabolized, driving a burst of mitochondrial reactive oxygen species (ROS) generation that triggers cell death. It is widely believed that the source of succinate-driven ROS in early reperfusion is reverse electron transfer (RET) at respiratory complex I (Cx-I). However, evidence for Cx-I RET ROS is largely based on experimental evidence from isolated mitochondria with succinate as the sole substrate. We hypothesized that numerous other conditions present during early reperfusion may impact Cx-I RET ROS. Thus, experimental perturbations were applied to mitochondria to model early reperfusion, with measurement of ROS via the fluorescent probe PHPA. These conditions included: (i) A high concentration of NADH; (ii) A high concentration of lactate; (iii) A mildly acidic pH; (iv) A non-zero concentration of ADP buffered by a creatine / creatine phosphate / creatine kinase system; (v) Presence of purine nucleotide breakdown products; (vi) Presence of calcium. These studies revealed that under early reperfusion-like conditions mitochondrial ROS generation still occurred via Cx-I RET, but at a magnitude only half that previously reported. Furthermore, residual ROS that was insensitive to a specific Cx-I RET inhibitor (S1QEL) was slightly elevated under early-reperfusion like conditions, and this residual ROS was mostly inhibited by S3QEL, a blocker of ROS from respiratory complex III (Cx-III). Together, these data suggest that the relative contribution of Cx-I RET ROS to reperfusion injury may be overestimated, and that of Cx-III ROS underestimated.

Modified Lactoperoxidase System as a Promising Anticaries Agent: In Vitro Studies on Streptococcus mutans Biofilms.

The lactoperoxidase (LPO) system shows promise in the prevention of dental caries, a common chronic disease. This system has antimicrobial properties and is part of the non-specific antimicrobial immune system. Understanding the efficacy of the LPO system in the fight against biofilms could provide information on alternative strategies for the prevention and treatment of caries. In this study, the enzymatic system was modified using four different (pseudo)halide substrates (thiocyanate, thiocyanate-iodide mixture, selenocyanate, and iodide). The study evaluated the metabolic effects of applying such modifications to Streptococcus mutans; in particular: (1) biofilm formation, (2) synthesis of insoluble polysaccharides, (3) lactate synthesis, (4) glucose and sucrose consumption, (5) intracellular NAD+ and NADH concentrations, and (6) transmembrane glucose transport efficiency (PTS activity). The results showed that the LPO-iodide system had the strongest inhibitory effect on biofilm growth and lactate synthesis (complete inhibition). This was associated with an increase in the NAD+/NADH ratio and an inhibition of glucose PTS activity. The LPO-selenocyanate system showed a moderate inhibitory effect on biofilm biomass growth and lactate synthesis. The other systems showed relatively small inhibition of lactate synthesis and glucose PTS but no effect on the growth of biofilm biomass. This study provides a basis for further research on the use of alternative substrates with the LPO system, particularly the LPO-iodide system, in the prevention and control of biofilm-related diseases.

Molecular characterization of azoreductase and its potential for the decolorization of Remazol Red R and Acid Blue 29

Azoreductase is a reductive enzyme that efficiently biotransformed textile azo dyes. This study demonstrated the heterologous overexpression of the azoreductase gene in Escherichia coli for the effective degradation of Remazol Red-R and Acid-Blue 29 dyes. The AzK gene of Klebsiella pneumoniae encoding a ≈22 kDa azoreductase enzyme was cloned into the pET21+C expression vector. The inoculum size of 1.5%, IPTG concentration of 0.5 mM, and incubation time of 6 h were optimized by response surface methodology a statistical tool. The crude extract showed 76% and 74%, while the purified enzyme achieved 94% and 93% decolorization of RRR and AB-29, respectively in 0.3 h. The reaction kinetics showed that RRR had a Km and Vmax value of 0.058 mM and 1416 U mg−1, respectively at an NADH concentration of 10 mM. HPLC and GC-MS analyses showed that RRR was effectively bio-transformed by azoreductase to 2-[3-(hydroxy-amino) benzene-1-sulfonyl and AB-29 to aniline and 3-nitrosoaniline. This study explored the potential of recombinant azoreductase isolated from K. pneumoniae in the degradation of toxic textile azo dyes into less toxic metabolites.

Photometric flow system for the determination of serum lactate dehydrogenase activity

The routine method for LDH (Lactate dehydrogenase) activity determination is to monitor the increase of NADH concentration at 340 nm. There are some inconvenience in taking measurements in the near-UV region, especially in the case of serum samples analysis. In this work, two modifications of the routine LDH activity assay based on the use of reducing properties of NADH have been compared. Both methods involved the reduction of compounds that can be easily determined by well-known methods, ferric ion (with ferrozine) and nitrotetrazolium blue (NBT). A fully-mechanized Multicommutated Flow Analysis-Paired Emitter Detector Diode (MCFA-PEDD) system based on solenoid devices was developed and applied for both methods. The linear ranges obtained for Fe-ferrozine and NBT methods are 6.0–200.0 U L−1 and 10.0–250.0 U L−1 with estimated detection limits at 0.2 U L−1 and 4.5 U L−1, respectively. The low LOQ values enabled 10-fold sample dilutions, which is advantageous for samples with limited available volume. The Fe-ferrozine method is more selective for LDH activity in the presence of glucose, ascorbic acid, albumin, bilirubin, copper and calcium ions than NBT method. To confirm the analytical usefulness of the proposed flow system, the analysis of real human serum samples was carried out. The statistic tests showed satisfactory correlation between the results obtained for both developed methods and those received using the reference method.